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Human MicroRNA Targets

MicroRNAs (miRNAs) interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. The specific function of most mammalian miRNAs is unknown. We have predicted target sites on the 3′ untranslated regions of human gene transcripts for all currently known 218 mammalian miRNAs to facilitate focused experiments. We report about 2,000 human genes with miRNA target sites conserved in mammals and about 250 human genes conserved as targets between mammals and fish. The prediction algorithm optimizes sequence complementarity using position-specific rules and relies on strict requirements of interspecies conservation. Experimental support for the validity of the method comes from known targets and from strong enrichment of predicted targets in mRNAs associated with the fragile X mental retardation protein in mammals. This is consistent with the hypothesis that miRNAs act as sequence-specific adaptors in the interaction of ribonuclear particles with translationally regulated messages. Overrepresented groups of targets include mRNAs coding for transcription factors, components of the miRNA machinery, and other proteins involved in translational regulation, as well as components of the ubiquitin machinery, representing novel feedback loops in gene regulation. Detailed information about target genes, target processes, and open-source software for target prediction (miRanda) is available at http://www.microrna.org . Our analysis suggests that miRNA genes, which are about 1% of all human genes, regulate protein production for 10% or more of all human genes.

Introduction The Functions of MicroRNAs In the past three years, several hundred novel genes encoding transcripts containing short double-stranded RNA hairpins, named microRNAs (miRNAs), were identified in plants and animals ( Lee et al. 1993 ; Reinhart et al. 2000 , 2002 ; Lagos-Quintana et al. 2001 , 2002 , 2003 ; Lau et al. 2001 ; Lee and Ambros 2001 ; Llave et al. 2002a ; Mette et al. 2002 ; Mourelatos et al. 2002 ; Park et al. 2002 ; Ambros et al. 2003b ; Aravin et al. 2003 ; Brennecke et al. 2003 ; Dostie et al. 2003 ; Grad et al. 2003 ; Houbaviy et al. 2003 ; Lai et al. 2003 ; Lim et al. 2003a , 2003b ; Palatnik et al. 2003 ). More recently, miRNAs have also been identified in a large DNA virus, the Epstein Barr virus, and are likely to be found in other viruses ( Pfeffer et al. 2004 ). The cellular functions of most animal miRNAs are unknown. More than ten years after the discovery of the first miRNA gene, lin-4 ( Chalfie et al. 1981 ; Lee et al. 1993 ), we know that miRNA genes constitute about 1%–2% of the known genes in eukaryotes. Investigation of miRNA expression combined with genetic and molecular studies in Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana have identified the biological functions of several miRNAs (recent review, Bartel 2004 ). In C. elegans, lin-4 and let-7 were first discovered as key regulators of developmental timing in early larval developmental transitions ( Ambros 2000 ; Abrahante et al. 2003 ; Lin et al. 2003 ; Vella et al. 2004 ). More recently lsy-6 was shown to determine the left–right asymmetry of chemoreceptor expression ( Johnston and Hobert 2003 ). In D. melanogaster, miR-14 has a role in apoptosis and fat metabolism ( Xu et al. 2003 ) and the bantam miRNA targets the gene hid involved in apoptosis and growth control ( Brennecke et al. 2003 ). In mouse, miR-181a modulates hematopoietic differentiation ( Chen et al. 2004 ), and miR-196 directs the cleavage of Hox-B8 transcripts ( Yekta et al. 2004 ). miRNAs have specificity. In a range of organisms, miRNAs are differentially expressed in developmental stages, cell types, and tissues ( Lee and Ambros 2001 ; Lagos-Quintana et al. 2002 ; Sempere et al. 2004 ). In particular, differential expression has been observed in mammalian organs ( Lagos-Quintana et al. 2002 ; Krichevsky et al. 2003 ; Sempere et al. 2004 ) and embryonic stem cells ( Houbaviy et al. 2003 ). Estimates in worm show that there are approximately 1,000 molecules of miRNA per cell, with some cells exceeding 50,000 molecules ( Lim et al. 2003b ). This dynamic range of regulation of miRNA expression underscores the regulatory functional importance of miRNAs. The Mechanism of miRNA Action How do miRNAs pair with their target messages? miRNAs cause the translational repression or cleavage of target messages ( Doench and Sharp 2004 ). Some miRNAs may behave like small interfering RNAs (siRNAs) that direct mRNA cleavage between the nucleotide positions 10 and 11 in the siRNA:mRNA target duplex ( Tuschl et al. 1999 ; Zamore et al. 2000 ; Elbashir et al. 2001 ; Hutvágner and Zamore 2002a ; Llave et al. 2002b ; Martinez et al. 2002 ; Bartel 2004 ; Yekta et al. 2004 ). It appears that the extent of base pairing between the small RNA and the mRNA determines the balance between cleavage and degradation ( Hutvágner and Zamore 2002a ). Recent examples of cleavage of target messages are, in mouse, mir-196 guiding cleavage of Hox-B8 transcripts ( Yekta et al. 2004 ) and, in Epstein Barr virus, miR-BART2, a virus-encoded miRNA, guiding the cleavage of transcripts for virus DNA polymerase (gene BALF5 ) ( Pfeffer et al. 2004 ). While cleavage of mRNA is a straightforward process, the details of the mechanism of translational repression are unknown. The following rules for matches between miRNA and target messages have been deduced from a range of experiments. (1) Asymmetry: experimentally verified miRNA target sites indicate that the 5′ end of the miRNA tends to have more bases complementary to the target than its 3′ end. Loopouts in either the mRNA or the miRNA between positions 9 and 14 of the miRNA have been observed or deduced ( Brennecke et al. 2003 ; Johnston and Hobert 2003 ; Lin et al. 2003 ; Vella et al. 2004 ). Recent experiments show some correlation between the level of translational repression and the free energy of binding of the first eight nucleotides in the 5′ region of the miRNA ( Doench and Sharp 2004 ). However, confirmed miRNA:mRNA target pairs can have mismatches in this region ( Moss et al. 1997 ; Johnston and Hobert 2003 ). (2) G:U wobbles: wobble base pairs are less common in the 5′ end of a miRNA:mRNA duplex, and recent work shows a disproportionate penalty of G:U pairing relative to standard thermodynamic considerations ( Doench and Sharp 2004 ). (3) Cooperativity of binding: many miRNAs can bind to one gene ( Reinhart et al. 2000 ; Ambros 2003 ; Vella et al. 2004 ), and the target sites may overlap to some degree ( Doench and Sharp 2004 ). Given the overlap between the siRNA and miRNA pathways, it is reasonable to assume that rules of regulation in the siRNA pathway will partly apply to miRNA target recognition ( Hutvágner and Zamore 2002b ; Boutet et al. 2003 ; Doench et al. 2003 ). Lately, detailed characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the3′ terminus, lack of inverted repeats, and strand base preferences (positions 3, 10, 13, and 19) ( Jackson et al. 2003 ; Reynolds et al. 2004 ). These observations may provide clues for better quantitative description of miRNA:mRNA interaction. Regions adjacent or near to the target site can be important for miRNA specificity. In lin-41, a 27-nucleotide (nt) intervening sequence between two consecutive let-7 sites is necessary for its regulation ( Vella et al. 2004 ). Because of lack of conservation of this 27-nt intervening sequence in C. briggsae, incorporation of a corresponding rule is premature. Maturation of miRNAs and Assembly in RNA-Induced Silencing Complex miRNAs are transcribed as longer precursors, termed pre-miRNAs ( Lee et al. 2002 ), sometimes in clusters and frequently in introns (25% of human miRNAs; Table S1 ). Upon transcription, miRNAs undergo nuclear cleavage by the RNase III endonuclease Drosha, producing the 60–70-nt stem-loop precursor miRNA (pre-miRNA) with a 5′ phosphate and a 2-nt 3′ overhang ( Lee et al. 2003 ). The pre-miRNA is subsequently transported across the nuclear membrane, dependent on the protein exportin 5 ( Lund et al. 2003 ; Yi et al. 2003 ). Dicer cleaves the pre-miRNA in the cytoplasm about two helical turns away from the ends of the pre-miRNA stem loop, producing double-stranded RNA. A helicase unwinds the cleaved double-stranded RNA in a strand-specific direction ( Khvorova et al. 2003 ; Schwarz et al. 2003 ). One of the unwound strands is subsequently incorporated into a ribonuclear particle (RNP) complex, RNA-induced silencing complex (RISC) ( Hutvágner and Zamore 2002a ; Martinez et al. 2002 ). Every RISC contains a member of the Argonaute protein family, which tightly binds the RNA in the complex ( Hammond et al. 2001 ; Hutvágner and Zamore 2002a ; Martinez et al. 2002 ; Mourelatos et al. 2002 ). There are at least eight members of the Argonaute family in mammals ( Sasaki et al. 2003 ), and only a small subset has been functionally characterized. The Argonautes and Dicer bind single-stranded RNA via their PAZ domains ( Lingel et al. 2003 ; Sasaki et al. 2003 ; Song et al. 2003 ; Yan et al. 2003 ), and the known structures of the PAZ domains may have implications for prediction of miRNA targets ( Lingel et al. 2003 ; Song et al. 2003 ; Yan et al. 2003 ). Association of mRNAs and miRNAs with Fragile X Mental Retardation Protein Among the prime candidates for miRNA control are the genes that are posttranscriptionally regulated. The mRNA-binding protein fragile X mental retardation protein (FMRP) is involved in the regulation of local protein synthesis ( Antar and Bassell 2003 ) and binds 4% of mRNAs expressed in the rat brain, as tested in vitro ( Brown et al. 2001 ). The loss of function of FMRP causes fragile X syndrome, the most prevalent form of mental retardation (one in every 2,000 children). Over the past three years a number of different groups have identified in vivo mRNA cargoes of FMRP. The Warren and Darnell laboratories have identified ligands by co-immunoprecipitation followed by microarray analysis, complemented by extraction of polyribosomal fractions ( Brown et al. 2001 ). They discovered that FMRP and one of its three RNA-binding domains specifically binds to G-rich quartet motifs ( Brown et al. 2001 ; Darnell et al. 2001 ; Denman 2003 ; Miyashiro et al. 2003 ). Three more studies found that mRNAs containing U-rich motifs bind recombinant FMRP in vitro and associate with FMRP-containing mRNPs in vivo ( Chen et al. 2003 ; Denman 2003 ). Lastly, antibody-positioned RNA amplification as a primary screen followed by traditional methods identified over 80 FMRP-regulated mRNAs, with a combination of G-quartet and U-rich motifs in their mRNA sequences ( Miyashiro et al. 2003 ). Independently, FMRP has been shown to be associated with RISC components and miRNAs ( Jin et al. 2004 ). The Drosophila homolog of FMRP (FXR) and the Vasa intronic gene were identified as components of RISC ( Caudy et al. 2002 ). More recent studies have proved that mammalian FMRP interacts with miRNAs and with the components of the miRNA pathways including Dicer and the mammalian orthologs of Argonaute (AGO) 1 ( Ishizuka et al. 2002 ; Jin et al. 2004 ). Given the association of FMRP with Argonaute-containing complexes, we propose and investigate the hypothesis that the cargoes carried by FMRP are also miRNA targets, and we derive hypotheses of specific pairing interactions. Here, we predict miRNA targets in five vertebrate genomes as a way of facilitating experiments and exploring a number of open questions. What proportion of all genes is regulated by miRNAs? How many genes are regulated by each miRNA? Are specific cellular processes targeted by specific miRNAs or by miRNAs in general? What is the extent of cooperativity in miRNA:mRNA binding? Results Prediction of miRNA Targets Using currently known mammalian miRNA sequences, we scanned 3′ untranslated regions (UTRs) from the human (Homo sapiens), mouse (Mus musculus), and rat (Rattus norvegicus) genomes for potential target sites. The scanning algorithm was based on sequence complementarity between the mature miRNA and the target site, binding energy of the miRNA–target duplex, and evolutionary conservation of the target site sequence and target position in aligned UTRs of homologous genes. We identified as conserved across mammals a total of 2,273 target genes with more than one target site at 90% conservation of target site sequence (Tables S2 and S3 ) and 660 target genes at 100% conservation. We also scanned the zebrafish (Danio rerio) and fugu (Fugu rubripes) fish genomes for potential targets using known and predicted miRNAs ( Figure 1 ; Tables S4 and S5 ) and identified 1,578 target genes with two or more conserved miRNA sites between the two fish species. Figure 1 Target Prediction Pipeline for miRNA Targets in Vertebrates The mammalian (human, mouse, and rat) and fish (zebra and fugu) 3′ UTRs were first scanned for miRNA target sites using position-specific rules of sequence complementarity. Next, aligned UTRs of orthologous genes were used to check for conservation of miRNA–target relationships (“target conservation”) between mammalian genomes and, separately, between fish genomes. The main results (bottom) are the conserved mammalian and conserved fish targets, for each miRNA, as well as a smaller set of super-conserved vertebrate targets. In addition to the analysis of 3′ UTRs, we also scanned all protein-coding regions for high-scoring miRNA target sites. For convenience, these results are reported both as hits in cDNAs (coding plus noncoding; Table S6 ) and as hits in coding regions ( Table S7 ), with cross-references in the UTR target tables (number of hits in the coding region for each UTR in Tables S2 and S3 ). The algorithm and cutoff parameters were chosen to provide a flexible mechanism for position-specific constraints and to capture what is currently known about experimentally verified miRNA target sites: (1) nonuniform distribution of the number of sequence-complementary target sites for different miRNAs; (2) 5′–3′ asymmetry (the complementary pairing of about ten nucleotides at the 5′ end is more important than that of the ten nucleotides at the 3′ end [ Doench and Sharp 2004 ], and the matches near the 3′ end can to a limited extent compensate for weaker 5′ binding); and (3) influence of G:U wobbles on binding ( Doench and Sharp 2004 ). In choosing these parameters, we drew on experience from careful analysis of target predictions in Drosophila ( Enright et al. 2003 ) as well as proposed human targets of virus-encoded miRNAs ( Pfeffer et al. 2004 ). To facilitate evaluation of predicted targets and design of new experiments, we provide methods and results in a convenient and transparent form. We make the miRanda software freely available under an open-source license, so that researchers can adjust the algorithm, numerical parameters, and position-specific rules. We also provide web resources, including a viewer for browsing potential target sites, conserved with or without positional constraints, on aligned UTRs, with periodic updates ( http://www.microrna.org , as well as links to these targets from the miRNA registry site RFAM ( http://www.sanger.ac.uk ; Griffiths-Jones 2004 ). We provide both high-scoring targets, as strong candidates for validation experiments, and lower-scoring targets, which may have a role in broader background regulation of protein dose. Expression information (see Table S3 ) for miRNAs and mRNAs provides an additional filter for validation experiments, in addition ranking target sites by complementarity and evolutionary conservation. Validation of Target Predictions Only a small number of target sites of target genes regulated by miRNAs have been experimentally verified, so we sought direct and indirect evidence to help validate or invalidate the proposed set of mammalian targets. (1) We compared predicted targets with experimentally verified targets in mammals, C. elegans, and D. melanogaster , as well as their mammalian homologs. (2) We compared predicted target numbers from real and shuffled miRNA sequences and estimated the rate of false-positive predictions. (3) We assessed the enrichment of miRNA targets in mRNAs that are known cargoes of FMRP, an RNA-binding protein known to be involved in translational regulation. Agreement with known targets We previously used known miRNA sites for the let- 7 and lin-4 miRNAs in Drosophila to develop the target prediction method and check for consistency ( Enright et al. 2003 ). More recent experimental target identification provides independent control data. Recent work in C. elegans ( Vella et al. 2004 ) has narrowed the originally reported list of six target sites for let-7 in the UTR of lin-41 down to three elements, two target sites, and a 27-nt intervening sequence (a possible binding site for another factor). The surviving two target sites have high alignment scores, S = 115 and S = 110, while the other four sites are below threshold ( Enright et al. 2003 ), fully consistent with the experimental results. As one of the confirmed sites has a single-residue bulge, target prediction methods that require a perfect run of base pairs near the 5′ end of the miRNA would not detect it, while our method does. lsy-6, a recently experimentally identified miRNA in C. elegans, controls left–right neuronal asymmetry via cog-1, an Nkx-type homeobox gene; the cog-1 gene has a target site in its 3′ UTR, which also has a high score ( S = 125) and passes the conservation filter. Experiments in D. melanogaster have identified six new miRNA–target gene pairs: miR-7 targets the notch signaling genes HLHm3, HLHm4, and hairy, and miR-2b targets the genes reaper , grim, and sickle ( Stark et al. 2003 ). Consistent with these experiments, our target predictions in D. melanogaster ( Enright et al. 2003 ) ranked HLHm3, hairy, and HLHm4 at positions 1, 3, and 7, respectively, in the list of 143 target genes for miR-7 ( Enright et al. 2003 ). Similarly, our predictions ranked reaper, grim, and sickle at positions 3, 11, and 19, respectively, among the other 120 predicted target genes for miR-2c . We also predicted miR-6 to target this group of pro-apoptotic genes, with sites that have lower scores than the miR-2 family but are conserved in D. pseudoobscura. Unfortunately, one cannot in general use these predicted and then validated target sites ( Stark et al. 2003 ) for the derivation of new prediction rules, as the set of targets tested is limited to the type predicted and is not exhaustive. Indirect validation comes from the prediction that mammalian orthologs of some of the known miRNA targets in C. elegans and D. melanogaster are miRNA targets. An example is the proposed conservation of the miRNA–target relationship lin-4:lin-28 (we use the notation miRNA:mRNA for a miRNA–target pair), first discovered in worm ( Moss and Tang 2003 ): we detect target sites in human lin-28 for the lin-4 miRNA homolog miR-125 . We also confirm the human analog of a let-7:lin-28 relation predicted in C. elegans ( Reinhart et al. 2000 ). In summary, the predicted target sites on human lin-28 are miR-125 (1 site), let-7b (2 sites; Moss and Tang 2003 ), miR-98 (2 sites), and miR-351 (1 site). Another known lin-4 and let-7 target in C. elegans is lin-41 . The human homolog of lin-41 (sequence provided by F. J. Slack, personal communication) and another closely related gene (encoding Tripartite motif protein 2) are predicted as high-ranking targets of let-7 and miR-125 (the human homolog of lin-4 ) (see Tables S2 and S3 ). Another known instance of miRNA target regulation in worms is the regulation of cog-1 by the lsy-6 miRNA ( Johnston and Hobert 2003 ). Although there is no obvious homolog of lsy-6 in mammals, the vertebrate homolog of the target gene cog-1, nkx-6.1, is a conserved target for five different miRNAs in our predictions (see Table S2 ). The comparison of our results with known targets shows that our method can detect most (but not all) known target sites and target genes at reasonably high rank. However, given the small number of experimentally verified miRNA–target pairs, additional validation tests are desirable, such as statistical tests using randomization of miRNA sequences to estimate false positives. Estimate of false positives As a computational control of the validity of the prediction method, one can perform a statistical test that attempts to estimate the probability that a predicted site is incorrect. Here, a “false positive” is a predicted target site of a real miRNA on a real mRNA that has passed all relevant thresholds but is incorrect in that it is not biologically meaningful. The statement “not biologically meaningful” is rarely clearly defined, but can reasonably be taken to mean that no functionally effective miRNA:mRNA interaction occurs under conditions of co-expression at physiological concentration, where “functionally effective” is defined in terms of detectable changes of phenotypic attributes. Technically, an estimate of the false-positive rate can be obtained by computing (directly or via randomization) the background distribution of scores for biologically non-meaningful miRNA target sites and then deriving the probability that a non-meaningful target site passes all score thresholds, i.e., for a single aggregate score, that the incorrect site has a score T > T c , where T c is a fixed threshold that may be, in general, different for each miRNA. We chose to estimate the background distribution using shuffled miRNAs obtained by swapping randomly selected pairs of bases of each given miRNA 1,000 times, keeping the nucleotide composition constant. The shuffled miRNA sequences were scanned against human, mouse, and rat 3′ UTR sequences exactly as for the prediction procedure for real miRNA sequences. In the procedure, a miRNA:mRNA match site is predicted to be a target site if it passes three thresholds, S > S c for match score, |Δ G | > |Δ G c | for free energy of duplex formation, and C > C c for conservation, where C reflects a binary evaluation of orthology of mRNAs, similarity of position of the site on the mRNA, and a threshold percentage of conserved residues in the two mRNA target sites. Finally, the predicted target sites for a set of shuffled miRNAs are counted and then averaged over a total of ten randomized runs. The percentage of false positives for target transcripts with more than two, three, and four sites is 39%, 30%, and 24%, respectively, using a non-permissive conservation threshold of 100% for target site sequences ( Figure 2 ). In addition, the false-positive rate for single sites with a score of more than 110 is approximately 35%. Figure 2 Distribution of Transcripts with Cooperativity of Target Sites and Estimated Number of False Positives Each bar reflects the number of human transcripts with a given number of target sites on their UTR. Estimated rate of false positives (e.g., 39% for ≥2 targets) is given by the number of target sites predicted using shuffled miRNAs processed in a way identical to real miRNAs, including the use of interspecies conservation filter. To provide a realistic estimate of false positives using randomization, the distribution of scores from random trials (“random-false”) should be similar to the distribution of incorrect (non-meaningful) hits from real trials (“real-false”). The difference between these two distributions is difficult to compute in principle, as very few validated correct predictions are known at present. For human sequences, without any conservation filter, we obtained a total of 2,538,431 predicted target sites for real miRNAs, and, for shuffled miRNAs, on average, 2,033,701 (± 82,172) target sites—a difference of 20%. This difference may be indicative of a biological signal in the raw score (S) and energy ( Δ G) calculated by the miRanda algorithm or may be due to different polynucleotide compositions of shuffled miRNAs compared to real miRNAs. Even if this difference represents a real effect, by far the most predictive criterion for accurate target detection is conservation of target sites across species, and not alignment scores or energies (20% compared to a factor of three, see Figure 2 ; Table S8 ). As a consequence, the current set of predicted targets rests heavily on the criterion of conservation of miRNA:mRNA match between different species. We believe this to be essentially true for all currently published target prediction methods. Indirect experimental support: FMRP-associated mRNAs An excellent opportunity to test our target predictions comes from experiments showing the association of mRNAs and miRNAs with proteins involved in translational control, even if these experiments do not provide information on specific miRNA:mRNA pairings. In particular, FMRP, which may regulate translation in neurons, not only associates with hundreds of mRNAs ( Brown et al. 2001 ; Chen et al. 2003 ; Denman 2003 ; Miyashiro et al. 2003 ; Waggoner and Liebhaber 2003 ) and with miRNAs ( Jin et al. 2004 ), but also associates with components of the miRNA processing machinery, Dicer, and the mammalian homologs of AGO1 and AGO2 ( Jin et al. 2004 ). If all FMRP-bound mRNAs are regulated by miRNAs, one should see a large enrichment of predicted targets among such mRNAs. We tested this hypothesis with 397 FMRP-associated mRNAs taken from a number of recent experiments ( Brown et al. 2001 ; Chen et al. 2003 ; Denman 2003 ; Miyashiro et al. 2003 ; Waggoner and Liebhaber 2003 ). Are FMRP-bound messages enriched in predicted targets? Using five different datasets ( Table S9 ), we predicted that 74% of FMRP-associated messages are miRNA target genes (294 of 397 mRNAs). This corresponds to an enrichment factor of about five compared to the 59 targets one would expect from our analysis in a randomly chosen set of 397 mRNAs, where 59/397 equals 4,462/29,785 (4,462 predicted mammalian target mRNAs pass the 90% conservation filter for one or more sites per transcript out of a total of 29,785 transcripts). This suggests that in the 397 FMRP target genes, 59 should pass the filters. The enrichment factor does not vary much with the cutoff parameters used in target prediction (data not shown), but is subject to some uncertainty because of potential false-positive predictions. The enrichment of miRNA:FMRP interaction is consistent with the hypothesis that translational control involving FMRP protein is executed in a complex that involves one or more miRNAs interacting with transcripts at specific sites. Note that this analysis supports the validity of target gene prediction, not the identity of the controlling miRNA or the accuracy of specific sites. An additional validation test involved FMRP cargoes that had been identified in more than one study, using independent experimental methods. For example, the mRNAs of 14 genes ( Brown et al. 2001 ) were overrepresented in both the polyribosome fraction of mouse fragile X cells and in co-immunoprecipitation with mouse brain mRNPs that contain FMRP. Almost all of the 14 genes are predicted targets with more than one conserved site (11 of 12 annotated UTRs; Table S9 ). In some cases, expression data provide additional support: postsynaptic density protein 95 (PSD95)–associated (SAPAP4), a neuron-specific protein, is regulated by many miRNAs highly expressed in rat brain primary cortical neurons ( Kim et al. 2004 ). In summary, the three validation approaches (retrospective, statistical, and indirect experimental) suggest that the current version of the miRanda algorithm, in spite of clear limitations, can predict true miRNA targets at reasonable accuracy, provided that (1) the targets are detected as conserved and (2) the gene contains more than one miRNA target site or a single high-scoring site ( S > 110, approximately, including sites with almost perfect complementarity suggestive of mRNA cleavage). Overview of Mammalian miRNA Target Genes More than 2,000 mammalian targets. We predicted 2,273 genes as targets with two or more miRNA target sites in their 3′ UTRs conserved in mammals at 90% target site conservation (see Tables S2 and S3 ). This means we predicted approximately 9% of protein-coding genes to be under miRNA regulation. In addition, we predicted another 2,128 genes with only one target site, but the false-positive rate for these is significantly higher ( Figure 2 ). Of these, the top-scoring 480 genes ( S > 110) have an estimated false-positive rate comparable to that of genes with multiple sites and thus also are good candidates for experimental verification. Some of the genes with single sites may contain additional sites that we cannot detect for a number of reasons, including truncated UTRs. A significant subset of the total number of single-site target genes (7%) has near complementary single sites. These near complementary sites may indicate cleavage, for which additional sites may not be necessary. The targets listed in Table 1 were selected for variety of function, variation in number of sites, and varied extent of conservation (some are also conserved in fish). Somewhat surprisingly, the number of predicted targets per miRNA varies greatly, from zero (for seven miRNAs) to 268 (for let-7b ), but the distribution is nonuniform (mean = 7.1, standard deviation = 4.7; Figure 3 ). This indicates a range of specificity for most miRNAs and suggests that regulation of one message by one miRNA is rare. Figure 3 Multiplicity and Cooperativity in miRNA–Target Interactions One miRNA can target more than one gene (multiplicity) (A), and one gene can be controlled by more than one miRNA (cooperativity) (B). The distributions are based on ordered (ranked) lists and decay approximately exponentially (approximate straight line in log-linear plot). (A) Some miRNAs appear to be very promiscuous (top left), with hundreds of predicted targets, but most miRNAs control only a few genes (bottom right). (B) Some target genes appear to be subject to highly cooperative control (top left), but most genes do not have more than four targets sites (bottom right). Although specific values are likely to change with refinement of target prediction rules, the overall character of the distribution may well be a biologically relevant feature reflecting system properties of regulation by miRNAs. Table 1 Selection of Predicted miRNA Targets Add “ENSG00000” to the beginning of the identifiers to derive Ensembl identifiers. All miRNA–target relationships shown here are conserved in mammals, i.e., homologous miRNAs target transcripts of homologous genes at similar UTR positions with similar local sequence. Genes that are predicted to be targets in both mammals and fish are in bold. Where the miRNA–target relationship is also conserved in non-mammalian vertebrates, the miRNA is in bold a Contains conserved CPE motif N.A., not available Functional analysis We analyzed the distribution of functional annotation for all targets of all miRNAs using Gene Ontology (GO) terms (see Materials and Methods ; Table S10 ) and domain annotations from InterPro ( Mulder et al. 2003 ). The target genes reflected a broad range of biological functions ( Figure S1 ). The most enriched GO term was “ubiquitin-protein ligase activity,” with 3.3-fold enrichment ( Table S10 ). Since ubiquitination is a process controlling the quantity of specific proteins in a cell at specific times, miRNA regulation of components of the ubiquitin pathway could increase protein levels. Other overrepresented functional terms were “neurogenesis” (3.2-fold), “protein serine/threonine kinase” (2.5-fold), and “protein-tyrosine kinase activity” (2.5-fold). The four domains most overrepresented in predicted targets relative to all genes were Homeobox domain, 5.3-fold; KH domain, 4.0-fold; and Guanine-nucleotide dissociation stimulator CDC25 domain, 3.4-fold ( Figure S1 ; Table S10 ). Interestingly, KH domains are RNA-binding domains found in a wide range of proteins such as hnRNPk, FMR1, and NOVA-1. In addition to the Homeobox domain, other DNA-binding domains and domains associated with chromatin regulation were also enriched, suggesting that miRNAs in animals target the transcription machinery disproportionately, as they do in plants. Another overrepresented domain was semaphorins (3.0-fold). The semaphorins and plexins (semaphorin receptors) are involved in axon guidance, angiogenesis, cell migration, the immune system, and the adult nervous system ( Pasterkamp and Verhaagen 2001 ). Many semaphorins and their receptors are predicted targets of brain-expressed miRNAs (e.g., let-7c, miR-125b, miR-153, miR-103, miR-323, miR-326, and miR-337 ). The plexins dimerize with Neuropilin (NP1) to signal the Semaphorin ligand attachment; neuropilin is a predicted high-ranking target of let-7g and miR-130, both brain-expressed miRNAs. A significant proportion of ephrin receptors (seven out of ten genes) and ephrin ligands (five out of seven genes) are predicted targets. The family of ephrins is involved in boundary formation, cell migration, axon guidance, synapse formation, and angiogenesis, and the ephrin ligand, EphA2, which contains a conserved cytoplasmic polyadenylation element (CPE) motif, is considered to be under translational regulation in axon growth cones ( Steward and Schuman 2003 ). Although many members of the ephrin family are predicted targets of brain-expressed miRNAs, they appear to be targeted by different miRNAs, consistent with differential regulation. In Drosophila, both ephrin and EphR, closest to the mammalian B class of the ephrin family, also are predicted miRNA targets. Do specific miRNAs target particular functional groups? We analyzed each miRNA individually for GO term and domain enrichment ( Table S10 ). The targets of some miRNAs were strongly enriched in certain categories, e.g., miR-105 in “small GTPase mediated signal transduction” (5-fold), miR-208 in “transcription factor” (6-fold), and miR-7, which lies in the intron of the hnRNPk (an RNA-binding protein) gene, in “RNA binding proteins.” Neuronal differentiation of embryonic carcinoma cells by retinoic acid in both mice and humans is coupled to induction of let-7b, miR-30, miR-98, miR-103, and miR-135 ( Sempere et al. 2004 ), and their targets are enriched in “neurogenesis” (3.5-fold). miR-124a and miR-125, both highly and specifically expressed in brain, preferentially target RNA-binding proteins. Thirty-one new miRNAs (miR - 322 – miR - 352) cloned from rat neuronal polyribosomes have a large number of neuronal target genes and share many targets, e.g., miR-352 and miR-327 target 5HT-2c, and miR-340, -328, -326, -331, and -333 potentially target beta-catenin, which is implicated in various stages of neural differentiation. Two highly expressed miRNAs in the thymus, miR-181a and miR-142–3p are key components of a molecular circuitry that modulates hematopoietic lineage ( Chen et al. 2004 ). Ectopic expression of miR-181a causes a 2-fold increase in the cells of the B cell lymphoid lineage. Some of our high-ranking targets for miR-181a may provide clues for the mechanism of this effect. Germ cell nuclear factor GCNF (NR6A1) (the second-highest-ranked target for miR-181a ) is expressed in the thymus and bone marrow. miR-181a itself is encoded on the antisense strand of an intron of GCNF . We also predict that the gene Bcl11b, known to affect B cell growth, is a target of miR-181a, ranking third, as well as Lim/homeobox protein LHX9, recently found expressed in developing thymus ( Woodside et al. 2004 ). FMRP cargo mRNAs regulated by miRNAs FMRP is composed of several RNA-binding domains (two KH and one RRG) that bind messages. The specific binding motifs for FMRP on messages are incompletely known, but are thought to include G-quartet patterns and/or U-rich sequences ( Dolzhanskaya et al. 2003 ; Ramos et al. 2003 ). We predicted 294 mRNAs known to be FMRP cargoes as miRNA targets (see Table S9 ). The most reliable of these ( Table 2 ) reflect high confidence in experimental identification of FMRP association or conservation of target site between mammals and fish. Table 2 Selected FMRP Cargoes Predicted as miRNA Targets Transcripts for genes (Gene and ID) are described as FMRP cargoes in several studies (DR) and predicted here as targets of specific miRNAs (miRNA). Selected from a total of 294 such targets a Reference from which data was extracted b Homologous miRNA–mRNA pair conserved in fish c Additional miRNAs are predicted to target the gene (number in parentheses): APP (9), BASP1 (4), Capicua (2), DLG3 (7), and DLG4 (5) d The miRNA has multiple target sites on the gene e The 3′ UTR of the gene contains a CPE motif (Table S11) Alzheimer's disease amyloid protein Amyloid precursor protein (APP) is an FMRP-bound protein that is translationally regulated. The APP transcript contains a 29-nt motif at position 200 in the 3′ UTR that is known to aid destabilization of the APP mRNA in certain nutrient conditions and that binds nucleolin, a protein associated with RNPs containing FMRP ( Rajagopalan and Malter 2000 ). In addition, there is an 81-nt sequence at position 630 in the APP 3′ UTR that is required for the TGFbeta-induced stabilization of the APP mRNA ( Amara et al. 1999 ). We predicted APP as a target, with a total score of S = 708 with a minimum of eight miRNA sites, including two let-7 top-ranking sites that are conserved in human, mouse, and rat. One of the predicted miRNA target sites in the APP UTR lies in the 81-nt region ( Figure 4 ), and another is within 30 nt of the motif at position 200. Figure 4 Potential miRNA Target Sites in the 3′ UTRs of Selected Genes Nucleotide sequence conservation between the 3′ UTRs of human and the closest mouse or rat orthologous genes is averaged for each block of 40 base pairs (long rectangles; white indicates 0% identical nucleotides, black indicates 100% identical nucleotides, and grey indicates intermediate values). The positions of target sites for specific miRNAs (triangles above rectangles, with numbers indicating miR miRNAs, e.g. “130” is “mir-130”) are, in general, distributed nonuniformly. Sequence motifs other than target sites (triangles below rectangles) are mRNA stability elements (APP), a G-quartet (DLG4), and an AU-rich element (ELAVL1), representing possible protein-binding sites. Detailed alignments between the miRNA and the predicted target sites (arbitrary selection) illustrate, in general, stronger match density at the 5′ end of miRNAs than at the 3′ end, as required by the algorithm and as observed in experimentally validated targets. The nonconserved nucleotides in the target sites are highlighted in red. Gene names map to the following Ensembl identifiers (142192 is ENSG00000142192, etc.): APP, 142192; CPEB2, 137449; DLG4, 132535; EFNB1, 090776; EIF2c1, 092847; ELAVL1, 066044; EPHB1, 154928; EPHB3, 182580; FMR1, 102081; FMR2, 155966; FXR1, 114416; FXR2, 129245; and PTEN, 171862. Other APP-interacting proteins, APP-binding family B member 1 (mir-9, miR-340, and miR-135b), APP-binding family member 2 (let-7 and miR-218), and APP-binding family 2 (miR-188 and miR-206) were also predicted targets, some of which had near exact target site matches. In summary, the APP gene appears to be subject to translational regulation by the combinatorial control of a number of different miRNAs. PSD95 and synaptic processes PSD95 and similar scaffolding molecules, link the NMDA receptor with intracellular enzymes that mediate signaling; this process is involved in the development and maintenance of synaptic function and synaptic plasticity, and interference in this process is implicated in schizophrenia and bipolar disorder ( Beneyto and Meador-Woodruff 2003 ). FMRP binds PSD95 and is required for mGluR-dependent translation of PSD95 ( Todd et al. 2003 ). PSD95 is a high-ranking target of miR-125, miR-135, miR-320, and miR-327, all of which are either exclusively expressed in brain or enriched in brain tissue ( Lagos-Quintana et al. 2002 ; Krichevsky et al. 2003 ; Sempere et al. 2004 ). In particular, large transcript numbers of miR-125b are found copurified with polyribosomes in rat neurons in ( Kim et al. 2004 ). PSD95 has one reported G-quartet in its 3′ UTR at position 648 ( Todd et al. 2003 ), further suggesting it as an in vivo FMRP target. We predicted an additional G-quartet site at position 205–235 in the 3′ UTR of PSD95 . One of the miRNA (miR-125) target sites overlaps with the G-quartets, raising the possibility that miRNAs directly compete with FMRP to bind the message in this location. Likewise, NAP-22, which has three miRNA target sites (see Table S9 ), has a miR-207 target site that overlaps with a G-quartet ( Darnell et al. 2001 ). Other PSD95 family members are also involved in synaptic processes, in particular, in the integration of NMDA signaling in the synaptic membrane. All PSD95 family members in mammals (also known as discs large 1–5 ), SAP90, and CamKII are predicted miRNA targets (see Table S9 ), as well as mGluR, the protein product of which is an agonist that induces the rapid translation of PSD95 ( Todd et al. 2003 ) and three NMDA receptor subunits (see Table S9 ). These results suggest that miRNAs may be involved in NMDA and glutamate receptor signaling to coordinate and integrate information, with specificity achieved through the combinatorial action of different miRNAs. Components of RNPs Regulated by miRNAs FMRP-associated proteins FMRP binds its own mRNA, implying negative feedback if the binding inhibits FMRP production ( Ceman et al. 1999 ) . The fact that miRNAs target transcripts for FMRP and FMRP-binding proteins suggests another negative feedback loop in which high levels of these proteins inhibit their own production (depending, of course, on the concentration of miRNAs and mRNAs) ( Figure 4 ). The genes for six FMRP-associated (not associated at the same time) proteins, hnRNP A1, Pur-alpha, Pur-beta, Staufen, AGO-2, and PABP, are predicted miRNA targets. This indicates that FMRP-containing RNPs are under miRNA regulation. FXR2, a gene similar to FMR1 is also a miRNA target in human, mouse, rat, and fish. Details of the implied feedback regulation and differential control of RNP action remain to be determined. RISC. Our data suggest that the RNAi–miRNA machinery itself is under miRNA regulation; for example Dicer appears to be controlled by let-7 and miR-15b; Ago-1 by let-7 and miR-29b/c; Ago-2 by miR-138; Ago-3 by miR-138, miR-25, and miR-103; and Ago-4 by miR-27a/b. Dicer and two of the Argonautes also are predicted to be targets in both zebrafish and fugu. The let-7 sites on the 3′ UTR of Dicer and Ago-1 ( Figure 4 ) will accommodate most of the let-7 variants with similar scores. The variants of let-7 are expressed in a wide range of tissues and developmental stages, suggesting broad regulation of Dicer and Ago-1 by miRNAs. In contrast, the only miRNA that targets Ago-2 is miR-138, which has so far been cloned only once in the cerebellum ( Lagos-Quintana et al. 2002 ). The target site for miR-138 has only one mismatch at position 8; this may induce a siRNA-like cleavage of the message ( Hutvágner and Zamore 2002a ; Doench et al. 2003 ). Ago-3 is also a top target for miR-138, with only two mismatches in its site. We suggest that some miRNAs targeting this machinery (e.g., let-7, miR-27, miR-29, and miR-103) are expressed fairly widely, while others (e.g., miR-138 and miR-25 ) have lower and more restricted expression. Other RNPs. The highly conserved RNA-binding proteins, ELAV-like proteins (HuR, HuB, HuC, and HuD), contain three RNA-recognition motifs, which bind AU-rich elements in 3′ UTRs of a subset of target mRNAs ( Good 1995 ). These AU-rich elements increase the proteins' cytoplasmic stability and increase translatability ( Perrone-Bizzozero and Bolognani 2002 ). Experiments have identified 18 mRNAs bound to HuB in retinoic-acid-induced cells; of the 14 we were able to map unambiguously, 12 are predicted miRNA target genes: Elavl1 (known to regulate its own mRNA), Gap-43, c-fos, PN-1, Krox-24, CD51, CF2R, CTCF, NF-M, GLUT-1, c-myc, and N-cadherin ( Tenenbaum et al. 2000 ). Three of the ELAV-like genes themselves are also targets of a large number of miRNAs (see Tables S2 and S3 ; Figure 4 ). This is yet another example of miRNAs predicted to target the bound messages of RNA-binding proteins and of the regulation of RNA-binding genes by miRNAs. Cytoplasmic Polyadenylation Binding Proteins Regulated by miRNAs We predicted all four human cytoplasmic polyadenylation binding proteins (CPEBs) known in mammals as miRNA targets ranked within the top 170 target genes with 6–20 sites in their UTRs ( Figure 4 ; Table S11 ). Indeed, CPEB2 is the highest-ranking gene of all transcripts . The orthologs to CPEB1 in fish and fly (known as orb in D. melanogaster ) are also predicted as targets. CPEB is an RNA-binding protein first shown to activate translationally dormant mRNAs by regulating cytoplasmic polyadenylation in Xenopus oocytes ( Hake and Richter 1994 ). It also regulates dendritic synaptic plasticity ( Mendez and Richter 2001 ; Richter 2001 ) and dendritic mRNA transport ( Mendez and Richter 2001 ; Huang et al. 2003 ) and facilitates transport of mRNAs in dendrites together with kinesin and dynein in RNPs ( Huang et al. 2003 ). CPEB binds to its target message through the CPE motif (UUUUAU), which must be within a certain distance of the hexanucleotide AAUAAA. CPEB keeps messages in their dormant state until phosphorylated, after which it activates polyadenylation ( Mendez et al. 2000 ), thereby activating translation or degradation ( Mendez et al. 2002 ). In addition, CPEB co-fractionates with the postsynaptic density fraction in mouse synaptosomes, consistent with translation of stored mRNAs in dendrites being part of the mechanism of synaptic plasticity. We have three more lines of evidence suggesting the notion that translational regulation by CPEB is linked to miRNA regulation. First, our target list and the list of genes regulated by CPEB significantly overlap. There are nine genes known to be CPEB-regulated, seven of which are predicted targets: alpha-CAMIIK, Map 2, Inositol 1, 4–5-Triphosphate Receptor type 1, Ephrin A receptor class A type 2, SCP-1, and CPEB3 ( Mendez and Richter 2001 ). Second, CPEB is known to self-regulate in D. melanogaster ( Tan et al. 2001 ). The CPEB1 homolog in fly, orb, and CPEBs in vertebrates are predicted miRNA targets. Third, the gene most correlated in expression to the CPEB homolog in D. melanogaster is a Piwi protein (Sting), a member of the Argonaute family ( Pal-Bhadra et al. 2002 ; Stuart et al. 2003 ) that is involved in translational regulation and in the RISC. Among the predicted miRNA targets, 115 genes also contained CPE motifs, which were conserved in at least two mammals in the same positions in the UTRs and are therefore candidates for CPEB regulation ( Table S11 ; see Materials and Methods ). Our predictions include HuB, HuR, Eif-4 gamma, DAZ associated protein 2, VAMP-2 (known to be posttranscriptionally regulated), Presynaptic protein SAP102, and brain-derived neurotrophic factor precursor. Taken together these data suggest that the CPEB genes, the known CPEB-regulated genes, and the predicted CPEB-regulated genes are strong miRNA target candidates and provide rich ground for experimentation. Targets of Cancer-Related miRNAs Deregulated expression of certain miRNAs has been linked to human proliferative diseases such as B cell chronic lymphocytic leukemia ( Calin et al. 2002 ; Lagos-Quintana et al. 2003 ) and colorectal neoplasia ( Michael et al. 2003 ). Recent analysis of the genomic location of known miRNA genes suggested that 50% of miRNA genes are in cancer-associated genomic regions or in fragile sites ( Calin et al. 2004 ). The miRNAs miR-15 and miR-16 are located within a 30-kb region at Chromosome 13q14, a region deleted in 50% of B cell chronic lymphocytic leukemias, 50% of mantle cell lymphomas, 16%–40% of multiple myelomas, and 60% of prostate cancers ( Calin et al. 2002 ). Furthermore, miR-15 and miR-16 are down-regulated, or their loci lost, in 68% of B cell chronic lymphocytic leukemias ( Calin et al. 2002 ). Similarly, miR-143 and miR-145 are down-regulated at the adenomatous and cancer stages of colorectal neoplasia ( Michael et al. 2003 ), and miR-155 is up-regulated in children with Burkitt lymphoma ( Metzler et al. 2004 ). Our method predicted cancer-specific (by annotation) gene targets of miR-15a, miR-15b, miR-16, miR-143, miR-145, and miR-155 . The target genes and their miRNA regulators are as follows: (1) CNOT7, a gene expressed in colorectal cell lines and primary tumors ( Flanagan et al. 2003 ) (miR-15a); (2) LASS2, a tumor metastasis suppressor ( Pan et al. 2001 ) (miR-15b); (3) ING4, a homolog of the tumor suppressor p33 ING1b, which stimulates cell cycle arrest, repair, and apoptosis ( Shiseki et al. 2003 ) (miR-143); (4) Gab1, encoding multivalent Grb2-associated docking protein, which is involved in cell proliferation and survival ( Yart et al. 2003 ) (miR-155); and (5) COL3A1, a gene up-regulated in advanced carcinoma ( Tapper et al. 2001 ) (miR-145). miR-16 has a tantalizing number of high-ranking targets that are cancer associated and specifically involved in the Sumo pathway There is increasing evidence that Sumo controls pathways important for the surveillance of genome integrity ( Muller et al. 2004 ). The first- and fifth-highest-ranked targets of miR-16 are Sumo-1 activating and conjugating enzymes, respectively. The top two single-site targets for miR-16 are an Activin type II receptor gene (TGFbeta signaling) and Hox-A5, both known to be dysregulated at the level of protein expression in colon cancers ( Wang et al. 2001 ). Both of these sites show near perfect complementary matching between miR-16 and the target genes (indicating possible cleavage). Both of these target genes are also targets for another cancer related miRNA, miR-15 . Targets Conserved between Mammals and Fish Roughly 55 miRNAs have identical mature sequences in fugu and mammals, and 80 have very similar sequences in the two species; additional fish miRNA sequences can be predicted with confidence based on sequence similarity. It is therefore reasonable to expect that the targets of these probably functionally homologous miRNAs are orthologous genes in the different species. To follow up on this hypothesis, we assessed conservation of mammalian miRNA–target pairs between the 2,273 mammalian and 1,578 fish miRNA targets (with more than one target site per UTR). The analysis yielded 240 target genes conserved between mammals and fish. The number 240 is probably an underestimate because of several factors, including: (1) unfinished annotation of genomes, particularly rat and fugu; (2) ambiguity in assigning orthologs; and (3) lack of UTR information . The full set of conserved target genes between fish and mammals indicates a wide functional range of conserved targets ( Table S12 ). Many Hox genes are conserved as targets, including the miR-196 targets, Hox-A4:miR-34a, Hox-C9:let-7b (near prefect complementary match), and Hox-B5:miR-27b . Examples from the notch signaling pathway include miR-30:hairy enhancer of split 1 (Hes1) and miR-152:noggin. Targets Conserved between Vertebrates and Flies Twenty-eight of the 78 identified miRNAs in flies have apparent mammalian homologs. Based on this remarkable conservation across hundreds of millions of years, it is reasonable to expect that there is some conservation of target sites, target genes, and target pathways between flies and humans. Most strikingly we can identify hox genes and axon guidance genes as common targets between vertebrates and flies, e.g., capicua and sex combs reduced (one of the vertebrate homologs of Hox-A5 ). The hox gene cluster in Drosophila contains high-ranking predicted targets ( Enright et al. 2003 ) of miR-10 and miR-iab-4, and the hox gene cluster in mammals contains high-ranking targets of miR-196 . These miRNAs are themselves located in the hox gene region. We predicted miR-iab-4–3p to target abd-B in Drosophila, a gene related to the ancestral hox-7 cluster, the ancestral parent of many of the predicted targets of miR-196 . Axon guidance receptors and ligands conserved as targets include Lar, ephrins, and slits . Human slit1 is a top target of miR-218, which itself is transcribed from the intron of slit2, suggesting down-regulation of slit1 by transcription of slit2 . We expect that there are many more conserved targets but we are hindered by the difficulty of mapping orthologous genes between human and fish. Future work will elucidate to what extent there are common pathways regulated by common miRNAs between vertebrates and invertebrates. Target Sites in Protein-Coding Sequences Experiments suggest that miRNA target sites in metazoans are preferentially in UTRs, not in coding regions. If this is true, a correct target site prediction method should predict a larger number of targets in UTRs than in coding regions. Alternatively, target sites in coding regions may so far have escaped experimental verification, especially in plants, in which targets of miRNAs in coding regions are the rule, not the exception. To investigate this issue we computed the average density of target sites for high-scoring targets ( S > 130) and before application of conservation filters. The statistical assessment of the influence of conservation filters in coding regions would have raised complicated issues, as nearly two-thirds of nucleotides in coding regions are conserved between mammalian genomes to preserve amino acid sequences. Interestingly, we found, on average, 11 pre-conservation target sites per 1 million nucleotides in coding regions, versus 15 such target sites per 1 million nucleotides in UTRs. This is consistent with a stronger “raw” prediction signal in UTRs and may indicate a lower number of biologically relevant target sites in coding regions in mammals, consistent with early experimental findings. As a guide to experimentation, we report all sites in coding regions with an alignment score above 110 for miRNAs of length up to 20 nt and an alignment score above 130 for miRNAs longer than 20 nt (scores depend on the length). These cutoff scores approximately correspond to a 75% complementary match between miRNA and target, leaving open the question of how many match pairs are needed to lead to translational inhibition in coding regions, by any mechanism. We identified 942 genes that contained such sites in their coding regions. Strikingly, there was only one site with a perfect match, and this was for the imprinted miR-127, known to be antisense to the reciprocally imprinted retrotransposon-like gene on the opposite strand ( Seitz et al. 2003 ). Of the 942 genes, 25% have been otherwise identified as targets based on conserved target sites in their UTRs. However, only five genes have targets sites in their UTRs complementary to the same miRNA that targets the coding region (see Table S3 , columns H and I). For example, miR-211 has a near perfect complementary site in the coding region of a gene of unknown function (Ensembl ID ENSG00000134030, containing an Eif-4 gamma domain) and also has two conserved “normal” sites in the UTR. Similarly, miR-198 has a site in the coding region, as well as conserved sites in the UTR region, of a sodium and chloride GABA transporter (Ensembl ID ENSG00000157103). However, we see no trend for miRNAs that have conserved sites in UTRs to have additional sites in the coding region; rather, stronger target sites for a given miRNA tend to be confined either to the UTR or the coding region and are rarely in both. Target Sites with Near Perfect Matches in cDNAs We scanned all cDNAs for high-scoring matches without using conservation to check for high-scoring targets, which we may have missed through strict conservation rules (see Table S6 ). Over 40 genes contain sites that have near perfect complementarity to a miRNA ( S >120), and these target genes may be cleaved rather than translationally repressed as in the case of miR-196 and Hox-B8 . For example miR-298, an embryonic-stem-cell-specific miRNA ( Houbaviy et al. 2003 ), has a near match with MCL-1, and miR-328 (neuronally expressed) has a near match with LIMK-1, which is known to be involved in synapse formation and function. miR-129, expressed in mouse cerebellum, has a near perfect complementary match with Musashi-1, which is an RNA-binding gene essential for neural development, regulated in the cerebellum, and up-regulated in medulloblastoma ( Yokota et al. 2004 ). Comparison of miRNA Target Prediction Methods Recently, several computational methods for the prediction of miRNA targets have been developed ( Enright et al. 2003 ; Lewis et al. 2003 ; Rajewsky and Socci 2003 ; Stark et al. 2003 ; Kiriakidou et al. 2004 ; Rehmsmeier et al. 2004 ). Two of these have been applied to mammalian miRNAs, as described in Lewis et al. (2003) and Kiriakidou et al. (2004) . We now compare and contrast these two methods with each other and with the current version of our method, as further developed from miRanda 1.0 and as applied to mammalian and vertebrate genomes ( Enright et al. 2003 ). We compare algorithms and target lists, as an aid to the design of experiments. The three prediction methods share the goal of identifying mRNAs targeted by miRNAs. All three use sequence complementarity, free energy calculations of duplex formation, and evolutionary arguments in developing a scoring scheme for evaluation of potential targets. Results are reported as lists of target sites and lists of target genes containing such sites. The three methods differ, however, in important technical details, such as the datasets of miRNA and UTR sequences and the algorithm and scoring scheme, as well as the report format. We now summarize these technical differences and compare the lists of resulting target genes for a common subset of miRNAs. The interpretation of such comparisons is hampered by the fact that selection criteria and the use of numerical cutoffs differ conceptually, and genomic coverage is nonuniform. In the first method, Lewis et al. used 79 miRNAs in human, mouse, and rat, seeking targets in a UTR dataset extracted from the June 2003 version of the Ensembl database. The UTR dataset had 14,300 ortholog triplets conserved between human, mouse, and rat and 17,000 ortholog pairs between human and mouse. All annotated UTRs were extended by 2 kb of 3′ flanking sequence. The algorithm required exact complementarity of a 7-nt miRNA “seed” sequence, defined as positions 2–8 from the 5′ end of the miRNA, to a potential target site on the mRNA, followed by optimization of mRNA–miRNA duplex free energies between an extended window of 35 additional bases of the mRNA and the rest of the miRNA. Target genes were ranked using a composite scoring function, which took into account all sites for a particular miRNA on a given mRNA. Conserved miRNA:mRNA pairs were required to involve orthologs of miRNA and mRNA in human, mouse, and rat, but there was no requirement for conservation of target site sequence (beyond the seed match) or position on the mRNA. Using shuffled miRNA sequences, with the constraint that shuffled controls match real miRNAs in relevant sequence properties, the false-positive rate of predictions was estimated to be 50% for target genes conserved between mouse and human, 31% for target genes conserved in human, mouse, and rat, and 22% for target genes identified in fugu as well as mammals. As a final result, Lewis et al. reported 400 conserved target genes for the 79 miRNAs. Among these targets, 107 genes were reported as conserved in the fish fugu. In the second method, Kiriakidou et al. used 94 miRNAs in human and mouse, seeking targets in a dataset of 13,000 UTRs conserved in mouse and human (from Ensembl, date not given). The algorithm used a 38-nt sliding mRNA window and calculation of miRNA–mRNA duplex free energies, keeping duplexes with energies below −20 kcal/mol. The duplexes were further filtered using a set of requirements regarding matches and loop lengths in certain positions, as derived and extrapolated from experimental tests involving a predicted target site for let-7b miRNA on the UTR of the human homolog of worm lin-28 . The target site sequence was engineered into a Luciferase reporter, followed by sequence variation of the target site and test of an initial set of 15 predictions in the same reporter assay. Using shuffled miRNA sequences, and applying the same rules and parameters, the false-positive rate of predictions was estimated to be 50% for targets conserved between human and mouse. As a final result, Kiriakidou et al. reported 5,031 human targets, with 222 reported as conserved in the mouse. In the third method (this work), we used 218 mammalian miRNAs and 29,785 transcripts derived from Ensembl ( Table 3 ) and, as a final result, report 4,467 target genes. What are the main differences between these three prediction methods? Comparison of the total number of predicted target genes is not very informative, as different datasets and cutoffs were used. We attempted to remove one of the technical differences, by explicitly comparing reported targets for the same set of 79 miRNAs used by Lewis et al. (although significant differences remained in the sets of UTR sequences used): the overlap of target genes between Kiriakidou et al. (out of 189) and Lewis et al. (out of 400) was 10.6%; the overlap between Lewis et al. (out of 400) and this work (out of 2,673) was 46%; and the overlap between Kiriakidou et al. (out of 189) and this work (out of 2,673) was 49%. In each case the totals (“out of”) are the number of target genes for the common set of 79 miRNAs and the percentage is relative to the smaller set of two compared. The obvious reason for the larger overlap with our results, 46% and 49% respectively, is the larger number of targets in our predictions, which in turn is primarily the result of choice of cutoff. Table 3 Number of Genes and 3′ UTR Sequences Used for Target Prediction 3′ Direct comparison of the three prediction methods is complicated by the fact that the noticeable differences between the target lists of the three methods are due to the aggregate effects of datasets, algorithm, including selection rules, use of conservation, and cutoffs. The following characteristics of the three methods underlie these differences and should be taken into consideration when choosing targets for experimentation. (1) As to UTR datasets, Lewis et al., with the earliest published report, used a smaller set of UTRs, with some likelihood of false positives as a result of UTR extension. The UTR sets used in this work, the third in terms of publication date, are the most comprehensive and plausibly the most reliable (as of February 2004). (2) As to miRNA datasets, there was an increase from 79 for Lewis et al. to 94 for Kiriakidou et al. to 218 miRNAs used in this work. (3) As to the cooperativity of binding, the scoring system of Lewis et al. evaluated cooperativity of multiple target sites by the same miRNA on a target gene, but disregarded multiple target sites from different miRNAs on one gene; that of Kiriakidou et al. focused on single sites; and that of this work gave high scores to multiple hits on a target gene, no matter whether these hits involved the same miRNA or different miRNAs. These tendencies are not exclusive where scores involve functions of several real numbers, with cutoffs applied to the aggregate score; e.g., our method also allows strong single target sites. (4) As to assessment of false positives using statistical methods based on shuffling, the comparison of percentages is inconclusive, as the statistics of the background distribution of true negatives is not well known. It appears certain, however, from both Lewis et al. and this work, that statistical confidence increases with the extent of conservation among increasingly distant species. (5) As to validation experiments, each of the methods used a different type and set, with mixed overall conclusions. On the reassuring side, there was direct validation of some of the predicted target sites of Lewis et al. and of Kiriakidou et al. using reporter constructs in cell lines. We found some agreement between the sites validated in this way and our predicted targets (details in Table S13 ), but in some cases we predicted different details of target sites for a given experimentally tested miRNA:mRNA pair. Also, Kiriakidou et al. used a series of such experiments to extrapolate from a set of specific sequence variants to general rules for identification of target sites. However, serious doubts about the validity of any set of rules persist as there is very little in vivo validation in which native levels of specific miRNAs are shown to interact with identified native mRNA targets with observable phenotypic consequences under normal physiological conditions. (6) As to differences in algorithm, one can state opinions about the strengths or weaknesses of each particular algorithm, but the relationship between each prediction method and the actual in vivo process by which miRNAs have functional interactions with their target mRNAs remains unclear or, at best, unproven. In summary, in our view, each of the three methods, including the one in this work, falls substantially short of capturing the full detail of physical, temporal, and spatial requirements of biologically significant miRNA–mRNA interaction. As such, the target lists remain largely unproven, but useful hypotheses. The predicted targets are useful in practice for the design of experiments as they increase the efficiency of validation experiments by focusing on target lists significantly enhanced in likely targets, compared to random. It is plausible that targets near the top of lists are the most likely to lead to successful experiments. Task-specific filtering of target lists for particular planned experiments is recommended, especially with respect to cooperativity of binding (more than one site for one or more miRNAs on one gene transcript) and coincidence of expression, as new data on expression patterns of miRNAs and mRNAs in different tissues become available. For example, a recommended conservative approach to the design of experiments would use all available expression information and restrict the predicted target genes to those with two or more target sites at normal threshold ( S > 90) or one target site with a higher threshold ( S > 110), counting only sites with up to one G:U pair in residues 2–8 counting from the 5′ end of the miRNA. To take into account the rapid development of this field and the likely close interaction of theory and experiment, we plan to periodically update our prediction method and parameters and make revised target lists available on http://www.microrna.org . Next, we discuss some conceptual consequences of the composition of our target list. Discussion How Widespread Is the Regulation of Translation by miRNA? With plausible parameters, we have predicted that close to 9% (2,273 out of 23,531) of all mammalian genes have more than one miRNA target site in their 3′ UTRs, with 1,314 being stronger candidates with more than two target sites. This could well be an underestimate of the total number of genes subject to miRNA regulation, as we have used a conservative conservation filter. On the other hand, not all predicted miRNA–mRNA pairs would have a biological consequence unless both miRNA and mRNA are expressed at the same time in the same cell and at sufficient concentration. The human genome has about 250 miRNA genes, compared to about 35,000 protein genes. Thus, the the determination that about 1% of genes (miRNAs) control the expression of more than 10% of genes is a reasonable first order estimate. It is currently not known if any miRNAs control the expression of miRNA genes, i.e., the progression from miRNA transcript to mature miRNA. How Conserved in Evolution Are miRNA Targets? As many miRNA sequences are detectably conserved across large evolutionary distances, they must be subject to strong functional constraints. These constraints are unlikely to come from single-site interactions with the target, as experimentally validated animal miRNAs rarely have perfectly matched target sites. Plausibly, the evolution of miRNAs is constrained by functional interactions with multiple targets. As a consequence, any compensatory mutation in the miRNA in response to mutations in a target site would be disruptive to the miRNA's interaction with other target sites. Co-evolution of the miRNA sequence and all of its target sequences is therefore a rare event. With these assumptions, the constraints on the local mRNA sequence of individual target sites are weaker than those on the miRNA sequence. We were therefore surprised to observe a substantial number of cases (28.6% of the 2,273 targets) with 100% conservation of target site sequence and with the target sites being within ten nucleotides of each other on the globally aligned UTRs of orthologous genes between mammals. Lacking more detailed knowledge of miRNA evolution, we draw two operational conclusions. (1) Conservation of target site sequence and position is a practical information filter for predicted target sites, reducing the rate of false positives. (2) It is very likely that new miRNAs have continuously appeared in evolution ( Lai 2003 ) at some non-negligible rate and that the set of targets for any given miRNA has lost or gained members, even between species as close as human and mouse. It is therefore important to develop prediction tools that do not rely on conservation filters or at least allow us to make them weaker. Work on this is in progress. Multiplicity and Cooperativity Regulation by miRNAs is obviously not as simple as one miRNA–one target gene, as perhaps the early examples (lin-4 and let-7) seemed to indicate. The distribution of predicted targets reflects more complicated combinatorics, both in terms of target multiplicity (more than one target per miRNA) and signal integration (more than one miRNA per target gene). The distribution of the number of target genes (and target sites) per miRNA is highly nonuniform, ranging from zero for seven miRNAs to 268 for let-7b, with an average of 7.1 targets per miRNA. It is difficult to describe in detail, beyond the examples discussed in this text and beyond the annotation of target genes in Figure 2 and Table S3 , which specific processes appear to be regulated by each miRNA or each set of co-expressed miRNAs. Groups of targets may reflect a reaction, a pathway, or a functional class (see Results ). Although all miRNA–target pairs are subject to the condition of synchrony of expression, it is likely that typically one miRNA regulates the translation of a number of target messages and that, in some cases, the target genes as a group are involved in a particular cellular process. This was already known for the case of lin-4 ( Ambros 2003 ). The number of miRNA target sites per gene is also nonuniform, with a mean of 2.4. Although we do list target genes with single miRNA sites, there is increasing evidence that, in general, two or more sites are needed in the context of repression of translation. Although the details of these distributions (see Figure 2 and Table S3 ) depend on technical details, such as uniform cutoff for all miRNAs and evaluation in terms of a particular, imperfect scoring system, the general features of the distributions (see Figure 3 ) may be generally valid. We conclude that multiplicity of targets and cooperative signal integration on target genes are key features of the control of translation by miRNAs. Neither multiplicity nor cooperativity is a novel feature in the regulation of gene expression. Indeed, regulation by transcription factors appears to be characterized, at least in eukaryotes, by analogous one-to-many and many-to-one relations between regulating factor and regulated genes ( Kadonaga 2004 ). We are, of course, aware that the control cycles and feedback loops involving miRNAs cannot be adequately described without more detailed knowledge of the control of transcription of miRNA genes, about which little is known at present. Mechanisms of miRNA Action The role of a few animal miRNAs as posttranscriptional regulators of gene expression and, in particular, as inhibitors of translation is well established. However, the molecular mechanism of action is not well understood. Posttranscriptional control of protein levels can be achieved, for example, by cleaving the mRNA, by preventing RNP transport to ribosomes, by stalling or otherwise inhibiting translation on ribosomes, or by facilitating the formation of protein complexes near ribosomes that degrade nascent polypeptide chains. What do our results imply regarding the mechanism of action? In analogy to plant miRNAs that have near perfect sequence complementarity and facilitate mRNA degradation, our predicted targets with near perfect complementarity between miRNA and mRNA plausibly are involved in mRNA cleavage (e.g., miR-196 and miR-138; see Results ). Most of these would involve single target sites. In the case of Hox-B8, cleavage has been experimentally shown in mammalian cells ( Yekta et al. 2004 ). We estimate that fewer than 5% of miRNA targets are cleaved as a result of miRNA binding. Multiple target sites of lesser complementarity are consistent with RNP formation leading to translational inhibition, not mRNA degradation. Although we did predict single miRNA target sites for some genes, most target genes have multiple sites, indicating that cooperative binding ( Doench and Sharp 2004 ) may be essential for formation of inhibitory RNP complexes. An interesting and somewhat paradoxical feature is seen with mRNAs bound by FMRP, some of which increased and some of which are decreased in polysome fractions in FMRP knock-out mice ( Brown et al. 2001 ). We see no bias in which of these two sets is most enhanced as predicted miRNA targets. This ambiguity not only raises questions about details of FMRP regulation but also raises the possibility that miRNA targets may not always be translationally repressed and may instead be translationally enhanced. Improvement of Prediction Rules Current methods for predicting miRNA targets rely on conservation filters to reduce noise. Although the miRNA–mRNA pairings of experimentally validated targets were carefully used to define prediction rules ( Enright et al. 2003 ; Lewis et al. 2003 ; Stark et al. 2003 ), the information content in sequence match scores and free energy estimates of RNA duplex formation appears to be low. What is missing? Perhaps the fine details of experimentally proven target site matches are incorrect, although in some experiments mismatches and insertions have been tested. More plausibly, the rules do not yet capture additional functionally relevant interactions of miRNAs, such as in maturation and transport. Such additional interactions remain to be described in molecular detail, such as interactions with the small RNA processing machinery (Drosha and Dicer) and with the components of RNPs (AGO and FMRP). A first step in this direction is the very recent analysis of the crystal structure of a PAZ domain of a human Argonaute protein, eIF2c1, complexed with a 9-mer RNA oligonucleotide in dimer configuration, which may represent three-dimensional interactions for the 3′ end of a miRNA (and siRNA) complexed, e.g., with Dicer or AGO ( Ma et al. 2004 ). In this structure, each PAZ domain makes close binding contact with nine nucleotides of a single-stranded RNA. The two 3′ terminal nucleotides bind in a pocket through RNA backbone and other contacts. The remaining seven nucleotides bind PAZ through a series of backbone contacts such that nucleotides 3 to 9 are in an RNA helical conformation with bases exposed for base pairing to the second single-stranded RNA. If a 20–21-nt single-stranded RNA is bound to a PAZ domain in the same fashion, the 5′ end would be free for other interactions, such as binding to another protein domain in the RISC or base-pairing to mRNA. The conformational entropy that results when the 3′ end binds to PAZ, because the RNA helix is pre-formed, is consistent with weaker base pairing between miRNA and mRNA at the 3′ end of the miRNA, and stronger base pairing at the 5′ end. The dimeric structure of the PAZ domain ( Ma et al. 2004 ) also raises the tantalizing possibility of cooperative binding of a dimer of two miRNA–PAZ combinations to two target sites on one or more mRNAs. In such an arrangement, seven residues at the 3′ ends of the two miRNAs (residues 3–9, but not the terminal two nucleotides) are paired in antiparallel fashion, with near perfect complementary pairing. As more details of molecular contacts become available, prediction rules will evolve and improve in accuracy. The following elements are worth considering in the next generation of target prediction rules: (1) details of strand bias as deduced from siRNA experiments ( Khvorova et al. 2003 ), (2) contribution of sequences outside of the mRNA target sites, (3) refinement of position-dependent rules, including different gap penalties for the mRNA and the miRNA, (4) energetics of miRNA–protein binding, starting with PAZ domain interaction, and (5) translation of systematic mutational profiling experiments into scoring rules ( Doench and Sharp 2004 ). Principles of Regulation by miRNAs Although the predicted targets are subject to error (see estimate of false positives) and the prediction rules in need of improvement, several general principles of gene regulation by miRNAs are emerging. (1) Except in cases where a highly complementary match causes cleavage of the target message, miRNAs appear to act cooperatively, requiring two or more target sites per message, for either one or several different miRNAs. (2) Most miRNAs are involved in the translational regulation of several target genes, which in some cases are grouped into functional categories. (3) miRNAs carried in the context of RNPs appear to be sequence-specific adaptors guiding RNPs to particular target sequences. miRNA regulation of cellular messages may therefore range from a switch-like behavior (e.g., cleavage of mRNA message) to a subtle modulation of protein dosage in a cell through low-level translational repression ( Bartel and Chen 2004 ). These aspects of miRNA regulation complicate the design of experiments aiming at testing target predictions, or, more generally, at discovering biologically meaningful targets. Straightforward experiments that test one target site for one miRNA on one UTR will not be able to disentangle the effects of multiplicity or cooperativity. Tests for multiple sites on one UTR for one miRNA capture aspects of cooperativity ( Doench and Sharp 2004 ), but still do not capture signal integration by diverse miRNAs. The most complicated situation is one in which multiple miRNAs affect multiple genes in combinatorial fashion, with fine-tuning depending on the state of the cell. We look forward to the results of ingenious experiments designed to deal with the complexity of miRNA regulation. The results of this genome-wide prediction for mammals and fish are meant to be a guide to experiments that will in time elucidate the genetic control network of regulators of transcription, translation/maturation, and degradation of gene products, including miRNAs. Materials and Methods miRNA sequences Mature human and mouse miRNA sequences were obtained from the RFAM miRNA registry ( Griffiths-Jones 2004 ). To cover cases of incomplete data, any mouse miRNA sequence not (yet) described in humans was assumed to be present in human, with the same sequence, and vice versa. Similarly, all mouse miRNAs were assumed to be identical and present in the rat genome. These assumptions are reasonable as sequence identity for known orthologous pairs in human and mouse is, on average, 98% (with 110 out of 146 orthologous sequences being identical). In total, 218 mammalian miRNAs were used. For human target searches, 162 native miRNA sequences were available plus 17 mouse and 39 rat miRNA sequences; for mouse, 191 native, 14 human, and 13 rat sequences; and for rat, 45 native, 159 mouse, and 14 human miRNA sequences. Mature miRNA sequences for zebrafish and fugu were predicted starting from known human and mouse miRNA precursor sequences ( Ambros et al. 2003a ). Each precursor sequence was used, in a scan against the zebrafish supercontigs (release 18.2.1) using NCBI BLASTN (version 2.2.6; E-value cutoff, 2.0) ( Altschul et al. 1990 ), to identify a sequence segment containing the potential zebrafish miRNA. The mammalian and fish segments were then realigned using a global alignment protocol (ALIGN in the FASTA package, version 2u65; Pearson and Lipman 1988 ). After testing the potential fish miRNA precursors for foldback structures ( Zuker 2003 ), the final set of 225 predicted zebrafish miRNAs was selected. The same set of sequences was used for fugu. 3′ UTR sequences The Ensembl database ( Birney et al. 2004 ) served as the source of genomic data. The Ensembl BioPerl application user interface was used to generate 3′ UTR sequences for all transcripts of all genes from each genome. Some transcripts are alternatively spliced from the same gene, so the total number of genes is smaller than the number of transcripts ( Table 3 ). When no Ensembl annotated 3′ UTR sequences were available, we predicted 3′ UTRs by taking 4,000 bp of genomic sequence downstream of the end of the last exon of a transcript ( Table 3 ). If this predicted region overlapped coding sequence on either strand, we halted 3′ UTR extension at that point. UTR orthology and alignment Orthology mappings between genes from different genomes were obtained using “orthologue tables” from the EnsMart ( Kasprzyk et al. 2004 ) feature of the Ensembl database. Pairs of orthologous UTRs were aligned with each other using the AVID ( Bray et al. 2003 ) alignment algorithm to facilitate analysis of conservation of position and sequence of target sites. In total, 26,205 human transcripts, representing 15,869 genes, were mapped to both mouse and rat transcripts. For zebrafish, 11,442 transcripts, representing 10,909 genes, were mapped to fugu transcripts and 11,306 transcripts mapped to human transcripts (10,063 genes). miRNA target prediction The miRanda algorithm (version 1.0; Enright et al. 2003 ) was used to scan all available miRNA sequences for a given genome against 3′ UTR sequences of that genome derived from the Ensembl database and—tabulated separately—against all cDNA sequences and coding regions. The algorithm uses dynamic programming to search for maximal local complementarity alignments, corresponding to a double-stranded antiparallel duplex. A score of +5 was assigned for G:C and A:T pairs, +2 for G:U wobble pairs, and −3 for mismatch pairs, and the gap-open and gap-elongation parameters were set to −8.0 and −2.0, respectively. To significantly increase the speed of miRanda runs, in calculating the optimal alignment score at positions i, j in the alignment scoring matrix, the gap-elongation parameter was used only if the extension to i, j of a given stretch of gaps ending at positions i–1 , j or j–1 , i (but not of stretches of gaps ending at i–k, j or j, i–k for k > 1) resulted in a higher score than the addition of a nucleotide–nucleotide match at positions i, j . Removal of this restriction with the availability of more computing power would result in a moderate increase in average loop length, but the advantages of this would probably be superceded by overall refinement of target prediction rules. Importantly, complementarity scores at the first eleven positions, counting from the miRNA 5′ end, were multiplied by a scaling factor of 2.0, so as to approximately reflect the experimentally observed 5′–3′ asymmetry; for example, G:C and A:T base pairs contributed +10 to the match score in these positions. The value of the scaling factor at each position is an adjustable parameter subject to optimization as more experimental information becomes available. Because of the ongoing discussion about the rules for target prediction, target genes (a total of 490) that contained target sites with more than one G:U wobble in the 5′ end are flagged in the Table S2 . The thresholds for candidate target sites were S > 90 and Δ G < −17 kcal/mol, where S is the sum of single-residue-pair match scores over the alignment trace and Δ G is the free energy of duplex formation from a completely dissociated state, calculated using the Vienna package as in Enright et al. (2003) . After finding optimal local matches above these thresholds between a particular miRNA and the set of 3′ UTRs in each genome, we asked whether target site position and sequence for this miRNA were conserved in the 3′ UTRs of orthologous genes, i.e., between human and mouse or rat, or between fugu and zebrafish. The alignments of target sites were generated transitively (UTR→miRNA→UTR) via a shared (or homologous) miRNA. We required that the positions of pairs of target sites in two species fall within ±10 residues in the aligned 3′ UTRs. Conserved target sites with sequence identity of 90% or more (human versus mouse or rat) and 70% or more (zebrafish versus fugu) were selected as candidate miRNA target sites and stored in a MySQL database. Using human as the reference species, we predicted 10,572 conserved target sites (conserved in either mouse or rat) in 4,463 human transcripts, of which 2,307 transcripts of 2,273 genes contained more than one target site. Similarly, using zebrafish as a reference species, we predicted 7,057 conserved target sites (conserved in fugu) in 4,820 zebrafish transcripts. To focus on the strongest predictions, conserved target sites for each miRNA were sorted according to alignment score, with free energy as the secondary sort criterion. In cases where multiple miRNAs targeted the same site on a transcript (or within 25 nt of a site), only the highest scoring, lowest energy miRNA was reported for that site. Functional analysis of targets. To facilitate surveys of target function and analysis of functional enrichment, InterPro domain assignments ( Mulder et al. 2003 ) and GO (molecular function hierarchy) mappings ( Ashburner and Lewis 2002 ) for all human genes were obtained using EnsMart. For each functional class derived from either source, we calculated its degree of under- or overrepresentation, F class , using the log-odds ratio of the fraction of annotated target genes with the same class (F 1 ) and the fraction of all annotated Ensembl human genes with that class (F 2 ): Here, N represents the number of genes of a given functional class for either target genes (N tar ) or all genes (N all ), and C represents the total number of functional classes. To eliminate bias from small counts we did not report assignments that were present in less than 1% of all annotated target genes ( F 1 ≤ 0.01 or F 2 ≤ 0.01). Randomized trials For each random experiment all miRNAs were shuffled by randomly swapping two bases of a miRNA 1,000 times. These shuffled sequences were then searched against human, mouse, and rat 3′ UTR sequences in the same way described for the main analysis, including analysis of conservation of target site sequence and position in orthologous 3′ UTRs. A total of ten randomized experiments were performed. Counts were averaged across all experiments, and the standard deviation and other statistical measures were calculated. Analysis of FMRP-associated mRNAs We compiled a list of 464 gene identifiers of FMRP-associated mRNAs from five different publications ( Brown et al. 2001 ; Chen et al. 2003 ; Denman 2003 ; Miyashiro et al. 2003 ; Waggoner and Liebhaber 2003 ). Among the 464 gene identifiers, 397 identifiers were mapped to the corresponding genes in our 3′ UTR dataset. The remaining 67 genes were not mapped because their published identifiers were obsolete, primarily because of their Affymetrix probeset identification numbers. To identify miRNA regulation of the 397 FMRP-associated mRNAs, these genes were then compared with the set of predicted miRNA targets. CPE motif prediction. We predicted CPE motifs in human, mouse, and rat UTRs. We used a search pattern using four criteria: (1) presence of the CPE motif UUUUAU, (2) presence of the hexanucleotide AAUAAA, (3) the CPE and the hexanucleotide motif being within 100 nucleotides of each other, and (4) the conservation of these motifs and the positions of the motifs in the mouse ortholog ( Mendez and Richter 2001 ). Supporting Information Figure S1 Overrepresentation of the GO and Interpro Domains (347 KB PDF). Click here for additional data file. Table S1 Human miRNAs in Introns (25 KB XLS). Click here for additional data file. Table S2 Predicted Mammalian miRNA Targets by Gene (8.0 MB XLS). Click here for additional data file. Table S3 Predicted Mammalian miRNA Targets by miRNA (17.0 MB XLS). Click here for additional data file. Table S4 Predicted Fish Targets by Gene (5.6 MB XLS). Click here for additional data file. Table S5 Predicted Fish Targets by miRNA (9.8 MB XLS). Click here for additional data file. Table S6 High-Scoring miRNA Matches in Human cDNAs (601 KB XLS). Click here for additional data file. Table S7 High-Scoring miRNA Matches in Human Coding Regions (512 KB XLS). Click here for additional data file. Table S8 Estimate of False Positives (23 KB XLS). Click here for additional data file. Table S9 Predicted Targets That Are Associated with FMRP (678 KB XLS). Click here for additional data file. Table S10 Function of Targets by Interpro and GO Mapping (357 KB XLS). Click here for additional data file. Table S11 Target Genes That Contain Predicted CPE Motifs (529 KB XLS). Click here for additional data file. Table S12 Conserved Vertebrate Target Genes (621 KB XLS). Click here for additional data file. Table S13 Overlap of the Predicted Targets with Validated Gene Targets from Lewis et al. (2003) and Kiriakidou et al. (2004) (68 KB XLS). Click here for additional data file.

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Functional effects of 17alpha-hydroxyprogesterone caproate (17P) on human myometrial contractility in vitro

Background 17alpha-hydroxyprogesterone caproate (17P) administration reportedly improves outcome for women with a previous spontaneous preterm delivery. This study, using in vitro strips of human uterine smooth muscle, aimed to investigate the direct non-genomic effects of 17P on spontaneous and induced contractions in tissues obtained during pregnancy, and in the non-pregnant state. Methods Biopsies of human myometrium were obtained at elective cesarean section, and from hysterectomy specimens, and dissected strips suspended for isometric recordings. The effects of 17P (1 nmol/L -10 micro mol/L) on spontaneous and agonist-induced (oxytocin 0.5 nmol/L for pregnant, phenylephrine 10 μmol/L for non-pregnant) contractions were measured. Integrals of contractile activity, including the mean maximal inhibition values (MMI) observed at the maximal concentration, were compared with those from simultaneously run control strips. Results There was no significant direct effect exerted by 17P on pregnant or non-pregnant human myometrial contractility. The MMI ± SEM for spontaneous contractions in pregnant myometrium was 4.9% ± 7.2 (n = 6; P = 0.309) and for oxytocin-induced contractions was 2.2% ± 1.3 (n = 6; P = 0.128). For non-pregnant myometrium, the MMI ± SEM for spontaneous contractions was 8.8% ± 11.0 (n = 6; P = 0.121) and for phenylephrine induced contractions was -7.9% ± 6.5 (n = 6; P = 0.966). Conclusions The putative benefits of 17P for preterm labor prevention are not achieved, even partially, by a direct utero-relaxant effect. These findings outline the possibility that genomic effects of 17P, achieved over long periods of administration, are required for its reported therapeutic benefits.

Background Preterm delivery constitutes a major problem in obstetric practice because of the large associated contribution to perinatal mortality and morbidity [ 1 , 2 ]. A significant proportion of all preterm deliveries are due to spontaneous preterm labor [ 2 ]. Despite much research effort, until recently, no effective method of preventing or treating preterm labor, and improving neonatal outcome, has been available. Meta-analysis of various tocolytic compounds in clinical practice has revealed that while they resulted in a delay in the interval to delivery, of time periods up to a week, they did not reduce the incidence of preterm delivery at different gestational ages [ 3 ]. More importantly, their use, in comparison to placebo, was not associated with any benefit in terms of objective measures of neonatal wellbeing or morbidity. It has however been recently reported that weekly injections of 17-alpha-hydroxyprogesterone caproate (17P), in women who have had a previous spontaneous preterm delivery, significantly reduces the risk of preterm delivery before 37, 35 and 32 week's gestation [ 4 ]. In addition, infants of women treated with 17P had significantly lower rates of necrotising enterocolitis, intraventricular haemorrhage and the need for supplemental oxygen. While evidence for the use of progestational compounds for prevention of preterm delivery, and recurrent miscarriage, has been conflicting [ 5 - 7 ], meta-analysis restricted to trials of 17P has suggested a significant reduction in the preterm delivery rate. This reported benefit of 17P, while being a welcome development in therapeutic strategies for preterm labor, has raised many questions in relation to whether the same benefit would apply to low risk groups, and the potential effects, if any, from the castor-oil injection of placebo used [ 8 , 9 ]. One of the most important questions relates to the mechanism by which the drug works and there are currently no data to answer this. Progestins have the potential to exert both genomic and non-genomic effects. The aims of this study were focused specifically on the latter mechanism i.e. to investigate the direct effects of 17P on contractions of isolated human myometrium, both spontaneous and agonist-induced, in tissue obtained during pregnancy and in the non-pregnant state. Methods Patient Recruitment and Tissue collection Patient recruitment took place in the Department of Obstetrics and Gynaecology, University College Hospital Galway. Ethical committee approval for tissue collection was obtained from the Research Ethics Committee at University College Hospital Galway and recruitment was by written informed consent. The biopsies were excised from the upper lip of the lower uterine segment incision in the midline i.e. upper portion of lower uterine segment. Women undergoing induction of labor were excluded from the study. For hysterectomy specimens, all women were pre-menopausal and undergoing abdominal hysterectomy without evidence of malignant uterine disease. Biopsies of myometrial tissue from hysterectomy specimens were obtained from the fundus. The biopsies were immediately placed in Krebs-Henseleit physiological salt solution (PSS), pH 7.4, containing: 4.7 mmol/L KCl, 118 mmol/L NaCl, 1.2 mmol/L Mg 2 SO 4 , 1.2 mmol/L CaCl 2 , 1.2 mmol/L KPO 4 , 25 mmol/L NaHCO 3 , and 11 mmol/L glucose. Tissues were stored at 4° C and used within 12 hours of collection. Tissue Bath Experiments Longitudinal myometrial strips were dissected measuring approximately 2 × 2 × 10 mm. The strips were mounted for isometric recording under 2 grams of tension in organ tissue baths, as previously described [ 10 - 12 ]. The tissue baths contained 10 ml of Krebs-Henseleit physiologic salt solution maintained at 37°C, pH 7.4 and gassed continuously with 95%O 2 /5%CO 2 . Myometrial strips were allowed to equilibrate for at least 60 minutes, during which time the Krebs-Henseleit physiologic salt solution was changed every 15 minutes. After the equilibration period, regular spontaneous myometrial contractions were allowed to develop. In separate experiments contractions were induced by the addition of oxytocin (0.5 nmol/L) to pregnant myometrial strips, and phenylephrine (10 μmol/L) to strips obtained from non-pregnant myometrium. Once regular phasic contractions had developed, the integrated tension for the first 20 minutes was calculated, and this value served as a control since no significant spontaneous reduction in myometrial contractility was observed over the duration of experiments in control strips (without addition of vehicle or 17P). The mechanical response of tissues was then measured by calculation of the integral of selected areas for 20 minute periods, corresponding to the cumulative exposure to 17P or vehicle, using the PowerLab hardware data acquisition system and Chart v3.6 software (AD Instruments, Hastings, UK). At 20 minute intervals 17P was added in a cumulative manner at concentrations of 1 nmol/L, 10 nmol/L, 100 nmol/L, 1 μmol/L, and 10 μmol/L (i.e., 1 × 10 -9 - 1 × 10 -5 M). Control strips (i.e. without exposure to 17P) were run simultaneously and for a similar duration, consisting of two separate groups of experiments as follows: 1. exposure of strips to PSS only; 2. exposure of strips to PSS and the vehicle for 17P. The overall duration of experiments was therefore 3 hours which is in accordance with standard in vitro myometrial experiments, allowing for an accurate drug exposure period of 20 minutes. The effects of 17P were assessed by subtracting the integrals of contractility measured after each bath exposure of 17P, from those obtained in control experiments (vehicle only). This allowed for calculation of the net effect of 17P on myometrial contractility. Potential effects of the vehicle were obtained by subtraction of the mean integrals measured after vehicle exposure, from those obtained without addition of vehicle i.e. spontaneous or agonist-induced contractions in PSS only. Percentage contractility was calculated by expressing the net integral measured after each 17P concentration addition, as a percentage of the integral calculated in the 20 minute period prior to any 17P addition. The mean maximal inhibition (MMI) refers to the mean percentage relaxation [i.e. 100% - mean contractility measured for each separate n sample of non-pregnant and pregnant myometrium] observed at the maximal bath concentration of 17P (i.e. 10 μmol/L), or corresponding concentration of vehicle for control strips. Drugs and Solutions Oxytocin, phenylephrine and 17P were purchased from Sigma-Aldrich, Dublin, Ireland. Because 17P is a lipid soluble compound it was necessary to dissolve it in a lipophilic vehicle. The vehicle used consisted of 75% Dimethyl sulfoxide (DMSO): 25% ethanol, to make a 1 mmol/L (10 -3 M) stock solution of 17P. The resultant final tissue bath concentrations of DMSO and ethanol, at the maximum concentration of 17P investigated (i.e. 10 μmol/L or 10 -5 M), were 0.83% and 0.28% respectively, allowing for the cumulative effect at each bath addition. DMSO and ethanol were purchased from Sigma-Aldrich, Dublin, Ireland. Fresh Krebs-Henseleit physiological salt solution was made daily. Fresh 17P solutions were prepared on the day of experimentation and were maintained at room temperature for the duration of the experiment. Statistical Analysis The integrals of contractile activity measured were compared to control values, and expressed as a percentage of that measured before drug addition. The effects of 17P on myometrial contractility were calculated for each 20-minute period of exposure from 1 nmol/L-10 μmol/L, and compared with the measurements obtained from control strips (ie. spontaneous, oxytocin-induced or phenylephrine-induced contractility, in the presence and absence of vehicle). The integrals of contractile activity were compared using a one-way ANOVA, which was followed by a Tukey HSD post-hoc. A value of P < 0.05 was accepted as statistical significance. Results There were 6 women recruited for the study at the time of elective cesarean section. The reasons for cesarean section included previous cesarean section (n = 2), breech presentation (n = 2), unstable lie (n = 1) and postmaturity with poor cervical Bishop score (n = 1). The mean age, ± standard error of the mean (SEM), was 36.3 ± 2.8 years (range 26–46). The median gestation at the time of cesarean section was 39 weeks (range 38–41). In relation to parity, 2 of the women were para 0, 1 was para 1, and 3 were of parity greater than 1, at the time of recruitment. There were 6 women recruited at the time of abdominal hysterectomy. The reasons for hysterectomy included menorrhagia (n = 4), irregular vaginal bleeding (n = 1), and fibroids with an ovarian cyst (n = 1). The mean age ± SEM was 42.2 ± 2.4 years (range 36–50). For non-pregnant myometrial tissue representative recordings are shown in Figure 1 . In Figure 1A a recording of spontaneous myometrial contractions is shown. In Figure 1B , the effects of vehicle for 17P on spontaneous contractions is demonstrated, and in Figure 1C the effects of addition of 17P are shown. The results of the calculated integrals are provided in Table 1 . Bath exposure of the strips from hysterectomy specimens to 17P did not result in any alteration in contractile activity, at any of the bath concentrations studied, in comparison to vehicle only experiments (spontaneous contractions: P = 0.121; phenylephrine-induced contractions: P = 0.966). Figure 1 Effects of 17P on myometrial contractility in non-pregnant tissue. Representative recordings of spontaneous contractions in PSS only (A), the effects of cumulative additions of 17P vehicle (B), and the effects of cumulative additions of 17P (C) are shown. Table 1 Mean Maximal Inhibition Values for Vehicle and 17P. Myometrial Contractility Non-Pregnant Pregnant Net Vehicle Relaxation ± SEM Net 17P Relaxation ± SEM Net Vehicle Relaxation ± SEM Net 17P Relaxation ± SEM Spontaneous -0.6% ± 12.1 (n = 6) 8.8% ± 11.0 (n = 6) 49.7% ± 11.9 (n = 6) ♦ 4.9% ± 7.2 (n = 6) *Agonist-Induced 31.8% ± 5.0 (n = 6) -7.9% ± 6.5 (n = 6) 56.2% ± 2.3 (n = 6) ¶ 2.2% ± 1.3 (n = 6) *Phenylephrine-induced in non-pregnant myometrium and oxytocin-induced in pregnant myometrium. ♦ P = 0.022 in comparison to controls with PSS only ¶ P < 0.001 in comparison to controls with PSS only The results obtained from myometrium during pregnancy are similarly shown in Figure 2 , and in Table 1 . Figures 2A , 2B and 2C demonstrate recordings of oxytocin-induced contractions, the effects of vehicle, and the effects of 17P respectively. There was no significant net relaxant or uterotonic effect exerted by 17P on pregnant myometrial contractility, at any of the bath concentrations studied experiments (spontaneous contractions P = 0.309; oxytocin-induced contractions: P = 0.128). No significant difference was observed between the effects of 17P on contractility in either pregnant or non-pregnant myometrium. Figure 2 Effects of 17P on myometrial contractility in pregnant tissue. Representative recordings of oxytocin-induced contractions in PSS only (A), the effects of cumulative additions of 17P vehicle (B), and the effects of cumulative additions of 17P (C) are shown. The vehicle for 17P independently exerted a uterorelaxant effect on spontaneous and agonist-induced contractions in pregnant myometrial tissue only (spontaneous contractions P = 0.022; oxytocin-induced contractions: P = 0.000), and this was not observed in non-pregnant myometrial tissue (spontaneous contractions P = 0.241; phenylephrine-induced contractions: P = 0.068) (Table 1 ). Discussion These results demonstrate that 17P does not appear to exert a direct relaxant effect on human myometrial contractions in vitro, in tissue obtained during the third trimester of pregnancy, or in the non-pregnant state. These findings are in contrast to the inhibitory effect of progesterone derivatives on spontaneous contractions in animal uterine tissues [ 13 , 14 ], and therefore suggest that the reported benefit of 17P in preventing preterm delivery in women who have had a previous preterm delivery, involves other mechanisms of action, presumably via its genomic effects. It seems likely that prolonged exposure of uterine smooth muscle to 17P, with resultant activation of the progesterone receptor isoforms, has the potential to modify gene transcription in order to maintain physiological uterine quiescence. However no direct effect on contractile activity was observed in our study over a period of hours of exposure. Previous studies, using various progestins, have yielded conflicting results in terms of the direct effects of these metabolites on human myometrial contractility in vitro. Progesterone metabolites, in some studies, have been reported to decrease both the frequency and amplitude of contractions [ 15 - 17 ], while other reports have outlined that progesterone stimulates the frequency and tonus of contractions in term human myometrium [ 18 , 19 ]. It has been hypothesized that progesterone addition to myometrial strips only enhances contractility if the tissue specimen was never deprived of progesterone i.e. placed immediately in a medium containing progesterone [ 19 ]. The studies to date have included various progesterone metabolites but we are unaware of any previous studies evaluating the effects of 17P on human myometrial contractions in vitro. The focus on 17P in this study has arisen from the recently reported randomized clinical trial outlining its benefits clinically for recurrent preterm labor. 17P is a naturally occurring progesterone which has been isolated from corpus luteum and adrenal gland. The synthetic caproate ester, like the naturally occurring compound, is a steroid, is highly lipophilic and is inactive when administered orally. To achieve solubility for clinical studies, the vehicle used for injection was castor-oil [ 4 ]. This led to significant controversy in relation to the potential effect of castor-oil on myometrial contractions in the placebo arm of the recently reported study [ 8 ]. For laboratory, or in vitro studies, achieving the required solubility of progestins can also be a difficult process. Efforts to achieve solubility in previous reports have included the use of Hepes buffer and ultrasound baths [ 19 , 20 ] with a resultant maximum 50% solubility. After numerous attempts at solubility for 17P in our studies, the most appropriate vehicle was a combination of DMSO and ethanol. While both of these compounds may exert an effect on uterine contractility, the maximal bath concentrations of both solvents was 0.83% and 0.28% respectively, which is acceptable for in vitro studies of this nature. In addition, control experiments, with PSS only, and PSS plus vehicle, were simultaneously run, to clearly evaluate any potential effect of vehicle. It is however apparent from our results, that addition of vehicle only did exert a significant relaxant effect on myometrium obtained during pregnancy, but no effect of vehicle was observed in myometrium obtained in the non-pregnant state. For both tissues, addition of 17P did not alter contractile activity. While the vehicle effects observed were sizeable, the experiment design, the numbers of patients recruited, and the reproducibility of the results, clearly indicate that 17P does not exert a direct effect on human myometrial contractility. In addition, while the experiments described here investigated the effects of 17P added cumulatively, separate experiments (data not shown) using a single dose (the highest dose) revealed similar results i.e. there was no evidence of tachyphylaxis over the duration of the experiments. There are some limitations to the methodology used in our study. The tissue biopsies from the non-pregnant uterus were obtained from the body of the uterus, while those obtained at the time of caesarean section were excised from the lower uterine segment. The results were similar for both tissue types, and there is reasonable evidence to suggest that the functional characteristics of lower and upper uterine segment myometrium are similar [ 21 ]. At present there are no data pertaining to differential progesterone receptor expression levels between upper and lower uterine segments. There are also obvious ethical constraints in obtaining biopsies from the upper segment of the uterus at cesarean section, and it is not feasible to dissect strips with certainty from the lower segment of the non-pregnant uterus. Secondly, the tissue samples obtained during pregnancy were all recruited from women at term. It could be that preterm myometrium displays a different responsiveness. Finally, in vivo administration of 17P could potentially result in the formation of a metabolite with a more efficient uterorelaxant effect. Conclusions Whether 17P injections become incorporated for routine use in the management of preterm labor remains to be seen. Questions surrounding its true benefits, the associated difficulties in terms of vehicle solubility and placebo, and the mechanism of action, remain. Our findings highlight the solubility problems for scientific evaluation as occurred in clinical studies. Whatever the possible mechanism of action of 17P in reducing the incidence and adverse sequelae of preterm labor, this study demonstrates that it does not exert a direct relaxant effect, unlike other conventional methods of tocolysis investigated, and raised the likeliehood of a genomic effect secondary to long term administration during pregnancy. Authors' contributions DJS performed the experiments and wrote the manuscript. MWO'R performed the experiments. AMF analysed the data and wrote the manuscript. JJM designed, supervised the study and wrote the manuscript. All authors read and approved the final manuscript.

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509420

Corneal topographic changes following retinal surgery

Background To study the effect of retinal/ vitreoretinal surgeries on corneal elevations. Methods Patients who underwent retinal/ vitreoretinal surgeries were divided into 3 groups. Scleral buckling was performed in 11 eyes (Group 1). In 8 (25%) eyes, vitreoretinal surgery was performed along with scleral buckling (Group 2). In 12 eyes, pars plana vitrectomy was performed for vitreous hemorrhage (Group 3). An encircling element was used in all the eyes. The parameters evaluated were best-corrected visual acuity (BCVA), change in axial length, and corneal topographic changes on Orbscan topography system II, preoperative and at 12 weeks following surgery. Results There was a statistically significant increase in anterior corneal elevation in all the three groups after surgery (p = 0.003, p = 0.008 & p = 0.003 respectively). The increase in posterior corneal elevation was highly significant in all the three groups after surgery (p = 0.0000, p = 0.0001 & p = 0.0001 respectively). The increase in the posterior corneal elevation was more than the increase in the anterior elevation and was significant statistically in all the three groups (group I: p = 0.02; group II: p = 0.01; group III: p = 0.008). Conclusions Retinal/ vitreoretinal surgeries cause a significant increase in the corneal elevations and have a greater effect on the posterior corneal surface.

Background Changes in corneal curvature and axial length have been reported following scleral buckling procedure using keratometer [ 1 - 7 ]. Videokeratography has also been used to assess the corneal surface changes after buckling procedure[ 8 ]. All these studies have reported a change in the corneal curvature in its anterior surface. However, no study has been performed to evaluate the posterior corneal topographic changes with scanning slit topography system following retinal surgeries. Hence, we made an attempt to study the effect of various retinal surgeries on posterior corneal surface using Orbscan II topography system. Methods A prospective study was performed by enrolling patients admitted in the Retina Services of Rajendra Prasad Centre for Ophthalmic Sciences, New Delhi for retinal/ vitreoretinal surgeries. Thirty one eyes of 31 patients who underwent retinal/ vitreoretinal surgery and came for regular follow-up as per schedule during the period between December 2001 and October 2002 were included in the study. Only those patients were included who had not undergone any previous ocular surgery & did not have any corneal pathology. An informed consent was obtained from all the patients. The patients were evaluated preoperatively on parameters of best-corrected visual acuity (BCVA), axial length (AL) measured by Ultrasound A-scan instrument (Sonomed, Inc., NY) and detailed corneal examination on slit-lamp biomicroscope and on Orbscan topography system II (Bausch and Lomb, Salt Lake City, Utah). The parameters that were evaluated by Orbscan topography system were anterior elevation, posterior elevation and simulated keratometry. All surgeries were performed by a single surgeon (LV) under local anesthesia by peribulbar injection of 6 ml of 2% xylocaine and 2 ml of 0.5% bupivacaine. Eyes with fresh retinal detachment with clear media and absence of advanced proliferative vitreoretinopathy underwent scleral buckling procedure (Group I, n = 11). In all these eyes, the break/ s were localized, cryotherapy was performed and subretinal fluid was drained. Only circumferential buckle of silicone of style 276 (Labtician Ophthalmics, Inc., Oakville, Canada) was used and radial buckle or sponge was not used in any eye. The size of the buckle was 90° to 360° depending upon the requirement in individual cases. Encircling element of style 240 (Labtician Ophthalmics, Inc., Oakville, Canada) was used in all the eyes undergoing scleral buckling. In eyes with associated vitreous hemorrhage or advanced proliferative vitreoretinopathy changes along with retinal detachment, vitreoretinal surgery was performed along with scleral buckling (Group II, n = 8). Pars plana vitrectomy was performed and vitreoretinal membranes were removed either by peeling or by segmentation or delamination. Air fluid exchange was performed followed by Air-Silicone oil exchange. In eyes with only vitreous hemorrhage without the presence of retinal detachment, only pars plana vitrectomy was performed (Group III, n = 12). An encircling element of style 240 (Labtician Ophthalmics, Inc., Oakville, Canada) was used in all these eyes to counter the anterior traction that could not be fully released by vitrectomy in order to avoid lens damage. The intraoperative details including the nature of surgery, size of the buckle, encircling element, drainage of subretinal fluid, vitrectomy & use of silicone oil or gas, were noted. Postoperative treatment included topical ciprofloxacin 0.3% QID, topical dexamethasone 0.1% QID and topical Homatropine 2% QID. The patients were evaluated at 12 weeks following surgery on similar (preoperative) parameters. Statistical analysis The data of all the patients were managed on an excel spreadsheet. All the entries were checked for any possible keyboard error. Preoperative and postoperative measurements in the three retinal surgery groups were summarized by mean and standard deviation. Changes following surgery within each group were assessed using paired 't' test. Preoperative and postoperative values in the three groups were compared using one way analysis of variance (ANOVA), followed by bon ferroni correction for multiple comparison. For the three retinal surgery groups, median was computed for increase in various parameters due to surgery. Kruskal Wallis one way analysis of variance was used to compare median increase in the three groups. STATA 7.0 statistical software was used for data analysis. In this study, p-value smaller than 0.05 was considered as statistically significant. Results The mean age of the patients was 45.96 ± 15.17 (range: 18–78) years and majority (83.87%) of the patients (N = 31) were males. Of these, right eye was operated in 19 (61.29%) patients. The mean preoperative best-corrected visual acuity (BCVA) was hand motion close to face in 26 eyes; counting finger near to face in 4 eyes and 1/60 on snellen's acuity chart in 1 eye. The mean decimal postoperative BCVA was 0.20 ± 0.12 at 12 weeks follow-up after surgery. The mean preoperative anterior corneal elevation as recorded by Orbscan II topography system in group I was 0.006 ± 0.007 mm, which increased to 0.024 ± 0.013 mm at 12 weeks after surgery (p= 0.003). In group II, it increased from 0.009 ± 0.006 mm preoperatively to 0.021 ± 0.010 mm at 12 weeks (p= 0.008) and in group III, it increased from 0.003 ± 0.004 mm preoperatively to 0.012 ± 0.007 mm at 12 weeks follow-up (p = 0.003). On comparative evaluation between the groups, the change in anterior corneal elevation was significant between group I and III (p = 0.04). The mean posterior elevation in group I increased from a preoperative value of 0.016 ± 0.010 mm to 0.043 ± 0.007 mm at 12 weeks after surgery (p= 0.0000). In group II, it increased from 0.014 ± 0.006 mm preoperatively to 0.043 ± 0.007 mm at 12 weeks (p= 0.0001) and in group III it increased from a preoperative value of 0.012 ± 0.005 mm to 0.029 ± 0.006 mm at 12 weeks after surgery (p = 0.0001). A comparative analysis between the groups indicated that the increase in posterior corneal elevation between groups I & III and groups II & III were found to be highly significant (I vs III: p= 0.001; II vs III: p= 0.001). Again, the increase in the posterior corneal elevation was greater than the increase in the anterior elevation in all the 3 groups and on comparative evaluation, the difference in the increase in posterior and anterior elevation was significant statistically in each group (group I: p = 0.02; group II: p = 0.01; group III: p = 0.008). The mean corneal astigmatism in group I increased from 0.89 ± 0.54D preoperatively to 2.50 ± 1.39D at 12 weeks follow-up (p= 0.004). In group II, the average corneal astigmatism increased from 0.87 ± 0.30D to 3.38 ± 2.15D at 12 weeks (p= 0.01) and in group III, the mean preoperative and postoperative corneal astigmatism was 0.85 ± 0.55D and 1.37 ± 0.87D respectively (p= 0.02). A comparative analysis of the change in corneal astigmatism following surgery between groups II & III was significant statistically (p= 0.02). The mean preoperative axial length in group I was 23.27 ± 0.79 mm which increased to 23.98 ± 0.76 mm at 12 weeks after surgery (p= 0.009). The mean preoperative and postoperative (12 weeks follow-up) axial length in group II were 23.92 ± 1.32 mm and 25.94 ± 2.96 mm respectively (p= 0.03). The mean preoperative and postoperative axial length in group III were 22.69 ± 0.87 mm and 22.71 ± 0.83 mm respectively (p= 0.79). Comparative analysis of increase in axial length following surgery between groups I & III and groups II & III were found to be significant statistically (I & III: p = 0.003; II & III: p = 0.003). Discussion Retinal surgery with or without the use of encircling and buckling elements for external tamponade can alter the shape of the globe. This may cause changes in the refractive status of the eye. Scleral buckling is known to cause a change in the shape of the sclera and can cause induced refractive changes, including astigmatic and nonastigmatic changes [ 5 - 10 ]. We have used Orbscan slit scanning system II to evaluate the corneal topographic changes following retinal/ vitreoretinal surgeries. The data accumulated by Orbscan may be limited by factors such as the accuracy of the system which is ± 20 μm, the measurement noise which leads to both positive and negative difference in the height of the posterior corneal surface and the necessity of aligning the posterior surface before and after surgery which may be a source of error[ 11 , 12 ]. However, this is the best tool available to study the posterior corneal elevation. In the present study, there was a significant increase in both anterior and posterior corneal elevation as detected by scanning slit topography (Orbscan II topography system) following surgery. This increase in the anterior and posterior corneal elevation is probably due to the use of encircling element and/ or buckle in retinal surgeries resulting in corneal steepening. We noted that the change in the posterior elevation was more significant than anterior elevation. A comparative analysis between the three groups indicated that there was no significant difference in the anterior elevation; however the posterior elevation was significantly more in eyes with buckle. It is possible that the buckle and the encircling element have a greater effect on the posterior corneal surface. The increase in the anterior and posterior corneal elevation might be one of the contributing factors for the non improvement of visual acuity in an eye that has undergone retinal/ vitreoretinal surgery. This change might escape detection by routine videokeratography. The anterior protrusion of the corneal back surface induces an increase in the negative power of the corneal surface. Assessing the corneal surface by keratometry or placido disc videokeratography may provide inadequate information regarding refractive change caused by corneal surface alteration that results in retinal/ vitreoretinal surgery. There was an increase in mean corneal astigmatism following surgery in all the groups. This is due to the effect of encircling element or buckle on the corneal surface. Studies have reported that induced astigmatism has been associated with radial scleral buckles[ 5 , 13 ], circumferential buckles[ 14 ], medial rectus disinsertion[ 15 ], anterior location of the scleral buckle[ 13 ], and use of sponge material rather than hard silicone[ 1 ]. In our study, a comparative analysis between the groups indicated that buckle causes more astigmatic changes than encircling element. In retinal/ vitreoretinal surgeries, the encircling band creates a circular indentation of the eye, thereby increasing its anteroposterior axial length; the myopic shift may be upto 3 diopters (D)[ 9 , 10 , 13 , 16 ]. An increase in axial length by 0.54 mm[ 9 ] and 1.7 mm[ 14 ] has been reported following scleral buckling in two studies. In the present study, there was an increase in the mean axial length of the eyes in all the three groups (Table 1). This increase in axial length may be attributed to an anteroposterior elongation of the eyeball secondary to the transverse compression by the buckle and/ or an encircling element. The postoperative increase in axial length was found to be more pronounced in eyes with buckle. Buckle being wider and thicker results in greater indentation and thereby a greater increase in axial length than encircling element. Conclusions Retinal/ vitreoretinal surgeries result in an increase in the elevation of the corneal surfaces. These changes are more pronounced on posterior corneal surface. Declaration of competing interest None declared. Individual contribution of authors RS designed the study and performed the data collection. NS wrote the manuscript. LKV performed the retinal surgeries. RMP performed the statistical analysis and RBV followed up the patients. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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509421

Brain choline concentrations may not be altered in euthymic bipolar disorder patients chronically treated with either lithium or sodium valproate

Background It has been suggested that lithium increases choline concentrations, although previous human studies examining this possibility using 1 H magnetic resonance spectroscopy ( 1 H MRS) have had mixed results: some found increases while most found no differences. Methods The present study utilized 1 H MRS, in a 3 T scanner to examine the effects of both lithium and sodium valproate upon choline concentrations in treated euthymic bipolar patients utilizing two different methodologies. In the first part of the study healthy controls (n = 18) were compared with euthymic Bipolar Disorder patients (Type I and Type II) who were taking either lithium (n = 14) or sodium valproate (n = 11), and temporal lobe choline/creatine (Cho/Cr) ratios were determined. In the second part we examined a separate group of euthymic Bipolar Disorder Type I patients taking sodium valproate (n = 9) and compared these to controls (n = 11). Here we measured the absolute concentrations of choline in both temporal and frontal lobes. Results The results from the first part of the study showed that bipolar patients chronically treated with both lithium and sodium valproate had significantly reduced temporal lobe Cho/Cr ratios. In contrast, in the second part of the study, there were no effects of sodium valproate on either absolute choline concentrations or on Cho/Cr ratios in either temporal or frontal lobes. Conclusions These findings suggest that measuring Cho/Cr ratios may not accurately reflect brain choline concentrations. In addition, the results do not support previous suggestions that either lithium or valproate increases choline concentrations in bipolar patients.

Background Bipolar disorder is a chronic severe mental illness affecting approximately 1% of the adult population. The most widely used mood stabilizer for this condition is lithium [ 1 ], although the exact mechanism by which it is clinically effective remains undetermined. One suggestion is that it acts via effects on choline metabolism. This is based upon findings that lithium can inhibit the membrane transport of choline in both animals [ 2 ], and human post-mortem brain tissue [ 3 ]. It also increases the accumulation of erythrocyte choline in lithium-treated patients [ 4 - 7 ]. Also of note is that choline concentrations increase significantly in rats following electroconvulsive shock [ 8 ]. Based upon this data choline has been used to treat mania in a some small pilot studies [ 9 ], with one open label study reporting that choline augmentation of lithium treatment helped rapid-cyclers [ 10 ]. Patients treated with choline also had increased basal ganglia concentrations of choline, suggesting that externally administered choline could alter brain concentrations [ 11 , 12 ]. The most appropriate method to measure brain choline concentrations in vivo utilizes proton magnetic resonance spectroscopy ( 1 H-MRS). Previous studies of bipolar patients utilizing this methodology have had mixed findings. Overall, while some studies have suggested there may be increased choline concentrations in specific situations [ 13 - 18 ], more have found no changes [ 19 - 27 ], and one found a trend towards a decrease in concentrations [ 28 ]. In both patients and volunteers lithium also doesn't appear to alter choline/creatine peak ratios concentrations [ 29 , 30 ]. Nonetheless, two reviews concluded that the evidence to date suggests that lithium increases brain choline concentrations [ 31 , 32 ], although as noted in these reviews previous studies have varied considerably in terms of patient populations, brain region studied, medications administered, and MRS methodology. Many studies have also examined differing patients (Type I and Type II) in differing mood states (mixed, depressed, manic, and euthymic). This may partially explain the varied results. Sodium valproate is also widely used as a mood stabilizer, both alone and in combination with lithium [ 33 ]. To date there have been few studies which have examined the effects of sodium valproate on choline concentrations or activity. An in-vitro study suggested that valproate may inhibit choline acetyltransferase activity [ 34 ]. In one study 9 patients taking either lithium or valproate were examined [ 35 ], and increased Cho/Cr ratios were seen in the bipolar patients compared to controls. There were no differences between the lithium and valproate treatment groups, although the sample sizes were small. However, another study in epilepsy patients treated with valproate found no changes in choline concentrations [ 36 ]. Nonetheless, given the lack of studies to date, the possibility that valproate and lithium may both increase choline concentrations warrants further investigation. Most of the previous studies have examined Cho/Cr ratios. However, it should be noted that the "choline" resonance peak seen in 1 H-MRS spectra is composed primarily of phosphocholine and glycerophosphocholine, along with free choline, acetylcholine, and cytidine diphosphate choline. Also, we have shown in animal studies that both lithium and valproate can both decrease creatine concentrations [ 37 ]. Therefore, when using Cho/Cr ratios it is not possible to be certain that any changes in this peak represent changes in brain choline concentrations. We were therefore interested to determine if there were any differences in results when using different methodologies, and more specifically to determine if studies using a ratio methodology may have different results from studies utilizing metabolite concentrations. Methods In the first part of the study patients taking either lithium or valproate were examined using the Cho/Cr ratio method, and both Bipolar Type I and Bipolar Type II patients were included who could also be taking other medications. In the second part of this study only Bipolar Type I patients on valproate monotherapy were included, and quantification of choline concentrations was made. Some of the data from the first part of this study has been reported previously [ 38 ]. Subjects and Study Design All subjects gave full informed consent, and both studies were approved by the ethics committee at the University of Alberta. Healthy controls were examined using a detailed, but non-standardized, psychiatric interview. They were excluded if there was any personal history, or immediate family history, of psychiatric disorder. For patients, diagnoses were made using DSM-IV criteria for Bipolar Disorder Type I or Type II following detailed psychiatric interview, with additional information being available in almost all cases from long-term psychiatric clinic records. They also had to be taking a dose of either lithium or valproate which maintained their blood levels within the ranges of 0.4–1.2 mmol/l for lithium and 200–700 μmol/l for sodium valproate. Serum lithium and valproate levels were also measured on the day of MRS scanning. Other medications taken by the patient were noted. In the second part of the study the same criteria were used, except that only patients meeting diagnostic criteria for Bipolar Disorder Type I were included, and they had to be on sodium valproate monotherapy. This was done to examine Bipolar Type I patients in more detail, and to remove a possible confounding variable. All patients had to be euthymic for the previous 3 months, as determined by interviews with the patient, and additional interviews with their relatives and bipolar clinic records when available. MRS scans were carried out within 24 hours of this interview. Magnetic Resonance Spectroscopy Methodology For both studies magnetic resonance experiments were performed using a Magnex 3 T scanner with 80 cm bore equipped with actively shielded gradient, and spectrometer control was provided by an Surrey Medical Imaging System (SMIS) console. The subjects head was immobilized with a restraint system. Signal transmission and reception were achieved using a quadrature birdcage resonator for 1 H measurements. Part 1 - Magnetic Resonance Spectroscopy Initially, MRI data were acquired using gradient echo imaging sequences to produce multiple slice images along both coronal and transverse planes. This allowed registration of a 2 × 2 × 3 cm volume-of-interest (VOI) to be selected in the temporal lobe. 1 H MR spectra were acquired using the PRESS localization method [ 39 , 40 ], with TE = 32 ms, TR = 3 s, and with 128 averages. Baseline correction and deconvolution of the spectra was accomplished using the Peak Research (PERCH) spectrum analysis software package. The metabolite peaks of interest [choline (Cho) and creatine (Cr)] in each spectrum were fitted to a Gaussian line-shape for peak area estimation. To determine changes in choline concentrations we examined the Cho/Cr ratio. Figure 1 shows an individual 1 H MRS spectra in which all the major metabolite peaks can be seen. Figure 1 A typical 1 H-MRS spectrum of the human brain at 3.0 T. A number of metabolites can be seen. 1: creatine (methylene) + phosphocreatine, 2: glutamate + glutamine, 3: myo -inositol + glycine, 4: taurine, 5: total choline compounds , 6: creatine (methyl) + phosphocreatine, 7: N-acetylaspartate. Study 2 - Magnetic Resonance Spectroscopy To accurately quantify the brain concentration of creatine we used a 125 ml glass sphere containing a solution of 4 mmol creatine as an external standard. The PRESS sequence was used to acquire proton MRS data with TE1 = 25 msec, TE2 = 25 msec, TR = 3000 msec, and 128 scan averages. The MRS data were acquired from three 2 × 2 × 2 cm 3 voxels placed in the cortex of the left frontal lobe, the cortex of the left temporal lobe, and in the external standard solution. The average coordinates [ 41 , 42 ] of the centers of the two brain voxels were determined: x = 0.5 mm (SD = 1.6), y = 63.5 mm (SD = 12.1), z = -25.5 mm (SD = 4.2) in the frontal lobe, and x= 32.2 mm (SD = 6.3), y = 20.5 mm (SD = 3.9), z = 10.7 mm (SD = 2.6) in the temporal lobe. In order to measure T 1 and T 2 values of the metabolites in the brain and external standard solution, MRS data were collected with different TE values at a constant TR and different TR values at a constant TE both for the healthy volunteers and the patients and also from external standard solution [ 42 ]. However, due to these constraints, the fact that the two studies used different populations at different times, and the size of the external 125 ml container (which limited voxel size to 2 × 2 × 2 cm 3 ), it was not possible to exactly match the voxel size or location between the two studies. MRS Data Analysis For quantitative measurement of brain metabolite concentrations we used previously described methodology [ 42 , 43 ]. In this, [Met] b , in millimoles per kg of wet brain, the CSF volume fraction, f csf , in the spectroscopic voxels must be corrected. Thus, brain metabolite concentrations were calculated as described in the following equation: where V voxel is the volume of a 8 cm 3 spectroscopic voxel [ 43 ], and N b represents the number of metabolite molecules per unit voxel in brain. Statistical Analysis for both MRS studies Means ± SEM were used in the statistical analysis. Sex differences were analyzed using chi-squared, and age differences with ANOVA with post-hoc Tukey tests. The MRS data was analyzed using Student's unpaired t -test using a significance level of p < 0.05 comparing diagnostic groups (patients vs controls) in each brain region (frontal and temporal). Results Study 1 Subjects A total of 18 healthy controls, 14 bipolar patients taking lithium, and 11 bipolar patients taking valproate completed this study. Of the 14 bipolar patients taking lithium, 7 were Type I and 7 were Type II. In the valproate group, 7 were Type I and 4 were Type II. These groups were studied both separately and together, but as there were no statistically significant differences between the Type I and Type II patients, the results for both types are presented together. Of the 14 bipolar patients taking lithium 12 patients were taking other psychotropic medications: these were benzodiazepines (7 patients), antidepressants (5 patients), and antipsychotics (2 patients). Of the 11 patients taking sodium valproate 10 patients were taking other psychotropic medications: these were benzodiazepines (5 patients), antidepressants (5 patients), and antipsychotics (4 patients). The mean age for the lithium group was 40.43 ± 2.96 years, for the valproate group 35.47 ± 2.27 years, and for the control group was 31.35 ± 2.89 years. These differences were statistically significant (F = 3.68, df = 2, p = <0.05), which was attributable to the lithium group being significantly older than the control group (Tukey post hoc , p < 0.05). There were no gender differences within the groups: 10 females and 8 males in the control group (χ 2 = 0.167, df 1, p > 0.05), 5 females and 9 males in the lithium group (χ 2 = 1.143, df 1, p > 0.05), and 6 females and 5 males in the valproate group (χ 2 = 0.474, df 1, p > 0.05). Mean serum lithium levels were 0.79 ± 0.06 mmol/l, and the range was 0.46–1.08 mmol/l. The mean serum valproate levels were 508 ± 42 μmol/l, and the range was 210–912 μmol/l. MRS Data 1 H MRS We utilized the ratio of the choline peak to creatine peak (Cho/Cr) as a primary correlate of Choline concentrations. This result has been reported briefly in a previous publication [ 38 ]. The mean Cho/Cr ratio with this measure was 1.46 ± 0.04 for controls, 1.18 ± 0.07 for lithium-treated patients, and 1.12 ± 0.08 for valproate-treated patients. These were statistically significant, with a reduction in ratios occurring in both the control vs. lithium comparison (t = 3.628, df = 30, p = 0.001) and the control vs. valproate comparison (t = 4.248, df = 27, p = 0.002). Study 2 Subjects A total of 11 healthy controls and 9 Bipolar Type I patients taking valproate as monotherapy were entered into this study. The mean age for the control group was 37.3 ± 2.2 years, and for the valproate patients 42.4 ± 3.0 years. These differences were not statistically significant (F = 1.49, df = 1, p = 0.27). There were no gender differences within the groups: 7 females and 2 males in the valproate group and 5 females and 6 males in the control group (χ 2 = 0.474, df 1, p > 0.05). The mean serum valproate levels were 472 ± 36 μmol/l, and the range was 284–728 μmol/l. In the frontal lobe the mean choline concentration for the healthy controls was 2.21 ± 0.17 mmol/kg wet brain and for the patients was 2.38 ± 0.12 mmol/kg wet brain. In the temporal lobe the mean choline concentration for the healthy controls was 2.35 ± 0.14 mmol/kg wet brain and for the patients was 2.40 ± 0.19 mmol/kg wet brain. There were no statistically significant differences between the controls and patients in either the frontal (t = 0.78, df = 18, p = 0.44) or temporal (t = 0.203 df = 18, p = 0.84) lobes (Table 1 ). Table 1 Concentrations (mmol/kg wet brain) and ratios (Cho/Cre) in frontal and temporal lobes in healthy volunteers and in patients chronically treated with valproate (Study #2) Choline (Cho) Creatine (Cre) Cho/Cre Frontal Temporal Frontal Temporal Frontal Temporal Healthy Controls Age Sex 1 50 M 3.51 2.95 6.67 8.53 0.53 0.35 2 45 M 2.19 3.03 10.1 9.11 0.22 0.33 3 43 F 3.01 2.31 9.97 9.52 0.30 0.24 4 39 M 2.11 2.72 7.94 7.60 0.27 0.24 5 37 F 2.47 2.34 9.98 9.89 0.25 0.24 6 36 F 1.91 1.76 8.28 8.19 0.23 0.22 7 35 M 1.76 2.36 7.93 8.36 0.22 0.28 8 32 F 1.88 1.51 9.56 9.56 0.2 0.16 9 32 M 1.94 2.14 7.04 7.79 0.28 0.28 10 30 F 1.82 2.52 7.8 8.63 0.23 0.29 11 28 M 1.72 2.23 7.16 8.51 0.24 0.26 Mean 37.00 2.21 2.35 8.40 8.70 0.27 0.26 Valproate Treated Patients 1 58 F 2.72 2.1 9.16 10.13 0.30 0.21 2 50 M 2.61 3.42 8.17 10.53 0.32 0.33 3 49 F 2.03 1.79 8.56 7.48 0.24 0.24 4 48 F 2.44 1.88 9.93 8.19 0.25 0.23 5 36 M 2.60 2.53 7.84 7.51 0.33 0.34 6 35 F 2.07 2.77 9.26 10.39 0.22 0.27 7 35 F 2.78 1.89 8.35 9.79 0.33 0.19 8 34 F 1.76 2.93 7.26 8.01 0.24 0.37 9 34 F 2.43 2.27 7.75 7.23 0.31 0.31 Mean 42.11 2.38 2.40 8.48 8.81 0.28 0.28 The Cho/Cr ratios in the frontal lobes were 0.27 ± 0.028 in controls and 0.28 ± 0.015 in patients. In the temporal lobes the Cho/Cr ratios were 0.26 ± 0.021 in controls and 0.28 ± 0.016 in patients. There were no statistically significant differences between the controls and patients in either the frontal (t = 0.367, df = 18, p = 0.72) or temporal (t = 0.539, df = 18, p = 0.59) lobes (Table 1 ). Discussion The results from the present study vary considerably between the two sections utilizing differing methodologies. This is despite the fact that both studies were carried out by the same group on the same scanner with bipolar patients coming from the same patient pool. This strongly suggests that the methodology used to determine choline concentrations can considerably alter the results. In the first part of the study we found that both the lithium-treated and valproate-treated patients had significantly reduced Cho/Cr peak ratios compared to controls. This is similar to the findings from one previous study which also suggested that there may be a trend towards decreased choline in grey matter [ 28 ]. This study was a frontal lobe study that measured metabolite concentrations in a 1.5 T scanner in bipolar type I patients hospitalized for manic (n = 9) or mixed (n = 8) states. In this study most patients were being treated with valproate and an atypical antipsychotic. These findings, however, differ from those in the second part of the present study in which we found no differences in choline concentrations between valproate-treated patients and controls in either frontal or temporal lobes. This second part of the study was much better controlled in terms of the patients receiving valproate monotherapy, only including bipolar Type I patients, and in using an external choline solution to accurately quantify choline concentrations. This finding of a lack of change is also in keeping with most previous studies. Several studies which have also previously measured metabolite concentrations with 1.5 T scanners also found no changes. These include a study of the hippocampus in 15 euthymic bipolar type 1 patients, of whom 10 were taking either lithium or valproate [ 19 ], a study of basal ganglia in 8 rapid cycling patients on lithium [ 22 ], a study of the anterior cingulate in 10 bipolar children [ 23 ], and a study in frontal lobes of 23 euthymic bipolar patients of whom 13 were on lithium [ 25 ]. Several other studies have examined metabolite ratios, mostly in patients on lithium, and those also found no changes in choline concentrations [ 20 , 21 , 26 , 27 ]. In a study using metabolite ratios in bipolar children who were off medication for at least one week there was also no change in choline concentrations [ 24 ]. In a double-blind placebo-controlled human volunteer study before and after one week of lithium administration we also found no changes in cholinein 10 volunteers [ 30 ], which is similar to a patient study which compared 7 patients on lithium to 6 non-lithium treated controls and in which no differences were seen [ 29 ]. In contrast, animal studies have suggested that lithium may increase brain choline concentrations, and in lithium-treated patients it also increases the accumulation of choline within erythrocytes [ 4 - 7 ]. Nonetheless, 1 H-MRS studies in patients examining this possibility is mixed. To date 6 studies have suggested some support for this [ 13 - 18 ], but in none of these studies were metabolite concentrations measured, and most of the studies measured choline/creatine ratios [ 14 - 18 ], the other one measuring metabolite intensity/tissue volume [ 13 ]. The first study to examine brain choline in basal ganglia studied only 4 patients, all of whom were on lithium [ 18 ]. Another study examined 19 euthymic inpatients and found increased choline/creatine ratios in basal ganglia, but only 10 of these patients were receiving lithium [ 17 ]. The third study to report an increase in this ratio (in this case in the left subcortical region) was in a mixed group of patients receiving a wide range of medications [ 16 ]. Two other studies have reported increased choline concentrations, but only in limited circumstances. In one study in 11 bipolar children patients were examined before and after lithium administration [ 14 ]. There were no significant findings before or after lithium administration, although there was a trend towards increased choline/creatine ratios in the patients before lithium treatment. This latter finding does not suggest that in patients lithium significantly alters the choline/creatine ratio. The final study examined 15 euthymic males who were on either lithium or valproate [ 13 ]. This study found that thalamic choline concentrations, determined by measuring metabolite intensity/tissue volume ratios, were significantly increased only if the right and left hemisphere were compared separately, but not if they were compared together. It is also conceivable that both lithium and valproate may increase Choline concentrations, but that the differences were not large enough for us to detect, or that without lithium or valproate treatment patients would have lower Choline concentrations. The cross-sectional nature of this study does not allow this to be examined. It is also important to recognize other limitations of the present study. Firstly, these MRS studies are not pre- and post-treatments, so may not accurately reflect changes that occur in individual patients. Secondly, part of the study used a ratio-method to assess choline concentrations, the limitations of which are increasingly clear (particularly since creatine concentrations may be altered by medication [ 37 ]). Thirdly, the sizes of all groups are small and it therefore possible that a larger study may have been fully powered to identify differences between groups. Fourthly, several patients in the first study (but not the second study) were on other drugs which may have affected the results of this study. Fifthly, we have not determined if age affects the results, and in the first part the groups were not all matched for age. In addition, the voxel locations were not the same in both studies due to the reasons discussed in the methodology section. Nonetheless, despite these limitations we believe the results add significantly to the literature in this under-researched area. We conclude that, taking all current evidence together including the findings from the present study, it is unlikely that either lithium or valproate significantly alter brain choline concentrations. However, given the large differences in patients populations, medications received, and MRS methodologies it is difficult to directly compare all these studies. In addition, the methodology used to measure choline concentrations can significantly alter the results. Future MRS studies in bipolar patients should, therefore, examine metabolite concentrations rather than a ratio of choline compared to other metabolites. Competing interests None declared.

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522869

Global nucleosome occupancy in yeast

A genome-wide study of nucleosome occupancy at yeast promoters shows that promoters that regulate active genes, contain multiple conserved motifs, or contain Rap1 binding sites tend to be depleted of nucleosomes.

Background Global gene-expression patterns are established and maintained by the concerted actions of transcription factors and the proteins that constitute chromatin. The global network of interactions between transcription factors and promoters in yeast is increasingly being characterized [ 1 ]. The role of chromatin in gene regulation is less clear, however. For example, the distribution of nucleosomes, the fundamental units of chromatin, is poorly understood on a gene-specific basis, much less a global basis [ 2 ]. The nucleosome consists of approximately 146 base-pairs (bp) of DNA wrapped around an octamer of histone proteins - two each of histones H2A, H2B, H3 and H4. Eukaryotic genomes are packaged into repeating units of nucleosomes separated by around 10-80 bp of linker DNA. High occupancy by nucleosomes is thought to be generally repressive [ 3 ], and extensive remodeling (and loss) of nucleosomes occurs in the promoters of genes undergoing activation [ 4 ]. In the case of the PHO5 promoter in yeast, this remodeling proceeds until essentially no nucleosomes are detected across a region of several hundred base-pairs [ 5 , 6 ]. Transcription factors and chromatin proteins each form complex regulatory networks that interact in a variety of ways [ 1 , 7 ]. Transcription factors modify chromatin structure by recruiting enzymes that remodel nucleosomes or posttranslationally modify histones (by acetylation or methylation, for example) [ 8 - 10 ]. The modifications can be maintained through cell division and propagated to proximal nucleosomes by positive-feedback mechanisms [ 7 , 11 , 12 ]. Hence, a signal such as the activation of a transcription factor can be temporally and spatially transmitted through chromatin. Conversely, chromatin can influence transcription factor function by modulating the accessibility of target binding sites in the DNA [ 13 , 14 ]. We used chromatin immunoprecipitation (ChIP) and DNA microarrays to evaluate nucleosome occupancy levels for essentially all promoters in yeast. Promoters that regulate active genes, contain multiple conserved motifs or recruit Rap1 tend to be relatively nucleosome-depleted. We also used real-time PCR and micrococcal nuclease digestion to show that nucleosomes are depleted in the vicinity of Rap1 consensus sites. This depletion can be partially reversed by the actions of the small molecule rapamycin or by removing Rap1-binding sites. We suggest that other transcription factors have less robust nucleosome-depleting activities than Rap1 and must therefore act collaboratively to gain access to their cognate sites in the DNA. Results ChIP-based assay for nucleosome occupancy Histones are essential components of the nucleosome and efficiently cross-link to nucleosomal DNA. Antibodies against invariant portions of histones have been used previously in ChIP assays to follow nucleosome loss at the yeast PHO5 promoter [ 5 , 6 ]. We extended this approach to evaluate relative nucleosome occupancy at essentially all promoters and other intergenic regions in yeast. DNA associated in vivo with histone H3 was isolated by ChIP using antibody against the carboxy terminus of histone H3 (no posttranslational modifications are thought to occur in this region). ChIP DNA and unenriched control DNA were amplified by in vitro transcription and evaluated using microarrays. DNA associated with histone H2B was evaluated in a similar fashion using anti-FLAG antibody and a FLAG-H2B strain. H3 and H2B datasets were compiled by averaging four and three independent biological experiments, respectively. These datasets are remarkably similar as shown by a genome-wide correlation of 0.83 (Figure 1a-c ). This correlation is comparable to that observed when comparing replicate H3 datasets (or H2B datasets), and suggests that both assays measure similar phenomena. In the H3 and H2B datasets, respectively, there are 347 and 214 regions depleted at least 1.5-fold relative to the average over all intergenics. In contrast, there are just 84 and 6 regions in the respective datasets enriched at least 1.5-fold relative to this average. The relatively narrow range of ChIP enrichment and the negative skew of the data (Figure 1b ) are consistent with the conventional view that the majority of the genome is packaged into nucleosomes with intervening stretches of free DNA such as the activated PHO5 promoter [ 5 , 6 ]. Despite these consistencies, a possible caveat to using ChIP to evaluate nucleosome occupancy is that immunoprecipitation efficiency can depend on epitope accessibility. Rather than having low occupancies, genomic regions depleted in the H3 ChIP might be inaccessible as a result of association with large protein complexes in chromatin. To investigate this possibility, we examined a published chromatin fractionation dataset in which cross-linked chromatin fragments were subjected to phenol-chloroform extraction and DNA that partitioned into the aqueous phase was quantified by microarrays [ 15 ]. Given the polar nature of DNA and the hydrophobic nature of denatured protein, aqueous extraction should generally enrich for free DNA. We found that regions depleted in the H3 ChIP assay overlap extensively with regions enriched by aqueous extraction, but not with regions depleted by aqueous extraction (Figure 1d ). Overall, there is a negative correlation of -0.54 between the H3 ChIP and aqueous-extraction datasets. Although the fractionation data may partially reflect differential cross-linking of lysines in the histone tails [ 15 ], this analysis suggests that regions depleted in the H3 ChIP experiment are relatively protein-free, as would be expected of non-nucleosomal DNA. Nucleosome occupancy correlates inversely with promoter strength As previous studies show that PHO5 activation is accompanied by marked nucleosome loss in the promoter region [ 5 , 6 ], we sought to determine whether nucleosome depletion is a general attribute of active promoters. A total of 4,365 intergenic regions that reside immediately upstream of one or more validated yeast genes were assigned as promoters. Relative transcription rates were determined for each yeast gene from transcript levels measured by array and previously collected mRNA half-life data [ 16 ]. We found an inverse correlation of -0.39 between the enrichment of promoters in the H3 and H2B ChIP assays and the transcription rates of downstream genes (Figure 2a ). Under the conditions examined, PHO5 is not induced and its promoter has an average nucleosome occupancy according to these datasets. To evaluate further the relationship between nucleosome depletion and transcription, we collated a set of 308 nucleosome-depleted promoters on the basis of their relative depletion across the replicate H3 and H2B experiments. Of these nucleosome-depleted promoters, 42% regulate highly active genes (Figure 2b ). These data suggest that there is a systematic relationship between promoter strength and nucleosome depletion. However, as this correspondence is not complete there are likely to be other determinants of nucleosome occupancy. Transcription factor binding motifs are over-represented in nucleosome-depleted promoters To identify additional determinants of occupancy, we sought sequence elements associated with nucleosome depletion. Specifically, we carried out an unbiased search for elements up to 10 bp in length that occur with higher frequency in nucleosome-depleted promoters. Two distinct categories of sequences emerged (Figure 3a ). The first includes poly(dA.dT) elements. Stretches of 10 or more dA.dT nucleotides appear in 38% of depleted promoters, compared with 26% of promoters overall (hypergeometric p < 10 -5 ). dA.dT stretches destabilize nucleosome formation in vitro and in vivo [ 17 , 18 ]. The enrichment of poly(dA.dT) elements in nucleosome-depleted promoters probably reflects, at least in part, this destabilizing influence. As a high proportion of the poly(dA.dT) elements identified in nucleosome-depleted promoters are more than 10 bp long (30% are at least 14 bp), these data do not address the minimum length required for destabilization. However, in vitro studies show that a 16-bp insertion leads to a 1.7-fold increase in accessibility of nucleosomal target sites [ 18 ]. The second sequence element enriched in nucleosome-depleted promoters corresponds to the consensus motif for the Rap1 transcription factor. This motif commonly occurs in the promoters of ribosomal proteins genes and is required for Rap1 binding in vitro and in vivo [ 19 , 20 ]. Some variant of this motif appears in 22% of nucleosome-depleted promoters, compared with just 8% of promoters overall (hypergeometric p < 10 -5 ). Furthermore, multiple Rap1 sites are found in 19% of nucleosome-depleted promoters with Rap1 sites, compared to 8% of promoters with Rap1 sites overall (hypergeometric p < 10 -3 ). These data suggest that Rap1 recruitment may lead to nucleosome loss. Because only the Rap1 consensus site was identified in an unbiased search, we sought to identify additional sequence motifs by incorporating species conservation data. Specifically, we evaluated a set of 71 conserved motifs identified by Kellis and colleagues, a majority of which function in transcription factor recruitment [ 21 ]. Nearly half of these 71 motifs are over-represented in nucleosome-depleted promoters relative to promoters overall, as defined by a hypergeometric p < 0.001. However, many of the implicated motifs appear in the same promoters. For example, nine of the over-represented motifs are associated with filamentation gene promoters [ 21 ]. We therefore considered the possibility that the total number of conserved motifs might be a more relevant predictor of nucleosome depletion. Indeed, we found that 31% of nucleosome-depleted promoters contain at least eight motifs, compared with 11% of promoters overall (hypergeometric p <10 -5 ; Figure 3b ). Furthermore, nucleosome-depleted promoters contain an average of 6.1 motifs, whereas the average promoter contains 3.1 (permutation p < 0.001; Figure 3c ). Next, we sought motifs associated with nucleosome depletion in the absence of multiple motifs, by confining our analysis to promoters containing a maximum of four motifs. This analysis identified just two over-represented motifs, which correspond to the Rap1 and Swi4 binding sites. Hence, although a large number of conserved motifs are enriched in nucleosome-depleted promoters, most appear to be relevant mainly when occurring in combination. Functionally cooperative transcription factors associate with nucleosome-depleted promoters As a majority of the conserved motifs recruit transcription factors [ 21 ], we examined the relationship between transcription factor binding and nucleosome occupancy more directly. Lee and colleagues combined ChIP and microarrays to identify target promoters for essentially all yeast transcription factors under the same conditions used here to evaluate nucleosome occupancy [ 1 ]. For each factor, we determined the significance of overlap between its target promoters and the set of nucleosome-depleted promoters. Of the 113 transcription factors in their database, 31 tend to associate with nucleosome-depleted promoters as defined by a hypergeometric p < 0.001. Rap1 has the most significant association (Figure 4a ), consistent with the enrichment of its binding motif (see above). Other top-ranked factors include Fhl1, which associates with many Rap1-bound promoters, and Swi4, whose binding motif is also enriched (Table 1 ). We sought an underlying binding mechanism or function common to the transcription factors we had identified. However, these factors utilize a variety of binding domains, regulate different pathways, and only a minority have significant associations with promoters of highly active genes. Nonetheless, a commonality does emerge when transcription factor cooperativity is considered. A recent informatics study by Banerjee and Zhang identified 31 functionally cooperative transcription factor pairs (representing a total of 33 factors) on the basis of comprehensive binding and expression data [ 22 ]. Only a fraction of these are known to interact physically, suggesting that other mechanisms also confer cooperative function. There is a remarkable correspondence between these functionally cooperative factors and those that preferentially associate with nucleosome-depleted promoters (see Table 1 ). Of the 31 factors we found to associate with nucleosome-depleted promoters, 17 were found to be functionally cooperative by Banerjee and Zhang ( p < 10 -5 ). Furthermore, an evaluation of nucleosome occupancy at promoters bound by both members of a cooperative pair revealed a significant association with nucleosome-depletion for 18 of the 31 pairs (hypergeometric p < 0.01). Together, these findings suggest that binding motifs and transcription factors act in combination to deplete nucleosomes and suggest a role for nucleosomes in transcription factor cooperativity [ 23 - 25 ]. Conditional nucleosome depletion at Rap1 consensus motifs Although a number of transcription factors appear to act in defining promoter nucleosome occupancy, only the Rap1 consensus motif was identified in an unbiased search of nucleosome-depleted promoters. Furthermore, there is a highly significant association between nucleosome-depleted promoters and promoters bound by this factor in vivo [ 1 ] (Figure 4a ). To investigate the relationship between Rap1 recruitment and nucleosome depletion further, we used ChIP and real-time PCR to evaluate nucleosome occupancy at several Rap1 binding sites in ribosomal protein promoters. We found that these regions are depleted 3- to 10-fold in H3 and FLAG-H2B ChIP assays, relative to a control promoter ( TUB2 ) with average occupancy by global analysis (Figure 4b ). We also used an orthogonal approach in which micrococcal nuclease digestion [ 26 ] was used to probe for nucleosomes at the TUB2 , RPS11B and RPS15 promoters (Figure 4c ). A pattern of nuclease protection indicative of a regular nucleosome array is evident at the TUB2 promoter, consistent with the average nucleosome occupancy attributed to this promoter by global ChIP analysis. In contrast, nuclease protection is not evident at the RAP1 sites in the RPS15 promoter, consistent with the marked nucleosome-depletion attributed to this region by global ChIP and real-time PCR analysis. The region surrounding the RAP1 sites in RPS11B exhibits weak nuclease protection, consistent with the modest nucleosome-depletion attributed to this region by global ChIP and real-time PCR. Although these focused analyses specifically addressed Rap1 sites in ribosomal protein genes, our global analyses indicate that approximately 30% of nucleosome-depleted promoters containing Rap1 motifs do not regulate ribosomal protein genes. Together these data confirm that nucleosomes are markedly depleted in the vicinity of Rap1 consensus sites in vivo , and thus extend previous studies showing that Rap1 induces local alterations in chromatin structure that, for example, result in increased nuclease sensitivity [ 27 - 29 ]. To gain further insight into the relationship between Rap1 and nucleosome depletion, we examined a mutant RPS11B promoter lacking its Rap1 consensus sites. We found that removal of these sites, which completely abrogates Rap1 binding [ 30 ], causes nucleosomes to return to the region, as reflected by a greater than twofold change in H3 ChIP enrichment (Figure 4d ). We also examined the effect of rapamycin treatment on nucleosome occupancy in the vicinity of these consensus sites. Although ribosomal protein gene expression is dramatically reduced by rapamycin [ 31 , 32 ], Rap1 remains bound to its target promoters ([ 30 , 33 ], and B.B., E.P. and S.S., unpublished results). We found that rapamycin treatment causes nucleosomes to return to the vicinity of Rap1 sites, as reflected by twofold and greater increases in H3 ChIP enrichment (Figure 4e ). Together these data show that Rap1 consensus sites are required for conditional nucleosome depletion at ribosomal protein gene promoters. Discussion To gain further insight into the role of nucleosomes in gene regulation, we systematically evaluated promoter nucleosome occupancy in yeast by immunoprecipitating nucleosomal DNA and quantifying enrichment with microarrays. Promoters that are inefficiently immunoprecipitated by general anti-histone antibodies, and are therefore presumed to be relatively nucleosome-depleted, tend to regulate active genes (Figure 2 ). This is consistent with the previous observation that the activated PHO5 promoter is largely devoid of nucleosomes [ 5 , 6 ]. However, as not all nucleosome-depleted promoters regulate active genes, there are most likely to be additional determinants of depletion. An unbiased search for sequence elements enriched in nucleosome-depleted promoters revealed poly(dA.dT) elements, previously shown to destabilize nucleosome formation [ 17 , 18 ], and the Rap1 consensus motif. By incorporating sequence conservation data [ 21 ], more than 30 other enriched motifs could be identified. However, most of these appear to be relevant mainly when occurring in combination. When we limited this analysis to promoters containing four or fewer motifs, all but two of these additional motifs drop out (only the Rap1 and Swi4 consensus sites remain). As the majority of conserved motifs incorporated in this analysis recruit transcription factors [ 21 ], these data suggest that multiple transcription factors act in combination to deplete nucleosomes. This possibility is further supported by our finding that functionally cooperative transcription factors tend to bind nucleosome-depleted promoters. These associations may reflect a mechanistic model in which transcription factors compete collaboratively to displace nucleosomes in order to gain access to target sites in the DNA [ 23 ]. This model was formulated to explain why certain pairs of transcription factors bind cooperatively to proximal target sites in vivo and on a chromatin template, but not to naked DNA [ 23 - 25 ]. This view invokes a broad role for nucleosomes as ubiquitous negative regulators of transcription factor binding and function. We speculate that by promoting synergy among multiple transcription factors and impeding the activities of individual ones, nucleosomes facilitate threshold behavior and filter noise (for example, genetic variation in motif sequence) in the transcriptional regulatory network. Although many factors appear to act in defining promoter nucleosome occupancy, our data indicate that Rap1 has a uniquely important role. Rap1 and its consensus motif are both markedly enriched in nucleosome-depleted promoters. Follow-up studies using real-time PCR and micrococcal nuclease digestion also demonstrate marked nucleosome depletion in the vicinity of Rap1 sites in the promoters of ribosomal protein genes. Moreover, nucleosomes appeared to return when the Rap1 consensus sites in one of these promoters were removed. These findings are consistent with previously described roles for Rap1 in opening chromatin and altering nucleosome positioning [ 27 , 28 ]. However, Rap1 recruitment is not equally associated with nucleosome depletion under all conditions. We find that nucleosomes partially return to the vicinity of Rap1 sites during a rapamycin-induced starvation response [ 34 ], even though Rap1 remains bound ([ 30 , 33 ], and B.B, E.P. and S.S., unpublished results). Hence, the nucleosome loss associated with Rap1 recruitment is most likely to require additional proteins, such as Esa1, a histone acetyltransferase recruited by Rap1 under exponential growth conditions but released in stress [ 30 ]. These findings may also offer insight into the barrier activity previously documented for Rap1 [ 35 ]. Heterochromatin propagation involves the sequential modification of histones in adjacent nucleosomes through positive-feedback mechanisms [ 7 , 11 ]. Certain factors such as Rap1 are able to block this propagation by largely unknown mechanisms [ 36 ]. One model speculates that these barriers create nucleosome-free 'holes' lacking the histone substrate required for heterochromatin propagation [ 29 , 35 ]. By identifying such a 'hole' in the vicinity of Rap1-binding sites in vivo our data support this model. Remarkably, the nucleosomal hole and the barrier function ascribed to Rap1 may be conditional, as nucleosomes return following treatment with the small molecule rapamycin, which activates a starvation response. Heterochromatic silencing has been shown previously to moderate under these conditions [ 37 ]. Hence, we speculate that dynamic influences on nucleosome occupancy may enable Rap1 to define chromatin domains and vary them in response to environmental cues. More broadly, the widespread nucleosome loss observed in the promoters of active genes provides a general caveat for ChIP studies examining posttranslational histone modifications, as a decrease in signal for a histone modification at a promoter undergoing activation may actually reflect nucleosome loss. Similarly, regions that appear relatively hypo-modified by ChIP may actually be nucleosome-depleted. However, this is not the case for low levels of acetylation [ 38 ] and H3 lysine 4 methylation [ 39 ] observed at yeast telomeres, as these regions have high occupancy. The data also provide insight into the maintenance of epigenetic information by histone modifications. Whereas epigenetic memory of a repressed state can be maintained on histones in promoters, memory of an activated state must be maintained on histones outside the promoters, for example in transcribed regions, which may not undergo significant nucleosome loss during activation [ 5 , 6 ]. Methylation of histone H3 at lysines 4 and 36, targeted to transcribed regions in yeast via interactions between RNA polymerase and the methylases [ 39 - 47 ], may represent such 'activating' marks. Materials and methods Chromatin immunoprecipitation (ChIP) DNA associated with histone H3 in vivo was immunoprecipitated with antibodies against the invariant H3 carboxy terminus using a ChIP protocol described previously [ 39 , 48 , 49 ]. Briefly, 45 ml log-phase w303a yeast (OD 600 ~ 1.0) growing in yeast extract/peptone/dextrose (YPD) were cross-linked in 1% formaldehyde for 15 min, washed twice in PBS, resuspended in 400 μl lysis buffer (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate) and lysed with glass beads. The resulting extract was sonicated to fragment chromatin (4 × 20 sec burst/30 sec rest with a Branson Sonifier 250 at 70% duty, power 3) and centrifuged for 15 min. Solubilized chromatin was then immunoprecipitated with polyclonal antibodies against the carboxy terminus of histone H3 (Abcam or Cell Signaling). A unenriched whole-cell extract sample (WCE) was also retained as a control. After enrichment, cross-links were reversed by incubating samples in 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1.0% SDS, 150 mM NaCl at 65°C overnight. DNA was purified from ChIP and WCE samples by proteinase K treatment, phenol/chloroform extraction, ethanol precipitation, and incubation with RNAse. DNA associated with histone H2B in vivo was isolated in a similar manner from yeast containing epitope-tagged H2B [ 50 ] using anti-FLAG M2 monoclonal antibodies (Sigma). DNA amplification and hybridization To obtain sufficient quantities for hybridization, immunoprecipitated DNA (from approximately 10 8 cells) and whole-cell extract DNA (unenriched control) were amplified in a linear fashion as described [ 51 ]. Briefly, terminal transferase was used to add poly(T) tails to DNA fragment and a T7-poly(A) adaptor primer was used to incorporate T7 promoters. The reaction products were used as template for an in vitro transcription reaction carried out with the T7 Megascript Kit (Ambion) and RNA samples were purified using an RNeasy Mini Kit (Qiagen). Amplified RNA was reverse-transcribed, incorporating amino-allyl dUTP, and the resulting DNA was fluorescently labeled by incubation with monofunctional reactive Cy5 (enriched sample) or Cy3 (unenriched control) dye as described [ 52 ]. Microarrays containing 6,438 PCR-amplified intergenic regions were prepared as described previously [ 39 , 53 , 54 ]. Mixed Cy5-/Cy3-labeled probe was hybridized to intergenic microarrays for 12-14 h at 60°C, washed and then scanned using a GenePix 4000A scanner with GenePix Pro software (Axon Instruments) as described [ 55 ]. In addition, transcript levels were determined by hybridizing Cy5-labeled mRNA extracted from log phase w303a yeast against Cy3-labeled genomic DNA on microarrays containing 6,218 open reading frames (ORFs), as described previously [ 16 ]. Microarray data processing Cy5 and Cy3 fluorescence were integrated for each feature using GenePix Pro Software (Axon). Data were processed and composite Cy5:Cy3 ratios determined according to protocols at the Stanford Microarray Database [ 56 ]. Correlations between replicate datasets were ~0.8 for all experiments. Composite datasets were log 2 transformed and zero centered before further analysis. The histone H3 ChIP dataset was determined from four independent immunoprecipitations and hybridizations (two each using antibodies from Cell Signaling or Abcam). The FLAG-H2B ChIP dataset was determined from three independent immunoprecipitations and hybridizations. The mRNA dataset was determined from three independent extractions and hybridizations of mRNA against genomic DNA. Relative transcription rates were determined by dividing transcript levels by half-life data collected by Wang and colleagues [ 16 ]. A set of activated promoters was defined as those in the top 10% by mRNA expression level of associated gene, with divergent promoters assigned to the more highly expressed gene. Complete datasets are available online [ 57 ]. Analysis of nucleosome-depleted promoters Z-scores were assigned to each intergenic that reflect depletion across the four H3 and three H2B ChIP experiments, using the formula Z = (x - μ)/σ where x is the average of the replicate measurements, μ is the average of all intergenics and σ is the standard error of the replicate measurements. We defined as nucleosome-depleted the 410 features with the highest Z-scores. This set, which includes 308 promoters, contains nucleosome-depleted outliers and is not inclusive of all promoters that immunoprecipitate with average or lower efficiency. The average aqueous enrichment ratio [ 15 ] for these 308 depleted promoters is 1.7-fold, significantly higher than expected by chance (permutation p < 0.001), consistent with the premise that these promoters are relatively free of nucleosomes. Sequence elements common to nucleosome-depleted promoters were identified by searching between 10 and 500 bp upstream of gene start sites for over-represented sequences up to 10 bp in length using the GeneSpring program suite (Silicon Genetics). Enrichment was confirmed by evaluating the significance of overlap between the set of nucleosome-depleted promoters and the set of promoters containing Rap1 consensus motifs (ACACCCATACAT with up to two mismatches) or poly dA.dT stretches at least 10 bp in length (identified using PatMatch, Saccharomyces Genome Database [ 58 ]). Statistical significances of overlaps between sets are expressed as P -values calculated by a hypergeometric probability model. The P -values reflect the extent to which observed overlaps exceed that expected under the null hypothesis that there is no relationship between the sets [ 59 ]. Where specified, permutation analyses were carried out by generating 1,000 random but representative promoter sets with an Excel macro and used to confirm statistical significance. Lists of promoters containing the 71 conserved motifs [ 21 ] were collated from gene sets available online [ 60 ]. Lists of promoters bound by transcription factors at a significance of p < 0.001 [ 1 ] were collated from data available at [ 61 ]. Real-time PCR Regions approximately 200 bp in size that span one or more Rap1 consensus sites in ribosomal protein gene promoters were amplified from ChIP and unenriched control samples using SYBR green PCR mix (Qiagen) in an MJ Research real-time PCR machine according to the manufacturers' instructions. Fold-ratios that reflect relative enrichment or depletion of a given region in the H3 or FLAG-H2B ChIP assays were determined using the 2 -ΔΔC T method described in the Applied Biosystems User Bulletin. For each region examined, the TUB2 promoter was used as the normalizer (this promoter is used as a control because its occupancy approximates that of the average promoter by global analysis), and the unenriched control sample was used as the calibrator. Each reported ratio represents the average of three independent ChIP experiments analyzed in duplicate by real-time PCR. The following primer pairs were used: RPS22A promoter: 5'-GCCTAAAACGCCCATAAGTT-3' and 5'-ACTGCAAACCCATATTCAAGA-3' RPS15 promoter: 5'-TACACCGCGCGTATAAATCA-3' and 5'-CCCAGCAAGGAGTTTCTCAG-3' RPS11B promoter: 5'-GAAGAAATATTTCCTTGCTGCACC-3' and 5'-AAGGGAAACGTAAAGCTATTGGAC-3' RPL23A promoter: 5'-ATTAACATCTGTACACCCCCAACT-3' and 5'-TACAGTTCGTTTCCTGCC ATATTA-3' TUB2 promoter: 5'-GGCCTAACAGTAAAGATATCCTCC-3' and 5'-GTTGTAGTAGCTGCTATGT CACTC-3' Centromeric vectors containing either a mutant RPS11B promoter lacking the two Rap1 consensus motifs [ 30 ] or an essentially wild-type allele were transformed into wild-type yeast and used in an H3 ChIP assay to evaluate the consequence of removing Rap1 binding sites on nucleosome occupancy. Enrichment was evaluated by real-time PCR using the following primer pair that selectively amplifies the plasmid alleles but not the endogenous RPS11B promoter: 5'-CTGGAAGAAATATTTCCTT GCTCTAG-3' and 5'-AAGGGAAACGTAAAGCTATTGGAC-3'. Micrococcal nuclease assay Log-phase cultures of W303a yeast grown in 450 ml YPD to OD 600 of 1.0 were spheroplasted with zymolase (10 mg in 40 ml volume of 1 M sorbitol, 50 mM Tris pH 7.4, 10 mM β-mercaptoethanol (β-ME), at 30°C for 38 min shaking at 300 rpm), divided into five aliquots, and digested with increasing concentrations (20 U to 320 U) of micrococcal nuclease (Worthington Biochem) in 600 μl 0.5 mM spermidine, 1 mM β-ME, 0.075% NP-40. DNA from digested samples was extracted with phenol twice and chloroform once and precipitated in ethanol. Samples were washed, resuspended in 10 mM Tris pH 7.5, subjected to RNAse treatment, cleaned up with the MinElute kit (Qiagen) and run out in a 1% agarose gel. Following depurination, denaturation and neutralization of the gel, DNA was transferred onto nylon membranes by capillary action and covalently linked to the membranes by UV irradiation. Southern blotting was carried out using a DIG Luminescent Detection Kit (Roche) and DIG-labeled probe generated by PCR using the TUB2 , RPS11B and RPS15 primers described above.

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC522869.xml

549213

Inter-species horizontal transfer resulting in core-genome and niche-adaptive variation within Helicobacter pylori

Background Horizontal gene transfer is central to evolution in most bacterial species. The detection of exchanged regions is often based upon analysis of compositional characteristics and their comparison to the organism as a whole. In this study we describe a new methodology combining aspects of established signature analysis with textual analysis approaches. This approach has been used to analyze the two available genome sequences of H. pylori . Results This gene-by-gene analysis reveals a wide range of genes related to both virulence behaviour and the strain differences that have been relatively recently acquired from other sequence backgrounds. These frequently involve single genes or small numbers of genes that are not associated with transposases or bacteriophage genes, nor with inverted repeats typically used as markers for horizontal transfer. In addition, clear examples of horizontal exchange in genes associated with 'core' metabolic functions were identified, supported by differences between the sequenced strains, including: ftsK , xerD and polA . In some cases it was possible to determine which strain represented the 'parent' and 'altered' states for insertion-deletion events. Different signature component lengths showed different sensitivities for the detection of some horizontally transferred genes, which may reflect different amelioration rates of sequence components. Conclusion New implementations of signature analysis that can be applied on a gene-by-gene basis for the identification of horizontally acquired sequences are described. These findings highlight the central role of the availability of homologous substrates in evolution mediated by horizontal exchange, and suggest that some components of the supposedly stable 'core genome' may actually be favoured targets for integration of foreign sequences because of their degree of conservation.

Background Helicobacter pylori is a bacterial pathogen associated with gastritis, peptic ulcers, gastric adenocarcinoma, and rare lymphomas [ 1 ]. It has a highly panmictic population structure in which homologous recombination makes the predominant contribution to sequence differences within a highly diverse population structure [ 2 ]. The acquisition of genes from other strains and species is by far the most rapid evolutionary process. This occurs frequently without loss of existing functions, is central to the evolution of niche-adaptive and pathogenic characteristics of bacteria, and greatly influences inter-strain differences in gene complement [ 3 - 5 ]. In this context, it is notable that none of the traits typically used to differentiate E. coli from Salmonella can be attributed to point mutation genes but are broadly attributable to horizontal exchange [ 6 ]. H. pylori is relatively unusual in that it is a naturally transformable Gram-negative species that does not appear to have a species-specific DNA uptake sequence and appears to rely upon its niche separation as a transformation barrier [ 7 ]. Disease associated H. pylori strains have been divided into two types, type I being those that carry the cag pathogenicity island [ 8 ] ( cag PAI), which has a foreign species origin, and are associated with more severe disease. Dinucleotide composition is highly stable within a genome and can distinguish between sequences from different species. Based upon its constancy the species composition is referred to as a 'genome signature' [ 9 , 10 ]. This characteristic has been applied to assessments of DNA metabolic processes such as methylation and base conversion, DNA structure, and evolutionary relationships. It has also become established as a method for the identification of sequences that have been acquired by inter-species horizontal transfer. For example, lateral transfer has recently been shown using these methods for a tryptophan pathway operon [ 11 ], the gain of additional metabolic functions in Pseudomonas putida [ 12 ], a determination that many gain of function genes have been acquired by E. coli rather than lost from S. typhi [ 13 ], and more recently developed Bayesian methods based upon similar premises have been used to assess global signatures and determine the origins of some lateral transfer events [ 14 , 15 ]. However there are problems associated with this and other methods that use progressive 'walking windows', and the larger the window the greater the problems. These result from the inclusion of intergenic sequence, the inability to distinguish divergences due to a single highly divergent gene from that from a cluster of less divergent ones, and an inability to identify the limits of the abnormal regions. In practice additional features are necessary to determine the ends of such regions, such as the location of repeats typical of pathogenicity islands in H. pylori [ 16 ], or comparisons with other sequences as in N. meningitidis strain MC58 [ 17 ]. In addition, divergence scores are influenced by the size of the sampling window used such that sampling effects limit analysis of sequences shorter than about 800 bp (data not presented), and the need to use fixed window sizes prevents gene by gene studies. We describe the use of a linear implementation of signature analysis that can efficiently address a range of walking window sizes using dinucleotide signatures (DNS) and longer signatures. In addition, use of a new approach based upon classical text analysis that allows analysis of genomes gene-by-gene is described. Analysis of H. pylori sequences, combined with comparisons of the identified genes between genomes, reveals complex changes that influence both niche-adaptive and core functions illustrating a previously unpredicted range of functions which are continuously undergoing variation and selection. Results and discussion Genes were ranked on the basis of their divergence from the mean genome composition. The degree of divergence that is indicative of acquisition from other species is not an absolute. The frequency with which genes are acquired, the untypicality of the donated material, and the rate at which they are ameliorated to the host sequence composition influence it. Strains J99 and 26695 had 53 (Table 1 ) and 60 (Table 4 ) genes respectively with DNS that were >2 SD from the mean. Those with annotated functions included genes from the cag pathogenicity island (6 and 5), vac and related toxins (3 and 4), and restriction-modification genes (2 and 4). On the basis of the similarities determined in the H . pylori strain J99 sequence annotation, 7 of the most divergent genes as determined by DNS are not present in strain 26695. Likewise, 2 of the 50 most divergent genes in strain 26695 are not present in strain J99. This is consistent with the identification of genes acquired from other species that have not extended to both sequenced strains. It also suggests that a significant proportion of the 6 to 7% of genes unique to one or other strain [ 18 ] are inherent to the Helicobacter gene pool, but are variably present in different strains rather than reflecting recent foreign origins. Comparisons of a selection of identified orthologous genes in the two strains are shown in Figure 1 . Table 1 The 53 most divergent (>2 SD) genes in H. pylori strain J99 by DNS showing their ranking in strain 26695 and in TNS and HNS analysis DNS order JHP # annotation 26695 # 26695 DNS order TNS order HNS order 1 JHP0952 hypothetical protein HP0427 14 3 1355 2 JHP0476 cag pathogenicity island protein (cag7) HP0527 1 2 2 3 JHP0556 vacuolating cytotoxin (vacA) paralog HP0609/10 4/13 5 4 4 JHP0274 vacuolating cytotoxin (vacA) paralog HP0289 2 6 5 5 JHP0305 hypothetical protein HP0322 3 8 10 6 JHP0942 hypothetical protein HP0996 5 13 27 7 JHP0856 vacuolating cytotoxin (vacA) paralog HP0922 6 9 6 8 JHP0050 hypothetical protein HP0058 88 7 84 9 JHP1300 hypothetical protein HP1408 15 1 1 10 JHP1044 hypothetical protein HP1116 8 14 8 11 JHP0928 hypothetical protein NAH - 12 9 12 JHP0074 hypothetical protein HP0080 9 32 125 13 JHP0440 hypothetical protein HP0488 7 16 17 14 JHP1042 hypothetical protein HP1115 20 25 694 15 JHP1321 histidine and glutamine-rich metal-binding protein HP1432 46 4 49 16 JHP0934 hypothetical protein NAH - 15 95 17 JHP0495 cag island protein (cagA) HP0547 31 20 12 18 JHP0931 topoisomerase I (topA 3) NAH - 18 20 19 JHP0693 hypothetical protein HP0756 24 59 1490 20 JHP0632 N-methylhydantoinase HP0696 19 44 36 21 JHP0471 cag pathogenicity island protein (cag3) HP0522 11 35 62 22 JHP0438 outer membrane protein HP0486 26 67 145 23 JHP0026 hypothetical protein HP0030 45 36 64 24 JHP1084 outer membrane protein (omp26) HP1157 34 17 24 25 JHP0481 cag island protein (cagT) HP0532 23 70 558 26 JHP0052 hypothetical protein HP0059 43 24 120 27 JHP0336 hypothetical protein HP1089 12 51 54 28 JHP1426 iron(III) dicitrate transport protein (fecA) HP1400 32 78 111 29 JHP0174 hypothetical protein HP0187 / 8 / 6 47&1127&596 88 90 30 JHP1297 type III restriction enzyme (res) NAH - 63 28 31 JHP0953 hypothetical protein NAH - 26 1463 32 JHP0067 urease beta subunit (urea amidohydrolase) (ureB) HP0072 21 37 70 33 JHP0941 integrase/recombinase (xerD) HP0995 25 100 541 34 JHP0548 flagellin A (flaA) HP0601 33 40 154 35 JHP0299 hypothetical protein HP061/2 230&765 11 275 36 JHP1033 hypothetical protein HP1106 59 262 342 37 JHP1409 type II restriction enzyme (methyltransferase) NAH - 55 15 38 JHP0626 iron(III) dicitrate transport protein (fecA) HP0686 62 89 47 39 JHP0940 hypothetical protein NAH - 53 393 40 JHP1253 hypothetical protein HP1333 40 75 384 41 JHP0132 cytochrome oxidase (cbb3 type) (fixN) HP0144 27 206 209 42 JHP0842 hypothetical protein HP0906 42 29 21 43 JHP0925 hypothetical protein NAH - 130 990 44 JHP0613 hypothetical protein HP0669 69 42 33 45 JHP0565 DNA mismatch repair protein (mutS) HP0621 22 227 82 46 JHP1363 DNA polymerase I (polA) HP1470 30 81 46 47 JHP0489 cag island protein (cagH) HP0541 71 137 398 48 JHP1260 siderophore-mediated iron transport protein (tonB) HP1341 85 1260 402 49 JHP0492 DNA transfer protein (cagE) HP0544 104 95 50 50 JHP1121 DNA-directed RNA polymerase, beta subunit (rpoB) HP1198 84 23 16 51 JHP1434 DNA repair protein (recN) HP1393 35 177 160 52 JHP0491 cag island protein (cagF) HP0543 82 170 828 53 JHP0191 hypothetical protein HP0205 57 33 7 Genes with > 2 SD divergence indicated in bold NAH indicates No Annotated Homologue in the other sequence Table 4 Top 60 most divergent (>2 SD) genes by DNS in H. pylori strain 26695 plus those additional genes in the top 50 genes from TNS and HNS DNS order annotation HP# J99 # J99 DNS order TNS order HNS order 1 cag pathogenicity island protein (cag7) HP0527 JHP0476 2 1 1 2 vacuolating cytotoxin (vacA) paralog HP0289 JHP0274 4 2 4 3 poly E-rich hypothetical protein HP0322 JHP0305 5 8 5 4 hypothetical protein HP0609 JHP0556* 3 6 9 5 hypothetical protein HP0996 JHP0942 6 14 46 6 vacuolating cytotoxin (vacA) paralog HP0922 JHP0856 7 5 3 7 hypothetical protein HP0488 JHP0440 13 10 12 8 hypothetical protein HP1116 JHP1044 10 11 13 9 hypothetical protein HP0080 JHP0074 12 18 122 10 hypothetical protein HP0489 JHP0441 115 36 582 11 cag pathogenicity island protein (cag3) HP0522 JHP0471 21 48 100 12 hypothetical protein HP1089 JHP0336 27 67 59 13 vacuolating cytotoxin (vacA) paralog HP0610 JHP0556* 3 12 17 14 hypothetical protein HP0427 JHP0952 1 3 737 15 hypothetical protein HP1408 JHP1300 9 4 738 16 type III restriction enzyme R protein (res) HP0592 NAH - 30 35 17 hypothetical protein HP0119 NAH - 7 2 18 vacuolating cytotoxin (vacA) HP0887 JHP0819 59 25 34 19 N-methylhydantoinase HP0696 JHP0632 20 35 43 20 hypothetical protein HP1115 JHP1042 14 33 866 21 urease beta subunit (urea amidohydrolase) (ureB) HP0072 JHP0067 32 38 87 22 DNA mismatch repair protein (MutS) HP0621 JHP0565 45 137 64 23 cag island protein (cagT) HP0532 JHP0481 25 87 693 24 hypothetical protein HP0756 JHP0693 19 71 1548 25 integrase/recombinase (xerD) HP0995 JHP0941 33 39 448 26 outer membrane protein HP0486 JHP0438 22 147 142 27 cytochrome oxidase (cbb3 type) (fixN) HP0144 JHP0132 41 102 168 28 type IIS restriction enzyme R and M protein (ECO57IR) HP1517 NAH - 42 14 29 DNA transfer protein (cagE) HP0441 JHP0492 49 51 22 30 DNA polymerase I (polA) HP1470 JHP1363 46 77 54 31 cag island protein (cagA) HP0547 JHP0495 17 15 7 32 iron(III) dicitrate transport protein (fecA) HP1400 JHP1426 28 99 129 33 flagellin A (flaA) HP0601 JHP0548 34 40 180 34 outer membrane protein (omp26) HP1157 JHP1084 24 17 25 35 DNA repair protein (recN) HP1393 JHP1434 51 154 207 36 type I restriction enzyme R protein (hsdR) HP0464 NAH - 90 26 37 cell division protein (ftsK) HP1090 JHP0335 67 181 90 38 hypothetical protein HP1003 NAH - 61 170 39 histidine-rich, metal binding polypeptide (hpn) HP1427 NAH - 26 1449 40 hypothetical protein HP1333 JHP1253 40 53 296 41 hypothetical protein HP0788 JHP0725 68 72 256 42 hypothetical protein HP0906 JHP0842 42 22 16 43 hypothetical protein HP0059 JHP0052 26 21 320 44 GMP reductase (guaC) HP0854 JHP0790 107 169 451 45 hypothetical protein HP0030 JHP0026 23 24 39 46 histidine and glutamine-rich metal-binding protein HP1432 JHP1321 15 9 1432 47 hypothetical protein HP0186 JHP0174 29 130 276 48 fucosyltransferase HP0651 JHP0596 105 43 75 49 translation elongation factor EF-Tu (tufB) HP1205 JHP1128 81 64 166 50 virulence associated protein homolog (vacB) HP1248 JHP1169 79 164 160 51 hypothetical protein HP0449 NAH - 81 449 52 type III restriction enzyme R protein HP1371 JHP1285 55 119 23 53 virB4 homolog (virB4) HP0459 NAH - 49 28 54 2',3'-cyclic-nucleotide 2'-phosphodiesterase (cpdB) HP0104 JHP0096 56 73 68 55 hypothetical protein HP1479 JHP1372 135 153 127 56 RNA polymerase sigma-70 factor (rpoD) HP0088 JHP0081 62 55 31 57 hypothetical protein HP0205 JHP0191 53 78 8 58 hypothetical protein HP1143 JHP1071 78 29 41 59 hypothetical protein HP1106 JHP1033 36 272 277 60 cag pathogenicity island protein (cag13) HP0534 JHP0482 71 225 1021 63 DNA topoisomerase I (topA) HP0440 NAH - 149 24 68 outer membrane protein (omp3) HP0079 JHP0073 796 45 99 69 hypothetical protein HP0669 JHP0613 44 60 42 74 cag pathogenicity island protein (cag8) HP0528 JHP0477 72 50 27 75 hypothetical protein HP0453 NAH - 58 10 84 DNA-directed RNA polymerase, beta subunit (rpoB) JHP1121 50 23 19 91 hypothetical protein HP1142 JHP1070 60 19 6 97 multidrug resistance protein (spaB) HP0600 JHP0547 75 41 30 103 type I restriction enzyme R protein (hsdR) HP1402 JHP1424 195 86 21 109 adenine/cytosine DNA methyltransferase HP0054 NAH - 120 20 119 preprotein translocase subunit (secA) HP0786 JHP0723 159 176 49 121 hypothetical protein HP0058 JHP0051 394 16 53 122 hypothetical protein HP0513 JHP0462 104 28 15 125 type I restriction enzyme M protein (hsdM) HP1403 JHP1423 299 340 44 132 hypothetical protein HP0731 JHP0668 110 80 32 139 hypothetical protein HP0508 JHP0458 84 32 77 142 hypothetical protein HP1187 JHP1113 274 31 38 167 hypothetical protein HP1520 NAH - 20 33 179 hypothetical protein HP0118 JHP0110 64 27 36 195 type III restriction enzyme R protein (res) HP1521 JHP1410 161 210 18 209 outer membrane protein (omp17) HP0725 JHP0662 257 47 101 224 hypothetical protein HP0733 JHP0670 769 222 48 230 hypothetical protein HP0611 JHP0299 35 37 1129 249 hypothetical protein HP0345 NAH - 46 1338 283 hypothetical protein HP0120 NAH - 44 50 291 translation initiation factor IF-2 (infB) HP1048 JHP0377 330 332 45 297 DNA polymerase III alpha-subunit (dnaE) HP1460 JHP1353 509 219 47 342 type I restriction enzyme R protein (hsdR) HP0846 JHP0784 244 101 37 363 adenine specific DNA methyltransferase (mod) HP1522 JHP1411 857 207 11 410 secreted protein involved in flagellar motility HP1192 JHP1117 614 13 1256 593 hypothetical protein HP1516 NAH - 34 1090 631 hypothetical protein HP0586 JHP0534 577 163 29 1080 type II restriction enzyme (methyltransferase) HP0478 JHP0430 953 220 40 * probably frame shifted components of the same vacA related gene Genes with > 2 SD divergence in each analysis are indicated in bold NAH indicates No Annotated Homologue in the other sequence Figure 1 Comparisons using LAlign between a representative selection of orthologous genes with divergent DNA present in both H. pylori strains J99 and 26695 (presented in descending order of divergence as determined in strain J99). It cannot be assumed that all genes identified in this manner have been recently acquired. It is necessary to assess the nature of the sequence to determine if its divergence might be accounted for on the basis of features of the encoded protein. For example, JHP0476/HP0527, JHP1300/HP1408 and JHP0074/HP0080 include repetitive sequences likely to account for their DNS divergence. This type of analysis cannot be used to determine the possible foreign origin of such genes. Notably, the most divergent cag PAI gene (the 1 st and 2 nd most divergent gene in the whole genomes of strain 26695 and J99 respectively, JHP0476/HP0527) has a highly complex repetitive structure and the size of the large divergent peak associated with this island using previous methods is largely due to the presence of this gene. While a significant proportion of the genes identified in this analysis are associated with regions including several such genes and which share characteristics of islands of horizontal transfer or pathogenicity islands, this is far from universally true. There are many instances of single genes or small numbers of genes that are present that are not associated with any features that might otherwise have been used as indicators of horizontal acquisition such as transposases and flanking repeats. Our initial goal was to identify recently acquired and exchanged genes as candidates likely to be important in niche-adaptation, host interactions, and alterations in bacterial fitness. It has been argued that essential genes are unlikely to be transferred successfully since recipient taxa would already bear functional orthologues, which would have experienced long-term co-evolution with the rest of the cellular machinery. In contrast, it is proposed that those under weak or transient selection – like those associated with nonessential catabolic processes, new operons, and those providing new niche-adaptive changes are likely to be successfully transferred and retained [ 19 ]. This leads to a model in which a stable 'core genome' comprised of essential metabolic, regulatory, and cell division genes provides a stable context for the more labile non-essential and niche adaptive genes. On this basis such genes are used for phylogenetic studies and are thought to provide a relatively constant background in which species evolution occurs. Many of the genes identified for which functions are known affect virulence or niche adaptive genes, including: the vacuolating cytotoxin and related toxins (2 and 3), urease and flagellar components, and genes involved in iron acquisition. However, we also find clear evidence, confirmed by differences between the two genome sequences, that recent, and therefore relatively frequent, horizontal transfer is not limited to genes associated with niche adaptation and virulence. Amongst the core function genes identified were mut S, fts K, xer D, and pol A. The comparisons of the latter three between the sequence strains are shown in Figure 1f,g & 1j . These comparisons support the results suggesting that these genes have been the substrates for horizontal exchange between species. Tetranucleotide composition has been used for the consideration of the presence of palindromic sequences that might be substrates for restriction systems and Chi sites and the presence of unstable repeats mediating phase variation [ 10 ], but the use of longer component signatures has not been used to identify horizontally acquired regions in bacterial genomes. Following analysis of eukaryotic sequences it was concluded that DNS captures most of the departure from randomness in DNA sequences and that longer component lengths correlate highly with the DNS results [ 20 ]. Also, analysis of dinucleotides separated by no, one, or two other nucleotides showed that separated pairs are more nearly random than adjacent pairs and were concluded to be relatively uninformative [ 9 ]. However, in preliminary analyses, while results using the typically long walking windows gave concordant results as previously reported, we found that the use of smaller walking windows generated progressively more different patterns of divergence with other length components. Using tetranucleotide (TNS) and hexanucleotide (HNS) signature analysis we find that, while in some instances there is significant overlap between the genes identified using the different component lengths, there are substantial differences that indicate additional horizontally transferred genes not identified by DNS alone (Tables 2 to 6 ). Table 2 Top 50 most divergent genes by TNS in H. pylori strain J99 plus those additional genes > 2 SD greater than the mean by DNS and the 50 most divergent by HNS TNS order Annotation JHP # 26695 # DNS order HNS order 1 hypothetical protein JHP1300 HP1408 9 1 2 cag pathogenicity island protein (cag7) JHP0476 HP0527 2 2 3 hypothetical protein JHP0952 HP0427 1 1355 4 histidine and glutamine-rich metal-binding protein JHP1321 HP1432 15 49 5 vacuolating cytotoxin (vacA) paralog JHP0556 HP0609/10 3 4 6 vacuolating cytotoxin (vacA) paralog JHP0274 HP0289 4 5 7 hypothetical protein JHP0050 HP0058 8 84 8 hypothetical protein JHP0305 HP0322 5 10 9 vacuolating cytotoxin (vacA) paralog JHP0856 HP0922 7 6 10 type I restriction enzyme (hsdS) JHP1422 NAH 319 3 11 hypothetical protein JHP0299 HP061/2 35 275 12 hypothetical protein JHP0928 NAH 11 9 13 hypothetical protein JHP0942 HP0996 6 27 14 hypothetical protein JHP1044 HP1116 10 8 15 hypothetical protein JHP0934 NAH 16 95 16 hypothetical protein JHP0440 HP0488 13 17 17 outer membrane protein (omp26) JHP1084 HP1157 24 24 18 topoisomerase I (topA 3) JHP0931 NAH 18 20 19 hypothetical protein JHP0318 NAH 286 293 20 cag island protein (cagA) JHP0495 HP0547 17 12 21 hypothetical protein JHP0110 HP0118 64 19 22 hypothetical protein JHP1208 HP1288 91 830 23 DNA-directed RNA polymerase, beta subunit (rpoB) JHP1121 HP1198 50 16 24 hypothetical protein JHP0052 HP0059 26 120 25 hypothetical protein JHP1042 HP1115 14 694 26 hypothetical protein JHP0953 NAH 31 1463 27 hypothetical protein JHP1070 HP1142 60 14 28 hypothetical protein JHP1113 HP1187 274 39 29 hypothetical protein JHP0842 HP0906 42 21 30 type II restriction enzyme JHP0630 NAH 173 588 31 histidine-rich, metal binding polypeptide (hpn) JHP1320 HP1427 70 1404 32 hypothetical protein JHP0074 HP0080 12 125 33 hypothetical protein JHP0191 HP0205 53 7 34 hypothetical protein JHP0376 HP1049 235 1128 35 cag pathogenicity island protein (cag3) JHP0471 HP0522 21 62 36 hypothetical protein JHP0026 HP0030 23 64 37 urease beta subunit (urea amidohydrolase) (ureB) JHP0067 HP0072 32 70 38 hypothetical protein JHP0939 HP0991 116 156 39 multidrug resistance protein (spaB) JHP0547 HP0600 75 18 40 flagellin A (flaA) JHP0548 HP0601 34 154 41 hypothetical protein JHP1071 HP1143 78 61 42 hypothetical protein JHP0613 HP0669 44 33 43 hypothetical protein JHP0623 HP0682 231 1186 44 N-methylhydantoinase JHP0632 HP0696 20 36 45 hypothetical protein JHP1049 NAH 278 470 46 vacuolating cytotoxin (vacA) JHP0819 HP0887 59 38 47 putative restriction enzyme JHP0164 NAH 88 43 48 type I restriction enzyme R protein (hsdR) JHP0784 HP0846 244 35 49 hook assembly protein, flagella (flgD) JHP0843 HP0907 103 175 50 hypothetical protein JHP0458 HP0508 84 44 51 hypothetical protein JHP0336 HP1089 27 54 53 hypothetical protein JHP0940 NAH 39 393 54 hypothetical protein JHP0462 HP0513 104 11 55 type II restriction enzyme (methyltransferase) JHP1409 NAH 37 15 58 hypothetical protein JHP1285 HP1371 55 25 59 hypothetical protein JHP0693 HP0756 19 1490 62 cag pathogenicity island protein (cag8) JHP0477 HP0528 72 31 63 type III restriction enzyme (res) JHP1297 NAH 30 28 64 hypothetical protein JHP0668 HP0731 110 32 67 outer membrane protein JHP0438 HP0486 22 145 70 cag island protein (cagT) JHP0481 HP0532 25 558 71 RNA polymerase sigma-70 factor (rpoD) JHP0081 HP0088 62 37 75 hypothetical protein JHP1253 HP1333 40 384 78 iron(III) dicitrate transport protein (fecA) JHP1426 HP1400 28 111 81 DNA polymerase I (polA) JHP1363 HP1470 46 46 85 type I restriction enzyme (hsdS) JHP0414 NAH 275 30 88 hypothetical protein JHP0174 HP0187/8/6 29 90 89 iron(III) dicitrate transport protein (fecA) JHP0626 HP0686 38 47 95 DNA transfer protein (cagE) JHP0492 HP0544 49 50 100 integrase/recombinase (xerD) JHP0941 HP0995 33 541 104 type III restriciton enzyme (mod) JHP1411 HP1522 857 13 105 type I restriction enzyme R protein (hsdR) JHP0416 HP0464 63 29 122 adenine specific DNA methyltransferase (mod) JHP0244 HP0260 236 48 130 hypothetical protein JHP0925 NAH 43 990 137 cag island protein (cagH) JHP0489 HP0541 47 398 138 type I restriction enzyme (hsdR) JHP1424 HP1402 195 22 158 hypothetical protein JHP0540 NAH 674 26 170 cag island protein (cagF) JHP0491 HP0543 52 828 177 DNA repair protein (recN) JHP1434 HP1393 51 160 190 type III restriction enzyme (mod) JHP1296 NAH 121 34 196 role in outermembrane permeability (imp) JHP1138 HP1215/6 208 45 206 cytochrome oxidase (cbb3 type) (fixN) JHP0132 HP0144 41 209 227 DNA mismatch repair protein (mutS) JHP0565 HP0621 45 82 230 hypothetical protein JHP0534 HP0586 577 40 258 type III restriction enzyme (res) JHP1410 HP1521 161 23 262 hypothetical protein JHP1033 HP1106 36 342 281 translation initiation factor IF-2 (infB) JHP0377 HP1048 330 42 290 type II restriction enzyme (methyltrasferase) JHP1284 NAH 750 41 1260 siderophore-mediated iron transport protein (tonB) JHP1260 HP1341 48 402 Genes with > 2 SD divergence in each analysis are indicated in bold NAH indicates No Annotated Homologue in the other sequence Table 3 Top 50 most divergent genes by HNS in H. pylori strain J99 plus those additional genes >2 SD greater than the mean by DNS and top 50 by TNS HNS order J99 annotation JHP # 26695 # DNS order TNS order 1 hypothetical protein JHP1300 HP1408 9 1 2 cag pathogenicity island protein (cag7) JHP0476 HP0527 2 2 3 type I restriction enzyme (hsdS) JHP1422 NAH 319 10 4 vacuolating cytotoxin (vacA) paralog JHP0556 HP0609/10 3 5 5 vacuolating cytotoxin (vacA) paralog JHP0274 HP0289 4 6 6 vacuolating cytotoxin (vacA) paralog JHP0856 HP0922 7 9 7 hypothetical protein JHP0191 HP0205 53 33 8 hypothetical protein JHP1044 HP1116 10 14 9 hypothetical protein JHP0928 NAH 11 12 10 hypothetical protein JHP0305 HP0322 5 8 11 hypothetical protein JHP0462 HP0513 104 54 12 cag island protein (cagA) JHP0495 HP0547 17 20 13 type III restriciton enzyme (mod) JHP1411 HP1522 857 104 14 hypothetical protein JHP1070 HP1142 60 27 15 type II restriction enzyme (methyltransferase) JHP1409 NAH 37 55 16 DNA-directed RNA polymerase, beta subunit (rpoB) JHP1121 HP1198 50 23 17 hypothetical protein JHP0440 HP0488 13 16 18 multidrug resistance protein (spaB) JHP0547 HP0600 75 39 19 hypothetical protein JHP0110 HP0118 64 21 20 topoisomerase I (topA 3) JHP0931 NAH – check 18 18 21 hypothetical protein JHP0842 HP0906 42 29 22 type I restriction enzyme (hsdR) JHP1424 HP1402 195 138 23 type III restriction enzyme (res) JHP1410 HP1521 161 258 24 outer membrane protein (omp26) JHP1084 HP1157 24 17 25 hypothetical protein JHP1285 HP1371 55 58 26 hypothetical protein JHP0540 NAH 674 158 27 hypothetical protein JHP0942 HP0996 6 13 28 type III restriction enzyme (res) JHP1297 NAH 30 63 29 type I restriction enzyme R protein (hsdR) JHP0416 HP0464 63 105 30 type I restriction enzyme (hsdS) JHP0414 NAH 275 85 31 cag pathogenicity island protein (cag8) JHP0477 HP0528 72 62 32 hypothetical protein JHP0668 HP0731 110 64 33 hypothetical protein JHP0613 HP0669 44 42 34 type III restriction enzyme (mod) JHP1296 NAH 121 190 35 type I restriction enzyme R protein (hsdR) JHP0784 HP0846 244 48 36 N-methylhydantoinase JHP0632 HP0696 20 44 37 RNA polymerase sigma-70 factor (rpoD) JHP0081 HP0088 62 71 38 vacuolating cytotoxin (vacA) JHP0819 HP0887 59 46 39 hypothetical protein JHP1113 HP1187 274 28 40 hypothetical protein JHP0534 HP0586 577 230 41 type II restriction enzyme (methyltrasferase) JHP1284 NAH 750 290 42 translation initiation factor IF-2 (infB) JHP0377 HP1048 330 281 43 restriction enzyme JHP0164 NAH 88 47 44 hypothetical protein JHP0458 HP0508 84 50 45 role in outermembrane permeability (imp) JHP1138 HP1215/6 208 196 46 DNA polymerase I (polA) JHP1363 HP1470 46 81 47 iron(III) dicitrate transport protein (fecA) JHP0626 HP0686 38 89 48 adenine specific DNA methyltransferase (mod) JHP0244 HP0260 236 122 49 histidine and glutamine-rich metal-binding protein JHP1321 HP1432 15 4 50 DNA transfer protein (cagE) JHP0492 HP0544 49 95 54 hypothetical protein JHP0336 HP1089 27 51 62 cag pathogenicity island protein (cag3) JHP0471 HP0522 21 35 64 hypothetical protein JHP0026 HP0030 23 36 70 urease beta subunit (urea amidohydrolase) (ureB) JHP0067 HP0072 32 37 82 DNA mismatch repair protein (mutS) JHP0565 HP0621 45 227 84 hypothetical protein JHP0050 HP0058 8 7 90 hypothetical protein JHP0174 HP0187/8/6 29 88 95 hypothetical protein JHP0934 NAH 16 15 111 iron(III) dicitrate transport protein (fecA) JHP1426 HP1400 28 78 120 hypothetical protein JHP0052 HP0059 26 24 125 hypothetical protein JHP0074 HP0080 12 32 145 Outer membrane protein JHP0438 HP0486 22 67 154 flagellin A (flaA) JHP0548 HP0601 34 40 160 DNA repair protein (recN) JHP1434 HP1393 51 177 209 cytochrome oxidase (cbb3 type) (fixN) JHP0132 HP0144 41 206 275 hypothetical protein JHP0299 HP061/2 35 11 342 hypothetical protein JHP1033 HP1106 36 262 384 hypothetical protein JHP1253 HP1333 40 75 393 hypothetical protein JHP0940 NAH 39 53 398 cag island protein (cagH) JHP0489 HP0541 47 137 402 siderophore-mediated iron transport protein (tonB) JHP1260 HP1341 48 1260 541 integrase/recombinase (xerD) JHP0941 HP0995 33 100 558 cag island protein (cagT) JHP0481 HP0532 25 70 694 hypothetical protein JHP1042 HP1115 14 25 828 cag island protein (cagF) JHP0491 HP0543 52 170 990 hypothetical protein JHP0925 NAH 43 130 1355 hypothetical protein JHP0952 HP0427 1 3 1463 hypothetical protein JHP0953 NAH 31 26 1490 hypothetical protein JHP0693 HP0756 19 59 Genes with > 2 SD divergence in each analysis are indicated in bold NAH indicates No Annotated Homologue in the other sequence Table 5 Top 50 most divergent genes by TNS in H. pylori strain 26695 plus those additional genes > 2 SD greater than the mean by DNS and the 50 most divergent by HNS TNS order annotation HP# J99 # DNS order HNS order 1 cag pathogenicity island protein (cag7) HP0527 JHP0476 1 1 2 vacuolating cytotoxin (vacA) paralog HP0289 JHP0274 2 4 3 hypothetical protein HP0427 JHP0952 14 737 4 hypothetical protein HP1408 JHP1300 15 738 5 vacuolating cytotoxin (vacA) paralog HP0922 JHP0856 6 3 6 hypothetical protein HP0609 JHP0556* 4 9 7 hypothetical protein HP0119 NAH 17 2 8 poly E-rich hypothetical protein HP0322 JHP0305 3 5 9 histidine and glutamine-rich metal-binding protein HP1432 JHP1321 46 1432 10 hypothetical protein HP0488 JHP0440 7 12 11 hypothetical protein HP1116 JHP1044 8 13 12 vacuolating cytotoxin (vacA) paralog HP0610 JHP0556* 13 17 13 secreted protein involved in flagellar motility HP1192 JHP1117 410 1256 14 hypothetical protein HP0996 JHP0942 5 46 15 cag island protein (cagA) HP0547 JHP0495 31 7 16 hypothetical protein HP0058 JHP0051 121 53 17 outer membrane protein (omp26) HP1157 JHP1084 34 25 18 hypothetical protein HP0080 JHP0074 9 122 19 hypothetical protein HP1142 JHP1070 91 6 20 hypothetical protein HP1520 NAH 167 33 21 hypothetical protein HP0059 JHP0052 43 320 22 hypothetical protein HP0906 JHP0842 42 16 23 DNA-directed RNA polymerase, beta subunit (rpoB) HP1198 JHP1121 84 19 24 hypothetical protein HP0030 JHP0026 45 39 25 vacuolating cytotoxin (vacA) HP0887 JHP0819 18 34 26 histidine-rich, metal binding polypeptide (hpn) HP1427 NAH 39 1449 27 hypothetical protein HP0118 JHP0110 179 36 28 hypothetical protein HP0513 JHP0462 122 15 29 hypothetical protein HP1143 JHP1071 58 41 30 type III restriction enzyme R protein (res) HP0592 NAH 16 35 31 hypothetical protein HP1187 JHP1113 142 38 32 hypothetical protein HP0508 JHP0458 139 77 33 hypothetical protein HP1115 JHP1042 20 866 34 hypothetical protein HP1516 NAH 593 1090 35 N-methylhydantoinase HP0696 JHP0632 19 43 36 hypothetical protein HP0489 JHP0441 10 582 37 hypothetical protein HP0611 JHP0299 230 1129 38 urease beta subunit (urea amidohydrolase) (ureB) HP0072 JHP0067 21 87 39 integrase/recombinase (xerD) HP0995 JHP0941 25 448 40 flagellin A (flaA) HP0601 JHP0548 33 180 41 multidrug resistance protein (spaB) HP0600 JHP0547 97 30 42 type IIS restriction enzyme R and M protein (ECO57IR) HP1517 NAH 28 14 43 fucosyltransferase HP0651 JHP0596 48 75 44 hypothetical protein HP0120 NAH 283 50 45 outer membrane protein (omp3) HP0079 JHP0073 68 99 46 hypothetical protein HP0345 NAH 249 1338 47 outer membrane protein (omp17) HP0725 JHP0662 209 101 48 cag pathogenicity island protein (cag3) HP0522 JHP0471 11 100 49 virB4 homolog (virB4) HP0459 NAH 53 28 50 cag pathogenicity island protein (cag8) HP0528 JHP0477 74 27 51 DNA transfer protein (cagE) HP0441 JHP0492 29 22 53 hypothetical protein HP1333 JHP1253 40 296 55 RNA polymerase sigma-70 factor (rpoD) HP0088 JHP0081 56 31 58 hypothetical protein HP0453 NAH 75 10 60 hypothetical protein HP0669 JHP0613 69 42 61 hypothetical protein HP1003 NAH 38 170 64 translation elongation factor EF-Tu (tufB) HP1205 JHP1128 49 166 67 hypothetical protein HP1089 JHP0336 12 59 71 hypothetical protein HP0756 JHP0693 24 1548 72 hypothetical protein HP0788 JHP0725 41 256 73 2',3'-cyclic-nucleotide 2'-phosphodiesterase (cpdB) HP0104 JHP0096 54 68 77 DNA polymerase I (polA) HP1470 JHP1363 30 54 78 hypothetical protein HP0205 JHP0191 57 8 80 hypothetical protein HP0731 JHP0668 132 32 81 hypothetical protein HP0449 NAH 51 449 86 type I restriction enzyme R protein (hsdR) HP1402 JHP1424 103 21 87 cag pathogenicity island protein (cag12) HP0532 JHP0481 23 693 90 type I restriction enzyme R protein (hsdR) HP0464 NAH 36 26 99 iron(III) dicitrate transport protein (fecA) HP1400 JHP1426 32 129 101 type I restriction enzyme R protein (hsdR) HP0846 JHP0784 342 37 102 cytochrome oxidase (cbb3 type) (fixN) HP0144 JHP0132 27 168 119 type III restriction enzyme R protein HP1371 JHP1285 52 23 120 adenine/cytosine DNA methyltransferase HP0054 NAH 109 20 130 hypothetical protein HP0186 JHP0174 47 276 137 DNA mismatch repair protein (MutS) HP0621 JHP0565 22 64 147 outer membrane protein HP0486 JHP0438 26 142 149 DNA topoisomerase I (topA) HP0440 NAH 63 24 153 hypothetical protein HP1479 JHP1372 55 127 154 DNA repair protein (recN) HP1393 JHP1434 35 207 163 hypothetical protein HP0586 JHP0534 631 29 164 virulence associated protein homolog (vacB) HP1248 JHP1169 50 160 169 GMP reductase (guaC) HP0854 JHP0790 44 451 176 preprotein translocase subunit (secA) HP0786 JHP0723 119 49 181 cell division protein (ftsK) HP1090 JHP0335 37 90 207 adenine specific DNA methyltransferase (mod) HP1522 JHP1411 363 11 210 type III restriction enzyme R protein (res) HP1521 JHP1410 195 18 219 DNA polymerase III alpha-subunit (dnaE) HP1460 JHP1353 297 47 220 type II restriction enzyme (methyltransferase) HP0478 JHP0430 1080 40 222 hypothetical protein HP0733 JHP0670 224 48 225 cag pathogenicity island protein (cag13) HP0534 JHP0482 60 1021 272 hypothetical protein HP1106 JHP1033 59 277 332 translation initiation factor IF-2 (infB) HP1048 JHP0377 291 45 340 type I restriction enzyme M protein (hsdM) HP1403 JHP1423 125 44 * probably frame shifted components of the same vacA related gene Genes with > 2 SD divergence in each analysis are indicated in bold NAH indicates No Annotated Homologue in the other sequence Table 6 Top 50 most divergent genes by HNS in H. pylori strain 26695 plus those additional genes > 2 SD greater than the mean by DNS and the 50 most divergent by HNS HNS order annotation HP# J99 # DNS order TNS order 1 cag pathogenicity island protein (cag7) HP0527 JHP0476 1 1 2 hypothetical protein HP0119 NAH 17 7 3 vacuolating cytotoxin (vacA) paralog HP0922 JHP0856 6 5 4 vacuolating cytotoxin (vacA) paralog HP0289 JHP0274 2 2 5 poly E-rich hypothetical protein HP0322 JHP0305 3 8 6 hypothetical protein HP1142 JHP1070 91 19 7 cag island protein (cagA) HP0547 JHP0495 31 15 8 hypothetical protein HP0205 JHP0191 57 78 9 hypothetical protein HP0609 JHP0556* 4 6 10 hypothetical protein HP0453 NAH 75 58 11 adenine specific DNA methyltransferase (mod) HP1522 JHP1411 363 207 12 hypothetical protein HP0488 JHP0440 7 10 13 hypothetical protein HP1116 JHP1044 8 11 14 type IIS restriction enzyme R and M protein (ECO57IR) HP1517 NAH 28 42 15 hypothetical protein HP0513 JHP0462 122 28 16 hypothetical protein HP0906 JHP0842 42 22 17 vacuolating cytotoxin (vacA) paralog HP0610 JHP0556* 13 12 18 type III restriction enzyme R protein (res) HP1521 JHP1410 195 210 19 DNA-directed RNA polymerase, beta subunit (rpoB) HP1198 JHP1121 84 23 20 adenine/cytosine DNA methyltransferase HP0054 NAH 109 120 21 type I restriction enzyme R protein (hsdR) HP1402 JHP1424 103 86 22 DNA transfer protein (cagE) HP0441 JHP0492 29 51 23 type III restriction enzyme R protein HP1371 JHP1285 52 119 24 DNA topoisomerase I (topA) HP0440 NAH 63 149 25 outer membrane protein (omp26) HP1157 JHP1084 34 27 26 type I restriction enzyme R protein (hsdR) HP0464 NAH 36 90 27 cag pathogenicity island protein (cag8) HP0528 JHP0477 74 50 28 virB4 homolog (virB4) HP0459 NAH 53 49 29 hypothetical protein HP0586 JHP0534 631 163 30 multidrug resistance protein (spaB) HP0600 JHP0547 97 41 31 RNA polymerase sigma-70 factor (rpoD) HP0088 JHP0081 56 55 32 hypothetical protein HP0731 JHP0668 132 80 33 hypothetical protein HP1520 NAH 167 20 34 vacuolating cytotoxin HP0887 JHP0819 18 25 35 type III restriction enzyme R protein (res) HP0592 NAH 16 30 36 hypothetical protein HP0118 JHP0110 179 27 37 type I restriction enzyme R protein (hsdR) HP0846 JHP0784 342 101 38 hypothetical protein HP1187 JHP1113 142 31 39 hypothetical protein HP0030 JHP0026 45 24 40 HP0478 JHP0430 1080 220 41 hypothetical protein HP1143 JHP1071 58 29 42 hypothetical protein HP0669 JHP0613 69 60 43 N-methylhydantoinase HP0696 JHP0632 19 35 44 type I restriction enzyme M protein (hsdM) HP1403 JHP1423 125 340 45 translation initiation factor IF-2 (infB) HP1048 JHP0377 291 332 46 hypothetical protein HP0996 JHP0942 5 14 47 DNA polymerase III alpha-subunit (dnaE) HP1460 JHP1353 297 219 48 hypothetical protein HP0733 JHP0670 224 222 49 preprotein translocase subunit (secA) HP0786 JHP0723 119 176 50 hypothetical protein HP0120 NAH 283 44 53 hypothetical protein HP0058 JHP0051 121 16 54 DNA polymerase I (polA) HP1470 JHP1363 30 77 59 hypothetical protein HP1089 JHP0336 12 67 64 DNA mismatch repair protein (MutS) HP0621 JHP0565 22 137 68 2',3'-cyclic-nucleotide 2'-phosphodiesterase (cpdB) HP0104 JHP0096 54 73 75 fucosyltransferase HP0651 JHP0596 48 43 77 hypothetical protein HP0508 JHP0458 139 32 87 urease beta subunit (urea amidohydrolase) (ureB) HP0072 JHP0067 21 38 90 cell division protein (ftsK) HP1090 JHP0335 37 181 99 outer membrane protein (omp3) HP0079 JHP0073 68 45 100 cag pathogenicity island protein (cag3) HP0522 JHP0471 11 48 101 outer membrane protein (omp17) HP0725 JHP0662 209 47 122 hypothetical protein HP0080 JHP0074 9 18 127 hypothetical protein HP1479 JHP1372 55 153 129 iron(III) dicitrate transport protein (fecA) HP1400 JHP1426 32 99 142 outer membrane protein HP0486 JHP0438 26 147 160 virulence associated protein homolog (vacB) HP1248 JHP1169 50 164 166 translation elongation factor EF-Tu (tufB) HP1205 JHP1128 49 64 168 cytochrome oxidase (cbb3 type) (fixN) HP0144 JHP0132 27 102 170 hypothetical protein HP1003 NAH 38 61 180 flagellin A (flaA) HP0601 JHP0548 33 40 207 DNA repair protein (recN) HP1393 JHP1434 35 154 256 hypothetical protein HP0788 JHP0725 41 72 276 hypothetical protein HP0186 JHP0174 47 130 277 hypothetical protein HP1106 JHP1033 59 272 296 hypothetical protein HP1333 JHP1253 40 53 320 hypothetical protein HP0059 JHP0052 43 21 448 integrase/recombinase (xerD) HP0995 JHP0941 25 39 449 hypothetical protein HP0449 NAH 51 81 451 GMP reductase (guaC) HP0854 JHP0790 44 169 582 hypothetical protein HP0489 JHP0441 10 36 693 cag island protein (cagT) HP0532 JHP0481 23 87 737 hypothetical protein HP0427 JHP0952 14 3 738 hypothetical protein HP1408 JHP1300 15 4 866 hypothetical protein HP1115 JHP1042 20 33 1021 cag pathogenicity island protein (cag13) HP0534 JHP0482 60 225 1090 hypothetical protein HP1516 NAH 593 34 1129 hypothetical protein HP0611 JHP0299 230 37 1256 secreted protein involved in flagellar motility HP1192 JHP1117 410 13 1338 hypothetical protein HP0345 NAH 249 46 1432 histidine and glutamine-rich metal-binding protein HP1432 JHP1321 46 9 1449 histidine-rich, metal binding polypeptide (hpn) HP1427 NAH 39 26 1548 hypothetical protein HP0756 JHP0693 24 71 * probably frame shifted components of the same vacA related gene Genes with > 2 SD divergence in each analysis are indicated in bold NAH indicates No Annotated Homologue in the other sequence The 50 most divergent J99 ORFs by HNS included 26 (52%) that were not in the 53 (>2 SD) most divergent by DNS, these included 11 restriction-modification system genes and 6 others that were not annotated within the strain 26695 genome sequence. The identification of genes of a type known to be horizontally exchanged, and different between the gene complements of the strains, is strong corroboration for the foreign origin of the additional genes identified by HNS. In several instances (Tables 2 to 6 ) the DNS did not detect these genes at all e.g. restriction enzymes that were the 3 rd , 13 th and 41 st most divergent genes by HNS, were 319 th , 857 th and 750 th most divergent by DNS, respectively. In some instances the TNS gave intermediate results and in others identified other genes as more divergent than the other methods. The TNS was most sensitive for the detection of rpoB (HP1198 / JHP1121) which is associated with a significantly different gene length in the two strains (Figure 1h ). One explanation for this observation is that while the DNS may initially be the most sensitive indicator of horizontal exchange it may become ameliorated to the new sequence characteristics more rapidly that the longer component features, which are probably detecting qualitatively different sequence characteristics. The differences in the analyses using different length components, and a comparison of the results from the two sequenced strains, suggest a complex evolutionary history for the cag pathogenicity island. These suggest that it probably has mosaic structure including sequences from more than one species background, in addition to sequence that is entirely typical of H. pylori . It is normally impossible to determine the chronology of events to distinguish insertions and deletions when comparing strains. In strain 26695 there are two open reading frames that are both good candidate coding sequences. There is only one gene in this location in strain J99 composed of the 5' gene from strain 26695 and the 3' end of the subsequent gene. This could have arisen from either a deletion or an insertion event. However, the normal DNS of the J99 gene (JHP0073, 799 th in divergence) and the 5' 26695 gene (HP0079, 751 st in divergence), and the high divergence of the 3' 26695 gene (HP0078, 68 th in divergence), indicate that the most likely event is an insertion into strain 26695 (Figure 1l ). Likewise HP0119 is likely to contain an insertion and JHP1113 probably reflects the original sequences (Figure 1k ). The inclusion of two DNA metabolism genes associated with recombination and repair is notable. Both mutS and recN were identified in both strains (22 nd and 35 th , and 45 th and 51 st most divergent genes by DNS in strains 26695 and J99 respectively). When the homologous genes were compared between the strains, extensive divergences were evident between more than one region of each protein. That these genes have divergent signatures in both strains suggests that neither has a wholly native composition. This observation is consistent with the models of rapid evolution which suggest that transient competitive advantages are enjoyed by organisms that are hypermutators under conditions of environmental stress and transitions, and that these states which can be produced by mutations in DNA repair genes [ 21 - 26 ]. However, such states have to be reversed so that an unsustainable mutational burden is not attained, and it has been proposed that this reversal is mediated by repair following horizontal transfer and homologous recombination, and that such strains are hyper-recombinogenic [ 27 - 29 ]. The untypicality of mutS and recN suggest that H. pylori is another species that can make use of this strategy for diversification under stressful conditions. The identification of RNA polymerase genes, with associated differences between the strains, is striking. The divergence of phylogenetic trees based upon different sequences has been highlighted, and particularly the differences between the trees associated with RNA polymerase genes and rRNA [ 30 , 31 ]. It has been argued that RNA polymerase is as essential to cell function as is rRNA and that there is no compelling reason to chose rRNA as the more reliable marker [ 32 ]. While the DNS analysis does not address the stability of rRNA (and specifically excludes the rRNA sequences because their differing coding requirements and evolutionary pressures generate a divergent signature for other reasons), it does indicate that RNA polymerase can be a substrate for horizontal transfer, and that trees based upon this gene, or other essential genes, need not necessarily be considered a challenge to rRNA based phylogenies. Conclusions The spectrum of recently horizontally acquired sequences identified emphasizes the two driving forces of horizontal exchange: the transfer of a phenotype which alters or enhances bacterial fitness resulting in increased competitive fitness or altered niche adaptation, and the presence of a substrate for homologous recombination. Because of the focus upon, and relative ease of identifying, large islands associated with readily identifiable features and phenotypes, the importance of the latter component has perhaps been underestimated. The genes that have been considered to code for 'core metabolic' 'house-keeping' functions are amongst those most likely to be changed by horizontal transfer events because of the presence of homologous substrates, and changes are likely to persist even when the change is phenotypically neutral. Equally, changes in the genes involved in core functions such as gene expression and DNA metabolism may have pleotropic effects and there may be significant differences in strain behaviour, that are not simply the consequence of differences in their respective gene complements. The selection of genes for phylogenetic analysis on the basis of their coding for conserved core functions is also problematic because these are also frequently the genes most likely to share the high homology that facilitates recombination and horizontal exchange. Methods A traditional nucleotide signature is generated by segmenting a sequence of DNA into k equal-sized subsequences (or 'windows'). The mathematical basis for the signature is an odds ratio – p i – calculated by dividing the frequency of a length- L oligonucleotide by its expected frequency. The odds ratios for each of the 4 L oligonucleotides in each window ( w ) are compared with the odds ratios for the overall sequence ( s ) [ 9 , 10 , 33 ]. The normalized difference δ is plotted and thus a nucleotide signature consists of a k -length sequence of δ values: δ ( w , s ) = (1/4 L )Σ(4 L , i : x )| p i ( w ) - p i ( s )|, where x is the set of all permutations of length L and i is one such permutation. There are interesting parallels between signature-style genome analysis and stylometric techniques previously used to determine the authorship of controversial literary texts. This is analogous with the biological problem and it is from this that our method is derived. Rather than using a fixed-window signature, signature scores are calculated for each coding open reading frame (ORF) and weighted with variance estimates so that the scores for shorter ORFs confer with their longer counterparts. Bissell's weighted cusum (cumulative sum) [ 34 ], , is modified so that n denotes the number of ORFs in the genome, X i the number of oligonucleotides in ORF i , and w i the number of nucleotides in ORF i . The results are scaled according to ORF size using the standard error σ = √( *# ORF ). In this way false positives are abrogated by normalizing for over-representation of lower order peptides. The method is implemented in Java and efficiency is maintained through an O ( N ) ( N = sequence length) refinement: probabilities for the complete sequence are calculated in O ( N ) steps for any length- L oligonucleotide, and maintain O ( N ) when 4 L > N through a hashing function; the second part of the program calculates σ for each ORF using a loop flattening technique, thereby avoiding the program having to recalculate overlapping sub-expressions. The program is available from and . Sequence alignments, as shown in Figure 1 , were performed and displayed using the programs: Lalign and viewed using Lalignview [ 35 ]. Abbreviations ORF, Open Reading Frame; DNS, Dinucleotide Signature; TNS, tetranucleotide signature; HNS, hexanucleotide signature. Authors' contributions NJS initiated the project, performed the genome sequence analyses, compared the two strains, interpreted the results, and prepared the biological aspects of the manuscript. PB was a DPhil student who worked on the coding aspects of the new methodology. JFP contributed to the bioinformatics discussions and planning stage of this project. SAJ directed and primarily developed the analysis strategy and the implementation of the new computational basis of the methodology, and prepared the computational aspects of the manuscript.

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CP-31398, a putative p53-stabilizing molecule tested in mammalian cells and in yeast for its effects on p53 transcriptional activity

Background CP-31398 is a small molecule that has been reported to stabilize the DNA-binding core domain of the human tumor suppressor protein p53 in vitro . The compound was also reported to function as a potential anti-cancer drug by rescuing the DNA-binding activity and, consequently, the transcription activation function of mutant p53 protein in mammalian tissue culture cells and in mice. Results We performed a series of gene expression experiments to test the activity of CP-31398 in yeast and in human cell cultures. With these cell-based assays, we were unable to detect any specific stimulation of mutant p53 activity by this compound. Concentrations of CP-31398 that were reported to be active in the published work were highly toxic to the human H1299 lung carcinoma and Saos-2 cell lines in our experiments. Conclusion In our experiments, the small molecule CP-31398 was unable to reactivate mutant p53 protein. The results of our in vivo experiments are in agreement with the recently published biochemical analysis of CP-31398 showing that this molecule does not bind p53 as previously claimed, but intercalates into DNA.

Background The tumor suppressor protein p53 protects organisms from malignancy by either inducing programmed cell death or by arresting the cell cycle in response to cellular stress. The intracellular concentration of p53 is tightly regulated at the posttranslational level and the protein is very unstable under physiological conditions. Upon stress, p53 is stabilized and can act as a potent transcription factor that activates a plethora of downstream target genes [ 1 , 2 ]. The p53 target genes can be grouped into classes according to their effect on a cell. One class is represented by p21 CIP , a cyclin dependent kinase inhibitor that is a potent inhibitor of the cell cycle. Another class of p53 target genes, of which bax is the most known representative, mediates p53-induced apoptosis. Other p53 target genes prevent the process of angiogenesis [ 2 ]. Not surprisingly, p53 is inactivated in a wide variety of human cancers [ 1 , 3 ]. Most mutations found in cancers are mis-sense mutations mapping to the central core domain of p53, which confers sequence-specific DNA binding activity to the protein. These mutations can cause destabilization of the core domain and loss of the DNA binding function. Thus, most mutant p53 proteins lack the ability to bind the DNA control elements of their target genes and fail to activate their expression. As a consequence, cells lacking functional p53 are unable to arrest the cell cycle or to undergo apoptosis in response to genotoxic stress. Since lack of p53 function plays such a central role in cancer development and in resistance to treatment, there has been much interest in the search of means and molecules to reactivate mutant forms of p53 [ 4 - 9 ]. A report by Foster et al. [ 7 ] generated special interest since it reported the discovery of a class of small molecules that was able to stabilize the p53 core domain. Not only were these compounds reported to stabilize the active conformation of wild type p53 but they were also shown to stabilize mutant p53 forms and enable them to activate transcription of p53 target genes. While the initial screening was conducted by an in vitro assay, activity of these compounds was subsequently confirmed in cell culture experiments and in a xenograft tumor mouse model [ 7 ]. One of their compounds, termed CP-31398, was reported to increase reporter gene activation by mutant p53 proteins about tenfold in the human p53-null lung carcinoma cell line H1299. We tested CP-31398 in a yeast cell-based assay and in human tissue culture cells. We could not detect any reactivation of mutant p53 in these cellular assays. Our results are in agreement with, and provide support to the results obtained by Rippin et al. [ 10 ], which indicate that CP-31398 intercalates with DNA rather than binding p53. Results The yeast Saccharomyces cerevisiae does not contain p53 homologous proteins. However, it has been demonstrated that p53 expressed in yeast can function as a potent transcriptional activator of artificial genes bearing its specific recognition sequence [ 11 ]. To test different mutant forms of p53 and the potential effect of various molecules on the activity of such mutants, we constructed a yeast strain carrying an integrated bi-directional reporter gene construct in which a single p53 binding site from the human p21 CIP1 promoter [ 12 ] was inserted between the divergent HIS3 and lacZ genes (figure 1A ). The p53-dependent expression of the yeast marker gene HIS3 allows growth selection on media lacking histidine and containing 3-amino-triazole (3-AT), which is a competitive inhibitor of the HIS3 gene product. The p53-dependent activation of this reporter gene is convenient for library screening, while expression of the bacterial lacZ gene allows verification and quantitation of the transcriptional activity of the various p53 forms and putative modulators. Figure 1 Human p53 protein activates transcription from a reporter construct in Saccharomyces cerevisiae . (A) Schematic representation of our yeast reporter construct integrated into our yeast strain. The black circle represents a single p53 responsive element from the human p21 promoter. (B) β-galactosidase assay to measure activation of the lacZ reporter gene. Wild type p53 and the indicated point mutant variants were transformed into the p53 responsive reporter strain and β-galactosidase activity in solution was determined. The activity of wild type p53 was arbitrarily set to 100%. p53R282W and p53V173A showed about 40% of activation compared to wild type p53. No activation of the reporter gene was detected in yeast cells containing the other point mutant variants. Average and standard deviation were determined from three independent experiments. (C) Growth on selective plates containing 20 mM 3-AT depends on expression of the HIS3 reporter gene and correlates with the activation of the lacZ reporter gene. Control plates consist of standard drop-out plates lacking the corresponding growth marker without 3-AT. Growth under selective conditions was dependent on activation of the p53 dependent reporter gene. Transformation of this strain with an episomal plasmid expressing human wild type p53 led to activation of the integrated lacZ and HIS3 reporter genes, which resulted in increased β-galactosidase activity (figure 1B ) and cell growth on plates lacking histidine and containing 20 mM 3-AT (figure 1C ). In contrast, expression of three mutant forms of p53 [ 1 ] with point mutations in their DNA-binding domain that completely abolish sequence-specific DNA-binding activity (p53R175H, p53R248W, p53R273H) did not activate transcription of the reporter genes (figure 1B and 1C , and data not shown). Expression of mutant forms that retain some DNA-binding activity in vitro and in mammalian cells [ 13 ] led to reduction of reporter gene expression compared to wild type p53 (figure 1B ). All p53 variants were expressed to comparable levels, as verified by western blot analysis (data not shown). Thus, the results of these transcriptional assays, taken together with published results of experiments performed in mammalian cells, indicate that the relative transcriptional activity of wild type p53 and the tested derivatives is comparable in yeast and in human cells. Since lack of p53 function plays such a central role in cancer development and in resistance to chemotherapeutic treatment, many efforts have been directed towards trying to reactivate mutant forms of p53 [ 4 - 9 , 14 ]. The report by Foster et al. [ 7 ] generated special interest since it presented the discovery of a small molecule (CP-31398) that was able to stabilize the core domain of p53 in vitro . In addition, this compound was reported to enable some otherwise silent p53 mutants to activate transcription from target gene promoters in cell culture experiments. We tested the effect of CP-31398 on human p53 activity in our p53-responsive yeast strain. Yeast cells expressing either wild type p53 or the mutant p53R173A were grown in media containing increasing concentrations of CP-31398. Activation of transcription of the p53-dependent reporter gene was assessed by measuring β-galactosidase activity in extracts from these cells (figure 2 ). No significant difference in lacZ reporter gene expression was observed between untreated cells and cells that were incubated with increasing concentrations of the compound. Very high concentrations of CP-31398 (500 μg/ml) reduced reporter gene activity, both in the case of wild type p53 expression and in the case of p5R173A expression. Results of growth assays on selective plates to indirectly measure HIS3 expression paralleled our data from the lacZ experiments (data not shown). Figure 2 Treatment with the p53 stabilizing compound CP-31398 shows no effect on reporter gene activity in yeast. Yeast cells expressing wild type p53 (lanes 1–5), p53V173A (lanes 6–10) or empty vector (-, lanes 11–15, white bars) were incubated with the concentrations of CP-31398 indicated (0–500 μg/ml) and expression of β-galactosidase was determined. β-galactosidase activity of wild type p53 without CP-31398 treatment was arbitrarily set to 100%. Yeast cells were treated with CP-31398 for 16 hours. Since these negative results regarding the lack of expected effects of CP-31398 on p53 could be due to our assay system in yeast, we tested CP-31398 in experiments with human tissue culture cells. We transfected the human p53-null H1299 lung carcinoma cell line that was also used for some of the experiments described by Foster et al. [ 7 ] with plasmid DNA expressing either human wild type p53 or the p53R173A mutant together with a reporter plasmid carrying a p53-responsive luciferase gene [ 12 ]. When we treated these cells with CP-31398 in concentrations that were shown to be effective by Foster et al. (5–20 μg/ml and higher concentrations), reporter gene signals decreased and massive cell death was observed (figure 3A and data not shown). Lower concentrations that showed no obvious toxicity to the cells had no significant effect on reporter gene activity. We observed very similar effects when we performed corresponding experiments in the osteosarcoma cell line Saos-2 (p53 null cell line) (data not shown). Figure 3 Treatment of H1299 lung carcinoma cells with CP-31398 provokes massive cell death and p53 independent decline of luciferase reporter gene activity. (A) H1299 cells were transfected with expression constructs for wild type p53 (lanes 1–3) and p53V173A (lanes 4–6). All the samples were cotransfected with a p53-responsive luciferase reporter (p21 luciferase, containing a single p53 responsive p53 binding site from the human p21 promoter, termed WWP-luc, see material and methods) and a constitutive reference β-galactosidase construct (CMV- lacZ ) for normalization. These cells were subsequently incubated with 0, 10, 15 μg/ml CP-31398 respectively and relative luciferase activities were determined. (B) H1299 cells were transfected with an expression construct for the synthetic activator GAL4 -VP16. All samples were cotransfected with a gal4p responsive luciferase reporter (UAS G luciferase) and a reference β-galactosidase plasmid (CMV- lacZ ) for normalization. The control cells were transfected with CMV- lacZ and UAS G luciferase only. These cells were subsequently incubated with 0, 10 and 15 μg/ml CP-31398 and relative luciferase activities were determined. The cells were treated with CP-31398 for 16 hours. Cell death and decreased reporter gene activity was not dependent on the expression of p53 since treatment with CP-31398 of the same cell lines expressing the unrelated activator GAL4-VP16 co-transfected with the respective reporter construct caused similar toxicity and lower reporter gene activity (figure 3B ). We next tested whether CP-31398 might have an effect on endogenous wild type p53 in the human cell line HeLa. These cells express wild type p53 protein, but p53 levels are low because of the presence of the viral HPV E6 protein, which targets p53 for degradation [ 15 ]. We transfected HeLa cells with the same p53-dependent luciferase reporter construct that was used with the other cell lines and treated the cells with increasing concentrations of CP-31398 (figure 4 ). To our surprise, there was a strong increase in reporter gene activation. When we expressed additional human wild type p53 from a transfection plasmid, the signal increased even more (data not shown). In contrast to the previous effect on other cell lines described above, we did not observe any significant cell death in the case of HeLa. Figure 4 Treatment of HeLa cervical carcinoma cells with CP-31398 leads to p53 dependent induction of the luciferase reporter. HeLa cells were transfected with a p53 responsive reporter gene (WWP-luc) and a reference β-galactosidase plasmid (CMV- lacZ ) for normalization. Control cells were transfected with CMV- lacZ alone. The cells were subsequently incubated with CP-31398 (0–10 μg/ml) and relative luciferase activities were determined. Cells were treated for 16 hours. We subjected extracts from HeLa cells treated with CP-31398 to western blot analysis. The p53 signals correlated with increasing CP-31398 concentrations, whereas the actin control signals did not (figure 5A ). These results are consistent with a classical response to genotoxic stress by compounds causing stabilization of p53 [ 16 ]. Figure 5 Western Blot analysis of HeLa cells treated with CP-31398 and daunorubicin. (A) HeLa cells were treated with increasing concentrations of CP-31398 and protein extracts were subjected to SDS-PAGE and subsequent detection with an anti-p53 antibody (DO-1). (B) HeLa cells were treated with the established p53 inducing agent daunorubicin. Protein extracts were subjected to SDS-PAGE and subsequent detection with an anti-p53 antibody (DO-1). Expression of actin is detected as a loading control in experiments 5A and B. We also measured changes in p53 levels in HeLa cells after treatment with increasing concentration of daunorubicin, a known anticancer agent that is highly cytotoxic by a number of proposed mechanisms – intercalation into DNA among them [ 17 ]. We found, as expected, that daunorubicin treatment led to a progressive stabilization of p53 in HeLa cells comparable to the response when cells were treated with CP-31398 (figure 5B ). Discussion We assessed the proposed p53 stabilizing action of CP-31398 in yeast cells and in human cells. CP-31398, a compound isolated in an antibody-based in vitro screen, was reported to stabilize the p53 DNA-binding core domain and to reactivate mutant p53 in vivo [ 7 ]. We were unable to detect any effect of CP-31398 on p53-dependent reporter gene activation by a mutant form of human p53 neither in human cells nor in yeast cells. In our hands, CP-31398 did not stabilize mutant p53 proteins so as to show differences in activation of p53-dependent reporter genes in yeast and in mammalian cells. In addition, concentrations that were shown to be effective in cell culture by Foster et al. [ 7 ] led to extensive cell death. Most importantly, such cell death was independent of p53 expression. The p53 protein expressed within yeast cells functions as a potent transcriptional activator. Reconstitution of transcriptional activation by p53 in a heterologous, yet cellular system such as a yeast cell should be suitable to assess DNA-binding and transcriptional activation activity regardless of posttranslational modifications and other influences that are inevitable when p53 is studied in the context of its regulatory network in mammalian cells. It has been proposed that such posttranslational modifications like acetylation and phosphorylation activate the latent DNA binding activity of p53 by allosteric mechanisms [ 18 ]. However, more recent in vivo and in vitro studies question whether DNA binding itself is regulated at all and suggest that induction of p53 activity primarily occurs at the level of increasing protein concentration within the nucleus [ 16 , 19 , 20 ]. The evident p53 activity in yeast cells, in which the proposed mammalian-specific p53 modifying enzymes are missing, seems to be more readily consistent with the conclusions of such studies. With our system in yeast, we should be able to detect stabilization of the p53 core domain as long as this leads to increased binding of p53 to its specific DNA recognition sequence and subsequent activation of reporter gene expression. Therefore, our yeast system provides a convenient means to screen compound libraries for identifying molecules that can reactivate mutant p53 proteins in a cellular environment. Thanks to the easy genetic malleability of yeast and the lack of endogenous p53-related pathways, cellular screens with this organism should allow not only identification of compounds that can permeate cellular membranes and be active in an intracellular environment but also rapid exclusion of molecules that are not specific for the chosen target. In contrast to the results obtained with the exogenous expression of wild type p53 in yeast cells or with the H1299 and Saos-2 human cells, we observed a strong increase in wild type p53-dependent reporter gene activation in HeLa cells. These cells showed no apparent cell death after treatment with CP-31398. Wang et al. [ 21 ] reported stabilization of wild type p53 and an increase in p53 levels in other cell lines. These observations are consistent with the results we obtained in HeLa cells. These authors also reported that ubiquitination and degradation of wild type p53 is blocked by CP-31398. This effect seems to be specific to mdm2-mediated p53 degradation since HPV (human papilloma virus) E6-mediated degradation of p53 was unaffected. We do not know why we do not see any stabilization of exogenous p53 in H1299 or Saos-2 cells, but it is possible that unspecific toxicity induced by CP-31398 masks the increasing p53-dependent reporter signal. While these results indicate that CP-31398 might stabilize wild type p53, they do not explain the mechanism. Direct interaction and stabilization of p53 is not excluded. However, other explanations seem plausible. Stabilization of the core domain structure by CP-31398 as proposed in the original article should presumably have no effect on p53 protein levels. But p53 levels increase after treatment with CP-31398. Such a response is in line with a classical stabilisation of p53 after genotoxic stress. In contrast, Wang et al. reported that no serine 15 or 20 phosphorylation was detected in their cells after treatment with CP-31398. Interaction with mdm2 was unaffected, but p53 degradation was nevertheless blocked [ 21 ]. Therefore, it remains unclear by which mechanism CP-31398 stabilizes p53; it seems unlikely that core domain stability and DNA binding are influenced by CP-31398 directly. It is interesting to note that CP-31398 can intercalate into DNA as reported by Rippin et al. [ 10 ]. This intercalation is probably toxic to the cell and likely induces a classical p53 response, similar to the known p53 inducer daunorubicin. Our results strongly suggest a classical p53 stabilization through reduced degradation due to genotoxic effects caused by CP-31398. In fact, wild type p53 levels changed quite dramatically in HeLa cells, which are resitant to the apoptotic effects of p53, whereas the other human cell lines did not survive the treatment, probably because they underwent apoptosis in response to CP-31398 [ 22 ]. In support to this interpretation, our control substance daunorubicin showed very similar and expected results as those obtained with CP-31398. Conclusions In contrast to the results reported by Foster et al. [ 7 ], we did not detect any stimulation of mutant p53 activity in vivo by CP-31398, a potential anti-cancer compound. Concentrations of CP-31398 that were reported to be active in the published work were highly toxic to human cells in our experiments. The results of our in vivo experiments are in agreement with the recently published biochemical analysis of CP-31398, which shows that this molecule does not bind p53 as previously claimed, but rather intercalates into DNA. Methods Yeast strains The yeast strain used in our experiments is a derivative of the S. cerevisiae strain JPY5 [ 23 ] ( MAT ura3-52 his3Δ200 leu2Δ1 trp1Δ63 lys2Δ385 ). The p53 responsive yeast strain was constructed by integration of the reporter construct described in the result section and in figure 1A into the HIS3 locus by homologous recombination. The integrating p21 reporter plasmid was linearized with AflII that cuts in the 3' untranslated region (3'UTR) of the S. cerevisiae HIS3 gene. Yeast growth and manipulations Yeast genetic techniques and media were as described in [ 24 ]. For selection of plasmids, dropout media containing all except the specified amino acids were used. Yeast transformation was performed by the lithium acetate procedure [ 25 ]. Recombinant plasmids All p53 forms tested in yeast were expressed from the vector pGAD424 (Clontech, Inc). Wild type p53 was subcloned from a mammalian expression vector with primers containing HinDIII restriction sites by polymerase chain reaction (PCR). The PCR product was introduced into the HinDIII sites of pGAD424, removing the GAL4 AD ORF from pGAD424. All the point mutant p53 variants were generated by assembled PCR with mismatched primer pairs and subsequent cloning into pGAD424 analogous to wild type p53. The yeast reporter plasmid was derived from pDE96 (yeast integrating plasmid, bi-directional HIS3 , lacZ ) [ 26 ] by introduction of a hybridised double stranded oligo containing the p53 responsive element from the p21 CIP1 promoter (p21_sense_SalI 5'-TCG AGC CGT CAG GAA CAT GTC CCA ACA TGT TGA GCT G-3' and p21_anti_XbaI 5'-CTA GCA GCT CAA CAT GTT GGG ACA TGT TCC TGA CGG C-3') into the XbaI and SalI sites of the vector backbone. The plasmid WWP-luc is described in [ 12 ]. The mammalian p53 expression plasmids were constructed by subcloning the HinDIII p53 fragments from the yeast expression vectors into the GAL4 expression plasmid pSCETV- GAL4 (1-93)RV, this resulted in p53 expression under the control of the CMV promoter. The mammalian GAL4 dependent reporter Gal5-luc contains five GAL4 responsive binding sites in front of the luciferase cassette [ 27 ]. Gal4-VP16 is described elsewhere [ 28 ]. Yeast β-galactosidase assay Yeast β-galactosidase assays in solution using permeabilized cells were performed as described in [ 24 ]. Activity was normalized to the number of cells assayed. Mammalian cell culture Cells were obtained from ATCC (American Type Culture Collection, Manassas, Virginia, USA) and cultured according to the recommendations of ATCC. Transient transfection and luciferase assays We used Polyfect ® transfection reagent (Qiagen, Inc) according to manufacturers recommendations for transfection of all cell lines. Cells for luciferase assays and western blotting were harvested by scraping 48 hours after transfection and subjected to three freeze thaw cycles in 100 mM potassium phosphate pH 7.8 1 mM dithiothreitol buffer. Supernatants were clarified by centrifugation (5 min, 13000 rpm) and resuspended in 100 μl extraction buffer. 10 μl of extract was mixed with 100 μl luciferase assay solution (Promega) and analyzed in a luminometer (EG&G Berthold Lumat LB 9507). β-galactosidase assays were performed according to standard methods using 50 μl of the extract and luciferase units were normalized according to β-galactosidase values. All measurements were performed from at least two independent transfections experiments. Western blot analysis and antibodies Protein extracts were prepared as described above. Proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and western blotting was performed according to standard procedures. Anti-p53 antibody DO-1 (Santa Cruz Biotechnology, Inc) reacts with an amino terminal epitope mapping between amino acid residues 11–25 of wild type and mutant p53. Anti-actin antibody (I-19; Santa Cruz Biotechnology, Inc) is an affinity purified goat polyclonal antibody raised against a peptide mapping to the carboxy terminus of human actin. Authors' contributions All experimental work was carried out by ST. AB conceived of the study and participated in its design and coordination. Both authors read and approved the final manuscript.

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539290

Antigenized antibodies expressing Vβ8.2 TCR peptides immunize against rat experimental allergic encephalomyelitis

Background Immunity against the T cell receptor (TCR) is considered to play a central role in the regulation of experimental allergic encephalomyelitis (EAE), a model system of autoimmune disease characterized by a restricted usage of TCR genes. Methods of specific vaccination against the TCR of pathogenetic T cells have included attenuated T cells and synthetic peptides from the sequence of the TCR. These approaches have led to the concept that anti-idiotypic immunity against antigenic sites of the TCR, which are a key regulatory element in this disease. Methods The present study in the Lewis rat used a conventional idiotypic immunization based on antigenized antibodies expressing selected peptide sequences of the Vβ8.2 TCR ( 93 ASSDSSNTE 101 and 39 DMGHGLRLIHYSYDVNSTEKG 59 ). Results The study demonstrates that vaccination with antigenized antibodies markedly attenuates, and in some instances, prevents clinical EAE induced with the encephalitogenic peptide 68 GSLPQKSQRSQDENPVVHF 88 in complete Freunds' adjuvant (CFA). Antigenized antibodies induced an anti-idiotypic response against the Vβ8.2 TCR, which was detected by ELISA and flowcytometry. No evidence was obtained of a T cell response against the corresponding Vβ8.2 TCR peptides. Conclusions The results indicate that antigenized antibodies expressing conformationally-constrained TCR peptides are a simple means to induce humoral anti-idiotypic immunity against the TCR and to vaccinate against EAE. The study also suggests the possibility to target idiotypic determinants of TCR borne on pathogenetic T cells to vaccinate against disease.

Introduction Experimental allergic encephalomyelitis (EAE) is an experimentally induced autoimmune disease mediated by T cells. It can be induced in susceptible animals either by immunization with myelin basic protein (MBP) or proteolipid protein PLP, or by immunization with synthetic peptides from the MBP sequence [ 1 ]. EAE can also be initiated by the passive transfer of encephalitogenic, MBP-specific T cell lines or clones [ 2 , 3 ]. In the Lewis rat, EAE is characterized by a self limiting, ascending, hind limb paralysis. Histologically, EAE is hallmarked by perivascular and submeningeal infiltration of inflammatory cells within the brain and spinal cord [ 4 ]. After recovery, animals become refractory to further induction of paralysis by immunization with MBP. Owing to similarities in clinical expression and histopathology, EAE has long been recognized as an animal model for multiple sclerosis, a demyelinating chronic inflammatory disease in humans of unknown origin. For this reason, studies on EAE are thought to elucidate aspects of the pathogenesis and indicate possible ways of immune intervention. EAE is mediated by MHC class II -restricted, MBP-specific CD4 + T lymphocytes bearing an antigen receptor (TCR) variable (V) regions belonging to a limited set of TCR V region gene families [ 5 , 6 ] and restricted Vα-Vβ gene combinations [ 7 ]. Several rational approaches have been used to prevent EAE, including passive transfer of monoclonal antibodies that interfere with the recognition of the MHC, TCR and MBP peptide complex [ 8 , 9 ], antibodies against CD4 [ 10 ] and T regulatory cells [ 11 - 14 ]. Active immunity against attenuated encephalitogenic T cells was shown to prevent the induction of disease [ 15 , 16 ] and vaccination with synthetic peptides of the complementarity-determining regions (CDR) of the TCR of ecephalitogenic T cells, confer resistance to EAE in the rat [ 17 - 20 ]. Together these facts indicated that T cells are crucial to the pathogenesis of EAE and, in converse, immunity to idiotypic determinants of the TCR of encephalitogenic T cells may be protective. Approaches to directly target the TCR of pathogenetic T cells are an attractive direction for therapy and immunointervention as well as an opportunity to further understand the immunological events involved in protection in vivo . However, limitations exist to methods available for TCR vaccination. Vaccination using attenuated encephalitogenic T cells requires that these are specifically expanded in vitro and can only be used in an autologous system. Synthetic peptides, albeit successful in several instances [ 17 - 20 ], offer no tri-dimensional conformation and may even yield to opposite effect, e.g ., worsening of disease [ 21 , 22 ]. Similarly, vaccination with single chain TCR was shown to either prevent or exacerbate EAE in mice [ 23 ]. In previous work from this laboratory we demonstrated the induction of anti-receptor immunity using immunoglobulins (Ig) expressing discrete peptide portions of human CD4 [ 24 ]. We refer to such Ig as antigenized antibodies, i.e ., Ig molecules in which foreign peptide sequences are conformationally-constrained and expressed in the complementority-determining region (CDR) loops [ 25 ]. Immunization with antigenized antibodies is an efficient method to focus the immune response against defined epitopes of foreign antigens. If CDR sequences of TCRs are functionally comparable to Ig idiotypes, antigenized antibodies provide a tool to induce anti-idiotypic responses against TCR. Here, we used antibodies antigenized with TCR sequences as vaccines to control disease. We engineered two antibodies encompassing in the CDR3 of the heavy (H) chain two synthetic peptides from the sequence of rat Vβ8.2 gene product, 39 DMGHGLRLIHYSYDVNSTEKG 59 (CDR2) and 93 ASSDSSNTE 101 (CDR3, VDJ junction), both reported to confer protection against EAE in the Lewis rat [ 17 - 20 ] when used as vaccines. The results show that vaccination with antigenized antibodies expressing sequences of encephalitogenic T cells induces anti-idiotypic immunity against the TCR and high level resistance against EAE. Material and Methods Animals Eight week old, weight-matched female Lewis rats were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed (three rats per cage) in the animal facility of the Universitiy of California, San Diego. Food and water were provided at libitum . Antigenized antibodies The peptide sequences 93 ASSDSSNTE 101 and 39 DMGHGLRLIHYSYDVNSTEKG 59 were engineered into the CDR3 loop of the murine V H 62 gene [ 26 ] according to our published methods [ 27 ]. The antigenized V H was then ligated in plasmid vector containing a human γ1 constant (C) region gene. Transfection of the plasmid DNA was performed on murine J558L cells, a H-chain defective variant of myeloma J558, carrying the rearrangement for a λ1 light (L) chain [ 28 ]. The resulting antigenized antibodies were termed γ1TCR-I and γ1TCR-II, respectively (Figure 1 ). Wild-type transfectoma antibodies γ1WT and γ2bWT [ 26 ] engineered to have the same C and V regions, but lacking the TCR peptides in the CDR3 of the H chain, served as controls. Transfected cells were incubated without selection for 24 hours and then selected in the presence of 1.2 mg/ml G418 (GIBCO). G418-resistant clones secreting high level of Ig were identified by enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP)-conjugated goat antibody to human Ig (Sigma) [ 29 ]. Cultures secreting 10–20 μg/ml were selected, expanded, and their supernatants precipitated by (NH 4 ) 2 SO 4 . Antibodies were purified by affinity chromatography on a Protein A-Sepharose column (Pharmacia-LKB, Alameda, CA) equilibrated with 3 M NaCl/1M glycine, pH 8.9. Elution was performed using glycine 0.1 M- HCl/0.5 M NaCl pH 2.8. The eluted fractions were neutralized using 1 M Tris-HCl, pH 8.0, and dialyzed against 0.15 M phosphate-buffered saline (PBS) pH 7.3. The purity of the antibodies was assessed by electrophoresis on a 10% Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel (PAGE). Figure 1 Schematic representation of the two V regions antigenized with TCR sequences. In each case the H chain of the antigenized antibody is formed of a murine V H 62 region in which the CDR3 has been engineered to express either 93 ASSDSSNTE 101 or 39 DMGHGLRLIHYSYDVNSTEKG 59 sequence between two Val-Pro (VP) doublets of the unique cloning site in the CDR3 loop of V H 62 . The complete H chain is the product of the fusion of the antigenized V H region with a human γ1C region. The light (L) chain (not shown) is the murine λ1 which is provided by the J558L host cell. (H chain not to scale). Synthetic peptides Synthetic peptide GSLPQKSQRSQDENPVVHF corresponding to amino acid residues 68–88 of guinea-pig MBP [ 30 ], DMGHGLRLIHYSYDVNSTEKG corresponding to amino acid residues 39–59 of rat Vβ8.2 (CDR2 peptide), ASSDSSNTE corresponding to amino acid residues 93–101 of rat Vβ8.2 (CDR3 peptide) rat [ 17 , 18 ], and the (NANP) 3 peptide of Plasmodium falciparum parasite [ 31 ] were all synthesized in the Peptide Synthesis Facility of the Universitiy of California, San Diego. After synthesis peptides were analyzed by HPLC for purity. Peptide KKSIQFHWKNSNQIKILGNQGSFLTKGPS corresponding to residues 21–49 of the extracellular domain of human CD4 was described previously [ 32 ]. Enzyme-linked immunosorbent assay (ELISA) Serum antibodies against antigenized antibodies and their control were determined by ELISA on 96-well polystyrene microtiter plates (Costar, Cambridge, MA) coated (5 μg/ml – 50 μl/well) with γ1TCR-I, γ1TCR-II, γ2bTCR-I proteins in 0.9% NaCl by drying at 37°C. The wells were blocked with a 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), and then incubated overnight at +4°C with individual rat sera diluted in PBS containing 1% BSA and 0.05% Tween 20 (PBSA). After washing, the bound antibodies were detected by adding peroxidase-conjugated goat antibodies to rat IgG (γ specific) (Biomeda, CA) at 1:500 dilution in PBSA for 1 hour at room temperature. After washing, the bound peroxidase was measured by adding o-phenylenendiamine (100 μl/well) and H 2 O 2 . After 30 minutes, the plates were read in a micro-plate reader (Vmax, Molecular Devices) at 492 nm. Tests were done in duplicate. Antibodies to TCR peptides were detected in ELISA on 96-well polystyrene microtiter plates coated (10 μg/ml) with the Vβ8.2 synthetic peptides 39 DMGHGLRLIHYSYDVNSTEKG 59 and 93 ASSDSSNTE 101 in 0.1M carbonate buffer, pH 9.6, by overnight incubation at +4 C. After blocking unreactive sites, sera (1:25 dilution in PBSA) were added to plates and incubated overnight at +4°C. The bound antibodies and reactive peroxidase were detected as detailed above. Ig reactive with synthetic peptide 21 KKSIQFHWKNSNQIKILGNQGSFLTKGPS 49 of human CD4 were determined on 96-well polystyrene microtiter plates coated (2.5 μg/ml) with peptide 21–49 in 0.9% NaCl by drying at 37 C as previously established [ 32 ]. Briefly, sera (1:400 dilution in PBSA) were incubated overnight at +4°C. After washing, the test was continued as specified above. Plates were read in a micro-plate reader (Vmax, Molecular Devices) at 492 nm. FACS analysis Autoantibodies reactive with the Vβ8.2 + TCR were sought by flowcytometry on the S23B1E11 T cell hybridoma [ 33 ], derived from the fusion of Vβ8.2 + CD4 T lymphoblasts specific for MBP with the murine TCR α/β - BW1100.129.237 thymoma cell line [ 33 ]. For FACS analysis the following procedure was utilized. 10 6 hybridoma T cells in 100 μl of RPMI-1640 containing 1% egg albumin, 0.01% NaN 3 and 10 mM Hepes, were incubated with rat sera (1:10 dilution) for 90 minutes at +4°C. Cells were washed three times with cold RPMI-1640 and subsequently incubated with a fluorescein-isotyocianate (FITC)-conjugated goat antibody (0.5 μg/10 6 cells) to rat Ig (H+L) (Caltag, So. San Francisco, CA) for 20 minutes at +4°C. After incubation, the cells were washed twice, resuspended in 1% paraformaldehyde, and analyzed in a FACS Scan (BD Biosciences). To stain for dead cells, 20 μl of propidium iodide in PBS were added to unfixed cells before FACS analysis. R-phycoerythrin conjugated mouse monoclonal antibody R78 (IgG1, k) specific for the rat Vβ8.2, the kind gift of Pharmingen (San Diego, CA), was used to control for the expression of the Vβ8.2 TCR on S23B1E11 hybridoma cells. In vitro proliferative response Poplyteal, inguinal and paraortic lymph nodes were removed from immunized animals at different times, dissociated and washed in RPMI-1640. Lymph node cells were plated in round-bottom 96-well plates at 2.5 × 10 5 cells/well in the presence of various (10–100 956;g/ml) amounts of antigen in 200 μl of RPMI containing 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM glutamine, 0.1 mM non-essential aminoacids, 1 mM sodium pyruvate and 0.5 μM 2-β mercaptoethanol. Cultures were incubated for 72 hours in a 10% CO 2 atmosphere. The evening before harvest 1 μCi/well of [ 3 H]-thymidine was added to each well. Cells were harvested onto glass fiber filters and counted on an automatic Beckman LS 6000IC β-counter. Vaccinations and immunizations schedule Animals were vaccinated with antigenized antibodies (100 μg/rat) in complete Freunds' adjuvant (CFA) divided equally between the posterior paws (25 μl each) and two points in the back subcutaneously. A booster injection (50 μg/rat) in incomplete Freunds' adjuvant (IFA) was given subcutaneously on day 21. EAE was induced on day 28 by immunization with MBP peptide 68 GSLPQKSQRSQDENPVVHF 88 (30 μg/rat) in the anterior paws (25 μl each) in CFA (H37RA 10 mg/ml). Control rats were similarly injected with transfectoma antibody γ1WT or γ2bWT. Rats inoculated with Freunds' adjuvant only served as additional control. Serum samples were collected from the retro-orbital sinus on day 0 before vaccination, day 21 before booster injection, day 28 before EAE induction, and day 50 after recovery from disease. Sera were stored at -20 C until use. Clinical evaluation of EAE EAE was monitored daily by two operators for clinical signs using the following scale: grade 0 = no appreciable symptoms; grade 1 = tail atony; grade 2 = paraparesis; grade 3 = paraplegia; grade 4 = paraplegia with forelimb weakness, moribund state. Typically symptoms of disease began to appear on day 11–13 from the injection of the encephalitogenic peptide. The Disease Index was calculated according to the formula: [(Maximum Score) × (Duration of Disease) × (Incidence)]. Statistical Methods Statistical analyses was performed using the Fisher's test. Results Vaccination with antigenized antibodies and effect on EAE Two antigenized antibodies were engineered to express the CDR3 93 ASSDSSNTE 101 and CDR2 39 DMGHGLRLIHYSYDVNSTEKG 59 sequences, and were termed γ1TCR-I and γ1TCR-II, respectively (Figure 1 ). Rats were vaccinated with an individual antigenized antibody and received a booster injection 21 days later. EAE was induced on day 28 by immunization with the encephalitogenic MBP peptide 68 GSLPQKSQRSQDENPVVHF 88 . As shown in Table 1 , vaccination with both γ1TCR-I and γ1TCR-II reduced disease severity. Rats immunized with γ1TCR-I (group I) had a disease index of 1.8. Within this group, two out of six rats (33%) did not develop disease, one had grade 1 and three had grade 2. None proceeded through grade 3 or 4. Rats immunized with γ1TCR-II (group II) had a disease index of 4.9. Within this group two out of ten rats (20%) did not develop the disease, two had grade 1, four had grade 2 and two had grade 3. In contrast, all fifteen control rats vaccinated with γ1WT or given CFA only (groups III and IV) developed EAE with a disease index ranging between 11.3 and 22.4. Unmanipulated rats immunized with the MBP peptide (group V) developed EAE with a disease index of 25.2. There was a direct correlation between the severity of the disease and its duration. In rats immunized with γ1TCR-I, the disease lasted on average for 2.5 days and in rats immunized with γ1TCR-II 3.8 days. In contrast, in all the other groups (groups III-V) the duration of the disease was significantly longer (6–7 days). Of note, although group III rats had an overall lower score than unmanipulated rats, they differed from rats in group I or group II by the above mentioned parameters and these difference were statistically significant (Table 1 ). CFA did not confer protection. Taken together, these data indicate that active immunity elicited with antigenized antibodies expressing rat Vβ8.2 TCR peptides was effective in markedly reducing the severity of EAE in the Lewis rat. Table 1 Vaccination against antigenized antibodies expressing TCR peptides protects from EAE Severity of Disease* Group No. Rats Immunogen Incidence Max Score (mean ± SD) Duration (mean ± SD) Disease Index I 6 γ1TCR-I 4/6 1.1 ± 0.9 a 2.5 ± 2.2 b 1.8 II 10 γ1TCR-II 8/10 1.6 ± 1.0 c 3.8 ± 2.3 d 4.9 III 10 γ1WT 10/10 2.2 ± 0.9 5.1 ± 1.0 11.3 IV 5 CFA 5/5 3.4 ± 0.9 6.6 ± 1.3 22.4 V 6 - 6/6 3.5 ± 0.5 7.2 ± 1.3 25.2 * EAE was scored according to incidence, severity and duration. Disease index was calculated as follows: Mean Maximum Score × Mean Duration Disease × Incidence. Significance: ( a ) Group I vs Group III p = 0.04 and Group I vs Group V p = 0.0002; ( b ) Group I vs Group III p = 0.009 and Group I vs Group V p = 0.0001; ( c ) Group II vs Group III p = 0.16 and Group II vs Group V p = 0.0005; ( d ) Group II vs Group III p = 0.12 and Group II vs Group V p = 0.001. Antibody responses after vaccination Antibodies in response to the immunogen were assessed by solid-phase ELISA at various times after immunization. As shown in Table 2 , antibody titers against the immunogen developed in each group (group I-III) irrespective of which antibody was used to detect the antibody response in sera. This suggests that the human constant region of the antigenized antibodies is immunogenic in the rat. Antibody titers increased after the booster immunization and after challenge with the encephalitogenic MBP peptide. Control rats (group IV-V) did not mount any antibody response. No reactivity was found on the 19 mer MBP peptide (GSLPQKSQRSQDENPVVHF) used as a control. Anti-TCR (anti-idiotypic) antibodies were tested using two approaches. In the first case, sera of immunized animals were tested on Vβ8.2 synthetic peptides by ELISA. A weak but distinct response was detected in both instances starting on day 21 or 28 (Figure 2 ). Sera from control animals did not react with TCR peptides. Together with the fact that these were tested at a 1:25 dilution it appears that the anti-idiotypic response is weak. In the second case, we tested anti-idiotypic antibodies for their reactivity with the TCR in its native configuration. This was done by flowcytometry using the Vβ8.2 + T cell hybridoma S23B1E11 as the cell substrate. Two out of six rats in group I had a bright cellular staining (Figure 3 ). Reactive antibodies were detectable on day 21, 28 and day 50. Rats immunized with γ1TCR-II (group II) as well control rats (group III-V) were negative. Interestingly, the two rats whose sera reacted with TCR by flowcytometry did not develop symptoms of EAE. Table 2 Detection of antibodies against γ1TCR-I and γ1TCR-II in vaccinated Lewis rats Days After Vaccination a Immunogen Rats (No.) Responders (No.) 0 21 28 50 γ1TCR-I 6 6/6 ≤2.3* 3.9 ± .2 4.2 ± .2 4.5 ± 0.2 γ1TCR-II 10 10/10 ≤2.3 3.7 ± 0.2 4.1 ± 0.1 4.5 ± 0.2 γ1WT 10 10/10 ≤2.3 3.2 ± 0.4 3.9 ± 0.2 4 ± 0.2 CFA 5 0/5 ≤2.3 ≤2.3 2.6 ≤2.3 - 6 0/6 - - ≤2.3 ≤2.3 b γ1TCR-I 6 6/6 ≤2.3 4 ± 0.2 4 ± 0.2 4.6 ± 0.3 γ1TCR-II 10 10/10 ≤2.3 4.1 ± 0.3 4.4 ± 0.3 4.7 ± 0.5 γ1WT 10 10/10 ≤2.3 3.3 ± 0.3 4 ± 0.2 4.2 ± 0.2 CFA 5 0/5 ≤2.3 ≤2.3 ≤2.3 ≤2.3 - 6 0/6 - - ≤2.3 ≤2.3 * Antibody titers are expressed in log10. Sera were tested on microtiter plates coated with each of the TCR antigenized antibody γ1TCR-I (panel a) or γ1TCR-II (pane b). End point dilutions were determined as the last serum dilution binding with an OD ≥ 0.200. Figure 2 Antibody response to TCR peptides following vaccination with antigenized antibody γ1TCR-I or γ1TCR-II 39 DMGHGLRLIHYSYDVNSTEKG 59 tested on the ASSDSSNTE (panel a) or (panel b). The number of rats in each group is that indicated in Table 1. Results are expressed as Log2 ± SD. Figure 3 Sera from rats vaccinated with γ1TCR-I bind Vβ8.2 + T cells by flowcytometry. Vβ8.2 + S23B1E11 T cell hybridoma were used as substrate. Sera were tested at 1:25 dilution. Bound antibodies were revealed using a FITC-conjugated goat antibody to rat Ig. Vaccination with a murine antigenized antibody To explore the importance of foreigness of the constant region on the immunogenicity of the Vβ8.2 peptides we engineered an antigenized antibody with a murine γ2b constant region. Homology search using the BLAST program of the NCBI gene bank indicated that the murine γ2b C region is 56.7% identical to the rat γ2b C region, with a homology of 71% between residues 106 and 333. Because significant protection was found in rats vaccinated with the antibody expressing the 93 ASSDSSNTE 101 peptide (γ1TCR-I), we engineered an antibody with the same V region (γ2bTCR-I). Rats vaccinated with γ2bTCR-I and subsequently immunized with MBP peptide, were protected only partially compared to rats vaccinated with γ1TCR-I (10.2 vs. 1.9) (Table 3 ). Notably, within the six rats immunized with γ2bTCR-I, two were grade ≤ 2 and four developed a grade 3 for an average of two days. On the other hand, three out of six rats immunized with control antibody γ2bWT proceeded through a grade 4 disease. Similarly, all five control rats (group III and IV) developed a grade 4 disease. Of note, although the severity of the disease in group I rats was less than in control group II, the difference was not statistically significant (Table 3 ). All rats developed antibodies to the respective immunogen. However, when compared with the total antibody titer of rats immunized with γ1TCR-I and γ1TCR-II the titers were on average lower at single time points (Table 4 ). All sera reacted with the synthetic peptide 93 ASSDSSNTE 101 starting from day 21 with a progressive increase over time (Figure 3 ). Table 3 Protection against EAE by vaccination with antigenized antibodies with a murine γ2b constant region Severity of Disease* Group No. Rats Immunogen Incidence Max Score (mean ± SD) Duration (mean ± SD) Disease Index I 6 γ2bTCR-I 6/6 2.5 ± 0.8 a 4.2 ± 0.4 b 10.5 II 6 γ2bWT 6/6 3.0 ± 1.3 5.7 ± 1.7 17.1 III 2 CFA 2/2 4 6 24 IV 3 - 3/3 4 7.3 ± 0.6 29.2 * EAE was scored according to incidence, severity and duration. Disease index was calculated as follows: Mean Maximum Score × Mean Duration of Disease × Incidence. Significance: ( a ) Group I vs Group II p = 0.394 and Group I vs Group IV p = 0.051; ( b ) Group I vs Group III p = 0.05 and Group I vs Group IV p = 0.026 . Table 4 Detection of antibodies against γ2bTCR-I in vaccinated Lewis rats Days After Vaccination Immunogen Rats (No.) Responders (No.) 0 21 28 50 γ2bTCR-I 6 6/6 ≤2.3 3.2 ± 0,2 3.7 ± 0.1 4 ± 0.3 γ2bWT 6 6/6 ≤2.3 3 ± 0.3 3.4 ± 0.3 3.6 ± 0.3 CFA 2 0/2 ≤2.3 ≤2.3 ≤2.3 ≤2.3 - 3 0/3 - - ≤2.3 ≤2.3 * Antibody titers are expressed in log10. Sera were tested on microtiter plates coated with γ2bTCR-I. End point dilutions were determined as the last serum dilution binding with an OD ≥ 0.200. Serum antibodies of vaccinated rats bind a synthetic peptide of human CD4 In the attempt to correlate the antibody response after vaccination with protection, the sera of vaccinated rats and their controls were tested on a synthetic peptide corresponding to amino acid residues 21–49 of the first extra-cellular domain of human CD4. This peptide binds Ig irrespective of antigen specificity and heavy chain isotype with an affinity of 10 -5 M (26). It also binds antigen:antibody complexes formed at molar equivalence with an affinity about 100 fold higher [ 31 ]. When the sera of vaccinated rats were assayed on plates coated with the synthetic peptide of human CD4, strong binding was observed by sera from all rats immunized with γ1TCR-I whereas sera from rats immunized with γ1TCR-II or γ1WT bound much less (Figure 5a ). Control sera of groups IV and V did not bind. Binding could be attributed either to a differential property of the two antigenized V regions or to differences in the immune response triggered by the V regions themselves. To distinguish between the two possibilities two experiments were performed. First, we assessed binding of γ1TCR-I and γ1TCR-II on the CD4 peptide. Both bound equally at saturating and non-saturating concentrations (data not shown). Second, we tested sera of rats immunized with γ2bTCR-I considering that, if the effect was due to the immune response against 93 ASSDSSNTE 101 , we would have found similar results. As shown (Figure 5b ), the sera of γ2bTCR-I vaccinated rats all bound to the CD4 peptide comparably to rats vaccinated with γ1TCR-I. This suggests that binding to the CD4 peptide may reflect differences in the type of V regions utilized by the antibodies generated in vivo in response to immunization with the TCR peptide 93 ASSDSSNTE 101 as compared with the TCR peptide 39 DMGHGLRLIHYSYDVNSTEKG 59 or the wild type V region. Further studies will be needed to clarify this issue. Figure 5 Sera from rats vaccinated with γ1TCR-I or γ2bTCR-I bind synthetic peptide 21–49 of human CD4. Proliferative response Spleen cells and draining lymph nodes of rats tested 15 or 30 days after the initial immunization were tested in a proliferative assay against the Vβ8.2 peptides. No proliferative response was detected (data not shown). Discussion In this report we demonstrate that the severity of EAE in the Lewis rat can be greatly attenuated, and in some instances completely prevented, by active immunization with antigenized antibodies expressing amino acid sequences of the rat Vβ8.2 gene product. Immunity against synthetic peptides of the TCR has been shown to be effective in preventing or reducing the severity of EAE in the rat [ 17 - 20 ], suggesting that autoimmunity against the TCR reacting with encephalitogenic sequences of MBP is key to immunoregulatory events. The control of pathogenetic T cells may involve both T cells and antibodies. Autoregulation via T cells in EAE is well established. Thus, spontaneous recovery from EAE is impaired by splenectomy or thymectomy [ 34 ] and EAE can be prevented by vaccination with "attenuated" pathogenic T cells [ 15 ]. Autoregulation in EAE may involve both CD4 + and CD8 + T cells [ 35 - 38 ] as well as suppression by cytolytic T-T interactions [ 39 ]. A prevailing idea has been that in the rat [ 40 ] and in the mouse [ 41 ] idiotypic determinants of the TCR may be autoimmunogenic and contribute to mechanisms of immune regulation leading to protection. On the other hand, at least in a few instances, monoclonal antibodies against these TcR Vβ region [ 9 , 42 , 43 ] or against TCR idiotype [ 44 ] have been shown to block or attenuate disease. Here we show that immunity against idiotopes of antibodies engineered to express TCR peptides is effective in generating anti-idiotypic immunity directed against rat Vβ8.2 TCR gene product. Importantly, this type of immunity protected from EAE. The new approach used herein to induce anti-TCR immunity is based on conventional idiotypic immunization in which antigenized antibodies mimic the immunogenic properties of soluble TCR functioning as a surrogate internal image [ 45 ] in much the same way as previously demonstrated for a non-self antigen [ 31 ]. The present approach is reminiscent of experiments in which induction of anti-idiotypic immunity against TCR with specificity for MHC was obtained by immunization with soluble alloantibodies of relevant specificity [ 46 , 47 ], or by immunization with autologous idiotype positive molecules that are shed from the cell surface in the serum [ 48 ]. Thus, antibodies purposely modified to express selected loops of the TCR obviate the necessity to purify the receptor, isolate idiotypic TCR molecules from the serum, or use antigen-specific T cell blasts. Antibodies reacting with TCR peptides were detected in every vaccinated rat indicating that immunization with antigenized antibodies is an efficient method to induce an anti-idiotypic response specific for a somatic receptor. The fact that only two out of sixteen vaccinated rats developed antibodies against the native receptor detectable by flowcytometry on Vβ8.2 + T cells suggests that cross-reactive anti-idiotypic antibodies may be very low titer. Alternatively, they may be adsorbed on T cells in vivo precluding their detection in the serum. The first possibility is consistent with the self nature of TCR peptides and a predicted paucity of self reactive clonotypes within the natural B cell repertoire. Interestingly, we noted that the anti-idiotypic response against a non-self peptide expressed in an antigenized antibody [ 31 ] is much greater than the one observed here against a self peptide. That only rats vaccinated with the antigenized antibody expressing the 93 ASSDSSNTE 101 sequence developed flowcytometry-reactive autoantibodies could reflect difference in conformation once the two peptides are embedded in the CDR3 loop of an antigenized antibody. For instance, 93 ASSDSSNTE 101 could be better surface exposed and more stably expressed 39 DMGHGLRLIHYSYDVNSTEKG 59 . A computer-assisted comparison of hydrophilicity profiles [ 49 ] of the 93 ASSDSSNTE 101 peptide in the parental. TCR Vβ8.2 gene product and in the antibody V region shows that in both instances the peptide is highly hydrophilic (Figure 5 ). On the other hand, the Vβ8.2 CDR2 region shows a highly hydrophilic profile alternating with large hydrophobic regions of poorly exposed amino acid residues, both in the parental TCR and in the antibody CDR (data not shown). Our data show that although the process of antibody antigenization allows one to conformationally-constrain and express discrete peptide sequences of somatic receptors, the induction of anti-receptor antibodies is not directly predictable. Previously, we demonstrated flowcytometry-reactive antibodies to human CD4 in a high proportion (75 %) of cases [ 24 ]. We conclude that the physical characteristics of a given receptor peptide (e.g., length, hydrophilicity, etc.) likely determine its ability to induce antibodies cross-reactive with the native receptor. Interestingly, rats immunized with the antigenized antibody expressing 93 ASSDSSNTE 101 but not 39 DMGHGLRLIHYSYDVNSTEKG 59 reacted immunologically with a synthetic peptide of human CD4 previously described to bind Ig [ 32 ]. Because the two antigenized antibodies reacted equally with the CD4 peptide and only differ by the composition of their CDR3, we suggest that binding to CD4 by anti- 93 ASSDSSNTE 101 serum antibodies underscores qualitative differences of the immune response between rats immunized with γ1TCR-I and 1TCR-II, respectively. Thus, it appears as if 93 ASSDSSNTE 101 induced a different immune response than 39 DMGHGLRLIHYSYDVNSTEKG 59 . Furthermore, since vaccination with γ1TCR-I also promoted greater protection from EAE, it is tempting to speculate that a component of the anti-idiotypic response against γ1TCR-I is associated with protection. In conclusion, three points have emerged from this study. First, antigenized antibodies expressing conformationally-constrained loops of the Vβ8.2 TCR can be used as vaccines in the prevention of EAE in the Lewis rat. Our new approach to generate anti-TCR immunity, confirms the relevance of anti-idiotypic regulation in controlling rat EAE [ 17 , 18 , 20 ]. Second, since a weak antibody anti-idiotypic response in the apparent lack of a cell proliferative response was associated with protection, it appears as if a humoral anti-TCR response is relevant to protection from disease. Although this contrasts the relevance of T cell immunity in the regulation of EAE in the rat, reports exist to support the idea that humoral immunity is also important [ 20 , 50 , 51 ]. EAE was shown to be prevented or attenuated by passive transfer of serum from rats recovering from EAE [ 52 ], or by passive transfer of monoclonal antibodies against these TCR Vβ region and its idiotypes [ 9 , 42 - 44 ]. However, whether anti-idiotypic antibodies against the TCR predispose to anergy, apoptosis or killing of pathogenetic T cells remains to be determined. Finally, our study indicates that antigenized antibodies can be used as vaccines in conditions where immunopathology and disease involve receptors on somatic cells, and anti-receptor immunity alone could prevent or mitigate a pathological condition. Competing interests The authors declare that they have no competing interests Figure 4 Antibody response to TCR peptide 93 ASSDSSNTE 101 following vaccination with antigenized antibody γ2bTCR-I. The number of rats in each group are not indicated in Table 3. Results are expressed as means of Log2 ± SD. Figure 6 Hydrophilicity profiles of TCR peptides-containing V regions. Hydrophilic profile of the rat Vβ8.2 TCR, amino acid residues 80–130, inclusive of the CDR3 sequence 93 ASSDSSNTE 101 . Figure 7 Hydrophilic profile of the mouse VH62, amino acid residues 80–125, engineered with the 93 ASSDSSNTE 101 peptide of the rat Vβ8.2 TCR-CDR3.

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524189

Analysis of Maxi-K alpha subunit splice variants in human myometrium

Background Large-conductance, calcium-activated potassium (Maxi-K) channels are implicated in the modulation of human uterine contractions and myometrial Ca 2 + homeostasis. However, the regulatory mechanism(s) governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset. Methods Myometrial biopsies were obtained at hysterectomy (non-pregnant, NP), and at Caesarean section, at elective (pregnant not-in-labour, PNL) and intrapartum (pregnant in-labour, PL) procedures. RNA was extracted from all biopsies and quantitative real-time RT-PCR was used to investigate for possible differential expression of the Maxi-K alpha subunit, and that of its splice variants, between these functionally-distinct myometrial tissue sets. Results RT-PCR analysis identified the presence of a 132 bp and an 87 bp spliced exon of the Maxi-K alpha subunit in all three myometrial tissue sets. Quantitative real-time PCR indicated a decrease in the expression of the Maxi-K alpha subunit with labour onset. While there was no change in the proportion of Maxi-K alpha subunits expressing the 87 bp spliced exon, the proportion of alpha subunits expressing the 132 bp spliced exon was significantly increased with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. An increased proportion of 132 bp exon-containing alpha subunit variants with labour onset is of interest, as channels expressing this spliced exon have decreased calcium and voltage sensitivities. Conclusions Our findings suggest that decreased Maxi-K alpha subunit mRNA expression in human myometrium at labour onset, coupled to an increased proportion of Maxi-K channels expressing the 132 bp spliced exon, may be linked to decreased Maxi-K channel calcium and voltage sensitivity, thereby promoting enhanced uterine activity at the time of labour.

Background The regulatory mechanisms for uterine smooth muscle contractility during human pregnancy and labour are poorly understood. Such information is essential to understanding the clinical problems associated with human parturition and particularly preterm or premature labour. It is clear however that the myometrium is transformed from a state of relative quiescence during pregnancy, to one of maximal contractile activity at the time of labour. It is also established that the state of contractility of uterine smooth muscle is intrinsically linked to cell membrane ion channel activity [ 1 , 2 ]. Potassium (K + ) channels are functionally important in the regulation of smooth muscle tone [ 3 ]. Among the diverse family of K + channels, large-conductance, calcium-activated K + (Maxi-K, also known as BK Ca ) channels are the predominant K + channels in myometrium, and thus have been implicated in the control of cellular excitability [ 4 ]. While evidence for an important role of Maxi-K channels is not particularly strong, it is thought that they play a pivotal role in the modulation of uterine contractility and myometrial calcium homeostasis. Pharmacological inhibition of Maxi-K channels, by the specific channel blocker iberiotoxin, increases contractile activity in human uterine tissue [ 5 ], whereas compounds that promote Maxi-K channel opening, such as NS1619, have a potent relaxant effect on pregnant human myometrium [ 6 ]. Structurally, Maxi-K channels are tetramers of a pore-forming α subunit of the slo gene family, and a regulatory β subunit [ 7 - 10 ]. The α subunit comprises 7 transmembrane regions (S0-S6) and 4 intracellular hydrophobic domains (S7-S10) [ 11 ]. The β subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology [ 12 ]. The α subunit is encoded by a single gene. However, it achieves molecular diversity by extensive alternative splicing of its gene transcript at several sites [ 7 , 13 - 15 ], which generates Maxi-K channel variants. There is a substantial body of evidence indicating that alternate splicing of the maxi-K transcript plays a major role in regulating potassium channel conductance [ 7 , 15 ]. These data include evidence for splice variation effecting calcium and voltage sensitivity, surface expression, and sensitivity to protein phosphorylation of the maxi-K channel [ 16 , 17 ]; [ 18 ]. Alternative splicing of the maxi-K channel α subunit is considered to be a molecular mechanism by which the channel is able to adjust and tune its response to a variety of regulatory and conductance requirements. Further evidence of the role of alternate splicing of the maxi-K transcript in altering maxi-K protein function in myometrium is provided by the finding of up-regulation of maxi-K splice variants known to alter channel current through alterations in calcium and voltage sensitivity in pregnant mouse myometrium [ 19 ]. What initiates alternative splicing of the α subunit transcript is incompletely understood, however there is evidence that expression of different alternatively spliced transcripts can be hormonally induced [ 20 , 21 ]. It appears that expression of different pore-forming α subunit isoforms, with associated regulatory β subunits, occurs in a tissue-specific manner, thereby providing functional specificity [ 22 ]. Maxi-K channels have been identified both in human non-pregnant [ 23 ] and pregnant [ 24 ] myometrium. For animal myometrial tissues, the data outlining Maxi-K α subunit mRNA expression in relation to labour are conflicting [ 19 , 25 , 26 ]. For human myometrium, it has more recently been reported that protein expression of both α and β subunits is down-regulated with labour onset [ 27 ]. Although multiple alternatively spliced exons of the Maxi-K α subunit have been identified [ 10 , 19 , 21 , 28 ], there is no information available to date pertaining to expression of α subunit splice variant mRNA transcripts in human myometrium during pregnancy or at the time of labour. Because phosphorylation sites can be introduced into the channel protein via alternatively spliced exons [ 19 ], alternative splicing may represent an important control mechanism regulating Maxi-K channel function during pregnancy and at labour. The aim of this study was to investigate the expression of alternatively spliced exons of the Maxi-K α subunit transcript in non-pregnant myometrium and in pregnant myometrium, prior to and after labour onset using quantitative real-time PCR. Methods Patient recruitment and tissue collection Patient recruitment took place in the Department of Obstetrics and Gynaecology, University College Hospital Galway (UCHG), Ireland, between October 2001 and August 2002. The study was approved by the Research Ethics Committee, UCHG, and recruitment was carried out by provision of information sheets and obtaining written informed consent. Biopsies of myometrium were excised from the midline of the upper lip of the uterine incision made at caesarean section, at elective (pregnant not-in-labour, PNL; n = 8) and intrapartum (pregnant in-labour, PL; n = 7) procedures. The mean age of the women was 33.3 years (range 26–42) of whom four were primagravida and eleven were multigravida. All women were delivered between 37 and 41 weeks gestation. There was no significant difference between those undergoing elective or emergency caesarean section in terms of age, parity or gestation. Women who had received prostaglandins or oxytocin were excluded from the study. Reasons for emergency section included breech presentation, previous caesarean section and abnormal foetal position. The criteria for inclusion in the intrapartum group were regular spontaneous uterine contractions, effacement of the cervix, and cervical dilatation >3 cm prior to caesarean section. Samples of non-pregnant myometrium (NP; n = 7) were excised from the body of the uterus of hysterectomy specimens from pre-menopausal women. The mean age of women undergoing hysterectomy was 42.5 years (range 34–48). Women with malignant conditions, and those receiving exogenous hormone therapy (e.g. progestagens), were excluded from the study. Immediately upon removal, tissue samples were rinsed in sterile saline, snap frozen in liquid nitrogen and stored at -80°C until RNA extraction. RNA preparation/Reverse Transcriptase-Polymerase Chain Reaction RNA was isolated from frozen tissue by homogenisation in TRIzol ® Reagent (Life Technologies, Paisley, UK) [ 29 ]. RNA concentration was determined by absorbance at A 260 . To eliminate any residual contaminating genomic DNA, all RNA samples were DNase-treated with the DNA-free™ DNA removal kit (Ambion, Huntingdon, Cambridgeshire, UK), as previously described [ 30 ]. RNA concentration was measured again by absorbance at A 260 , after removal of DNA, and adjusted to a final concentration of 500 ng/μL. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) was performed to check for mRNA expression of all potential spliced exons of the Maxi-K α subunit in non-pregnant and pregnant myometrium, prior to and after labour onset. Purified RNA samples were reverse transcribed using oligo (dT) 15 primer and 200 IU M-MLV reverse transcriptase (Promega, Madison, WI, USA), as previously described [ 30 ]. PCR amplification was performed with 20 pmol of each specific oligonucleotide primer pair (Table 1 ), and 1.25 IU Taq DNA Polymerase (Promega, Madison, WI, USA) as previously described [ 30 ]. Primer pairs were designed to flank predicted splice sites, allowing spliced exon expression in these different regions to be assessed. PCR products were separated by electrophoresis on a 1.5% agarose gel and visualised after ethidium bromide staining by UV illumination. Bands identified were purified by gel extraction using Qiagen Gel Extraction kit (Qiagen, West Sussex, UK), and sent for sequencing (MWG-Biotech Ltd., Milton-Keynes, UK). Table 1 Primer Pairs Primer name Primer sequence (5' – 3') Primer Tm (°C) Maxi 0F CGGAGGCAGCAGTCTTAG 58.2 Maxi 0R AAGAAAGTCACCATGGAGGAG 57.9 Maxi 1F CTCCTCCATGGTGACTTTCTT 57.9 Maxi 1R TTACAAGTGCACCGATGCTG 57.3 Maxi 2F GGAAACCGCAAGAAATAC 53.1 Maxi 2R ACCTCATGGAGAAGAGGTTG 57.3 Maxi 3F GGTCTGTCCTTCCCTACTGT 59.4 Maxi 3R CAAAGATGCAGACCACGACA 57.3 Maxi 4F GTGCCAGCAACTTTCATTAC 55.3 Maxi 4R TCAGGGTCATCATCATCGTC 57.3 Maxi 5F ACAGCATTTGCCGTCAGTG 56.7 Maxi 5R GGTCCGTCTGCTTATTTGCT 57.3 β-actin F CAACTCCATCATGAAGTGTGAC 55.8 β-actin R GCCATGCCAATCTCATCTTG 59.3 Splice variant-specific cDNA synthesis Splice variant-specific cDNAs were prepared for each RNA sample using reverse primers as shown in Table 2 . 3 μg of purified RNA (1 μg for β-actin) was reverse transcribed to cDNA for each amplicon of interest using 500 nmol/L specific reverse primer and 200 IU M-MLV reverse transcriptase (Promega, Madison, WI, USA), as previously described [ 30 ]. These cDNAs were stored at -20°C until required for real-time PCR. New forward primers were designed upstream of the spliced exon sequences, to ensure that product size was approximately 200 bp, the optimum product length for use with hydrolysis TaqMan probes. Table 2 Amplicon-specific primer and probe sequences Primer name Primer/Probe sequence (5' – 3') Primer/Probe Tm (°C) Conserved F TGCACAAAGAGGTATGTCATCAC 58.9 Conserved R GTTTGCTGTGGATGGGATGGA 59.8 Conserved Probe 6F * -CCCACTCGTCGCAGTCCTCCAGCAAGAAGA XT♣ 68.9 132 bp splice F ACGCTCAAGTACCTGTGGACCGT 64.2 132 bp splice R TGTGGTTCCAGTTGAGTCACCA 60.3 132 bp Probe 6F-CTCCAGGGTGGAGTGATTGGCTGTATGTT XTCAC 71.5 87 bp splice F CATCGCAAGTGATGCCAAAGAA 58.4 87 bp splice R TCAACTGGCTCGGTCACAAGC 61.8 87 bp Probe 6F-TTGCAGCTAGATCACGCTATTCCAAAGATCCA XT 68.9 β-actin F CAACTCCATCATGAAGTGTGAC 55.8 β-actin nested R GTCAAGAAAGGGTGTAACGCA 55.4 β-actin Probe 6F-TGGCACCCAGCACAATGAAGATCAAATCA XT 70.3 * 6F = FAM reporter dye; ♣ XT = TAMRA quencher dye Synthesis of cDNA standards Standards (1 × 10 9 to 1 × 10 4 cDNA copies, in 10-fold increments) were created for each spliced exon and conserved region amplicons, and for β-actin, to enable accurate quantitation of product-specific cDNA copy numbers. Amplicon-specific PCR products were generated by RT-PCR (as described above) using primers shown in Table 2 . Products were purified using Qiagen PCR purification kit (Qiagen, West Sussex, UK), quantified by absorbance at A 260 , and ligated into TA cloning vector pCR ® 2.1 (TA cloning ® kit, Invitrogen Ltd, Paisley, UK), according to manufacturers' instructions. Vector-ligated PCR products were transformed into One Shot ® TOP10 cells, which were plated on Luria-Bertani (LB) agar plates containing 50 μg/mL kanamycin antibiotic, and incubated overnight at 37°C. Plasmid templates containing inserts in the desired orientation to transcribe sense RNA, as determined by colony PCR, were linearized by HindIII digestion. 2 μL of digestion products were electrophoresed on 1% agarose gels and visualised to ensure complete plasmid linearisation. Sense cRNA transcripts were generated by in vitro transcription using the MAXIscript™ In vitro Transcription Kit (Ambion, Huntingdon, Cambridgeshire, UK). 1 μg of linearized plasmid DNA was in vitro transcribed in a final volume of 20 μL containing 0.5 mmol/L each of ATP, CTP, GTP, and UTP, 2 μL 10X Transcription buffer, and 2 μL T7 Enzyme mix for 1 hr at 37°C. After transcription, samples were treated with DNA-free™ DNA removal kit (Ambion, Huntingdon, Cambridgeshire, UK) to remove plasmid DNA. The supernatant, containing purified cRNA, was pipetted onto pre-hydrated NucAway™ spin columns (Ambion, Huntingdon, Cambridgeshire, UK) to remove free nucleotides from the transcription reaction and further purify the cRNA samples. These columns were centrifuged at 1200 g for 2 min. Eluted cRNA concentration was determined by absorbance at A 260 . Copy number/μL of cRNA was calculated according to the following formula, available from the Roche Lightcycler™ website: Once the total amount of cRNA copies/μL had been calculated, serial dilutions of cRNA standards were produced (from 1 × 10 9 cRNA copies/μL to 1 × 10 4 cRNA copies/μL, in 10-fold increments) for each product-specific cRNA molecule generated. Serially-diluted cRNA standards were reverse transcribed in a 20 μL final volume as described above, using transcript-specific reverse primers (Table 2 ), thereby generating product-specific cDNA standards. These cDNA standards were stored at -20°C until required for real-time PCR. Quantitative expression analysis using real-time PCR Real-time PCR amplification was performed on the Lightcycler™ instrument using the Lightcycler™ FastStart DNA Master Hybridization Probes kit (Roche Diagnostics, Mannheim, Germany). Hydrolysis TaqMan probes were synthesized for each amplicon to be quantified (TIB MolBiol Syntheselabour, Berlin, Germany), and are shown in Table 2 . Probes were designed with consideration taken for the design parameters outlined by Bustin [ 31 ]. Probes were resuspended in PCR-grade water to a working stock concentration of 4 μmol/L, and stored in the dark at 4°C. Prior to quantitative analysis, several titration experiments for cDNA, probe, primer, and MgCl 2 concentration were performed to determine optimum reaction conditions for amplification. The following master mix of the reaction components was prepared to the indicated end-concentration: 10.6 μL water, 2.4 μL MgCl 2 (3 mmol/L), 1.0 μL forward primer (0.5 μmol/L), 1.0 μL reverse primer (0.5 μmol/L), 1.0 μL specific probe (200 nmol/L) (see Table 2 ) and 2 μL Hybridization Master Mix. The master mix (18 μL) was aliquoted into Lightcycler™ glass capillaries (Roche Diagnostics) and 2 μL cDNA (samples and standards) was added to respective capillaries. Capillaries were centrifuged at 3000 rpm for 5 s, and loaded into the Lightcycler™ instrument. The experimental protocol used for TaqMan probe quantitative analysis consisted of two stages: initial denaturation (95°C for 10 mins), followed by 45 cycles of denaturation (95°C for 0 s) and annealing/extension (59–61°C for 50 s). The annealing temperature used in each experiment was dependent on the melting temperature of both the primers and probe involved. Fluorescence data were acquired at the end of each annealing/extension cycle. Data analysis was performed using Lightcycler™ Second Derivatives Method software. This method automatically determines the threshold cycle (C T ) values for each individual sample using a software algorithm, which allows initial mRNA concentration in each sample to be accurately quantified based on the standards used. Using this method removes user influence, as well as any influence of background fluorescence on the data. The fluorescence display mode used was F1/F2, which is the optimal setting for use with hydrolysis TaqMan probes. PCR products were isolated from capillaries after each program had finished and were visualised by electrophoresis on 1.5% agarose gels. Statistical analysis The SPSS computer software package was used for all statistical analyses (Statistical Package for the Social Sciences, v.10, SPSS Inc., Chicago, IL, USA). Multiple group comparisons were made using analysis of variance (ANOVA), which were followed by individual group comparisons using the Tukey HSD test, where appropriate. Results Analysis of expression of alternatively spliced exons of the Maxi-K α subunit in human myometrium RT-PCR analysis of the Maxi-K α subunit gene, using primers designed to flank predicted splice sites, produced a variety of bands in samples of non-pregnant (NP), pregnant not-in-labour (PNL) and pregnant in-labour (PL) myometrium (data not shown). From this analysis, only two potential alternatively spliced exon-containing PCR products were identified in the three tissue sets assayed (Figure 1 ). Sequence analysis of these bands confirmed the presence of two alternatively spliced exons, both of which had previously been identified in human myometrium. The first was a 132 bp spliced exon located in the S0-S1 linker region identified by Korovkina et al. [ 26 ], and the second was an 87 bp spliced exon located in the S8-S9 linker region identified by Wallner et al. [ 10 ]. PCR of reverse transcriptase negative controls (RT-) and a water control (no cDNA template) did not generate any products, confirming the absence of genomic DNA contamination (data not shown). Figure 1 Detection of alternatively spliced exon-containing RT-PCR products . Ethidium bromide stained agarose gels (2%) showing ( A ) 132 bp spliced exon-containing (437 bp) and exon-less (305 bp) PCR products and ( B ) 87 bp spliced exon-containing (622 bp) and exon-less (535 bp) PCR products, in non-pregnant (NP), pregnant not-in-labour (PNL), and pregnant in-labour (PL) myometrial tissues. M = 100 bp marker (Promega, US); M 2 = 2-log ladder (New England Biolabs Inc., UK). Quantitative analysis of alternatively spliced exon expression using real-time PCR Quantitative analysis of alternatively spliced exon expression was performed using sequence-specific hydrolysis TaqMan probes to analyse mRNA expression of these exons in non-pregnant myometrium and pregnant myometrium, prior to and after labour onset, as outlined. In order to correct for random errors from sources such as pipetting inaccuracies, separate real-time PCR reactions were performed in triplicate for each amplicon involved. Agarose gel electrophoresis, as well as sequencing analysis, confirmed the specificity of PCR products formed, yielding single product bands of the expected size (data not shown). Quantitative results for each amplicon were obtained by determination of the threshold cycle (C T ) values for each sample, as determined mathematically by the "Second Derivatives" method. Mean absolute cDNA copy number values for each probed amplicon involved, in each myometrial sample, were calculated and grouped per tissue set (i.e. NP, PNL, PL), as shown in Figure 2 . All data were normally distributed, as determined by Normality plots for each group (P > 0.05). Analysis of the expression of the housekeeping gene, β-actin, showed no significant differences between the three tissue sets assayed (P > 0.05). The 3' conserved region of the Maxi-K α subunit was analysed as a measure of overall α subunit expression. The results of this analysis indicated a decrease in expression of the α subunit transcript with labour onset (Figure 2A ). Although this did not reach statistical significance (P = 0.052), the observed decrease in α subunit mRNA expression at labour is in agreement with the decrease seen in α subunit protein levels at labour, reported recently [ 27 ]. Quantitative analysis of the expression of the 132 bp and 87 bp spliced exon transcripts indicated no significant differences in expression, in absolute terms, between NP, PNL, and PL tissues (P > 0.05)(Figure 2B ). Figure 2 Maxi-K α subunit mRNA expression analysed by quantitative real-time PCR . Results shown represent mean (± standard error of the mean, SEM) copy number values for ( A ) total Maxi-K α subunit (represented by the 3' conserved region), and β-actin, and ( B ) the 132 bp and 87 bp alternatively spliced exons of the Maxi-K α subunit. Copy number values were obtained based on product-specific serially-diluted cDNA standards, generated individually for each amplicon of interest. Tissue sets are indicated by striped columns (non-pregnant, NP), black columns (pregnant not-in-labour, PNL) and open columns (pregnant in-labour, PL). The results of expression analyses for the 87 bp and 132 bp spliced exons as a proportion of the total Maxi-K α subunit (i.e. the 87 bp and 132 bp variants) are demonstrated in Figure 3 . Analysis of mRNA expression of the 87 bp variant indicated no significant differences between the three tissue sets assayed (Figure 3A ). Expression of this variant mRNA accounted for only 1% of total Maxi-K α subunit expressed. However, the proportion of Maxi-K channels expressing the 132 bp spliced exon was significantly increased with labour onset (PL), compared to both non-pregnant (NP)(P < 0.05) and pregnant not-in-labour myometrial tissues (PNL) (P < 0.01)(Figure 3B ). Duplicate RT and quantitative PCR analysis confirmed these data. The increase in proportion of this 132 bp variant could be equated to approximately 1.7 fold, from 9% to 15% of total α subunit mRNA expressed. Figure 3 Expression of 132 bp and 87 bp variant mRNAs of the Maxi-K α subunit . The histograms show the mean (± standard error of the mean, SEM) of the ratios of spliced exon to total maxi-K α subunit mRNA for both ( A ) the 87 bp and ( B ) the 132 bp variants in NP, PNL and PL tissues. Results indicate significantly higher expression of the 132 bp exon as a proportion of the total α subunit with labour onset, compared to non-pregnant and pregnant not-in-labour samples. There is no change in expression of the 87 bp variant between the three tissue sets. Tissue sets are indicated by striped columns (non-pregnant, NP), black columns (pregnant not-in-labour, PNL) and open columns (pregnant in-labour, PL). *P < 0.05 versus PL; + P < 0.01 versus PL. Discussion In this study RT-PCR analyses were performed to identify mRNA expression of the Maxi-K α subunit, and alternatively spliced exons of this subunit, in human myometrium in its non-pregnant state, and at term pregnancy, prior to and after labour onset. This was followed by quantitative real-time PCR, which was performed to determine the overall pattern of expression of the Maxi-K α subunit as well as expression of alternatively spliced exons of this subunit identified in these tissue sets. Our findings indicate a trend towards a decrease in α subunit mRNA levels with human labour onset. In order to maintain the uterus in a quiescent state during pregnancy, K + channels provide a potent repolarizing current through the efflux of K + ions, thereby dampening cell excitability and promoting cell relaxation [ 32 ]. Previous studies on murine and rodent myometrium have reported conflicting results for Maxi-K α subunit mRNA and protein expression during pregnancy and with labour onset. Song et al. [ 26 ] identified a decrease in Maxi-K α subunit protein levels in rats at term pregnancy, whereas Benkusky et al. [ 19 ] indicated an increase in protein levels of this subunit in mouse term myometrium. However, a recent report has outlined significant down-regulation in the protein levels of both α- and β-subunits of the Maxi-K channel in human myometrium at labour onset, suggesting that the loss of Ca 2+ and voltage sensitivity is at least partly due to decreased levels of the Maxi-K channel [ 27 ]. Our findings are in agreement with this report, with the highest levels of mRNA expression in the PNL group, and decreased mRNA expression of the Maxi-K α subunit with labour onset (Figure 2 ). Although the decrease in mRNA expression between PNL and PL tissues was ~50%, it was found not to be statistically significant (P = 0.052). A reduction in expression of the Maxi-K α subunit could allow for enhanced myometrial contractility, as reduced α subunit expression would permit an increase in intracellular Ca 2+ levels without the activation of an opposing K + conductance [ 32 ]. Maxi-K channels derive their molecular diversity by alternative splicing of their α subunit transcript at several key sites, which generate channel variants with distinct phenotypes [ 7 , 15 , 16 ]. Previous studies provide evidence that alternate splicing effects calcium and voltage sensitivity of the maxi-K channel and thus channel function in myometrium [ 16 ], surface expression [ 17 ], and sensitivity to protein phosphorylation of the maxi-K channel [ 18 ]. Further direct evidence for the role of alternate splicing of the maxi-K transcript in altering maxi-K protein function in myometrium is provided by the finding of up-regulation of maxi-K splice variants known to alter channel current through alterations in calcium and voltage sensitivity in pregnant mouse myometrium [ 19 ]. Our results from RT-PCR analysis indicate the presence of only two spliced exons, both of which had been identified previously, in human myometrium. The 132 bp exon, previously identified by Korovkina et al. [ 28 ], encodes a 44 amino acid peptide that is inserted into the first intracellular loop of the Maxi-K α subunit, and contains four potential consensus sites for post-translational modification. The 87 bp exon, isolated by Wallner et al. [ 10 ], encodes a 29 amino acid peptide that is introduced into the loop region between hydrophobic regions S8 and S9 of the α subunit protein. Protein sequence analysis of this exon using PROSITE revealed a potential cAMP-/cGMP-protein kinase phosphorylation site (KKeT). Quantitative real-time PCR was used to determine whether identified spliced exons displayed altered expression in pregnancy and/or with labour onset. The use of hydrolysis TaqMan probes provided high reaction specificity and sensitivity, and allowed for highly accurate quantification of target sequences. Our results indicate that there was no significant change in expression of the 87 bp spliced exon, in absolute terms, in non-pregnant (NP), pregnant not-in-labour (PNL) and pregnant in-labour (PL) myometrium. Furthermore, analysis of the expression of this spliced exon as a proportion of the total α subunit expressed (i.e. the 87 bp exon-containing α subunit splice variant) also showed no differences between the three tissue sets assayed. The proportion of Maxi-K α subunits expressing this variant was very low, accounting for only ~1% of Maxi-K channels expressed in the three tissues sets. Little is currently known about the physiological effects of expression of this variant of the Maxi-K channel. However, as described above, it contains a consensus sequence for protein kinase phosphorylation; therefore, it may have important consequences for post-translational modification of channel function. Also, the region into which this exon is inserted, between hydrophobic regions S8 and S9 of the α subunit protein, is thought to be involved in determining the Ca 2+ sensitivity of the Maxi-K channel [ 16 ]. In contrast, while there was no significant change in the mRNA levels of the 132 bp spliced exon in absolute terms between the three tissue sets, there was a significant increase (~1.7 fold) in the proportion of Maxi-K α subunits expressing this exon with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. Messenger RNA for this 132 bp variant was expressed at much higher levels in comparison to the 87 bp variant, accounting for 9% of total Maxi-K channels in NP and PNL tissues, increasing to 15% at labour onset. An increased proportion of myometrial Maxi-K α subunits expressing the 132 bp spliced exon with labour onset in the human is of interest, as the presence of this exon has been shown to decrease both the Ca 2+ and voltage sensitivities of the Maxi-K channel [ 28 ]. Thus, our findings may provide an additional explanation for the observation of decreased Ca 2+ and voltage sensitivities of Maxi-K channels after labour onset [ 24 ] to reduced expression levels of the α- and β-subunits reported recently [ 27 ]. The exact mechanism by which the presence of this exon causes decreased sensitivity of the Maxi-K channel is unknown. It is possible that post-translational modifications at the four consensus sites present in the exon, perhaps in combination with conformational changes in the intracellular loop due to its increased length, may bring about the observed changes. Determination of the precise mechanism by which the 132 bp exon causes decreased channel sensitivity is an interesting question and is the subject of ongoing investigations in our laboratory. Conclusions Following the onset of labour, the putative disabling of the link between Ca 2+ and Maxi-K channel activation would permit Ca 2+ levels in the cell to rise without the activation of an opposing K + conductance, hence increasing the availability of Ca 2+ for myometrial contraction [ 30 ]. Our findings here suggest that, in human myometrium at labour onset, in addition to decreased Maxi-K α subunit mRNA expression, the increased proportion of Maxi-K α subunits containing the 132 bp spliced exon that are insensitive to Ca 2+ and voltage levels, is responsible for enhanced uterine activity at the time of labour. Whether these variants are assembled as homo- or hetero-tetramers at the plasma membrane remains to be determined. Further investigations are required to assess factors such as the role of β-subunit attachment and modulation of the calcium bowl, and their input to regulation of Maxi-K channels during human pregnancy and labour. This study provides the first quantitative analysis of Maxi-K α subunit mRNA expression in human myometrium, and also highlights alternative exon splicing as a potentially important control mechanism by which myometrial Maxi-K channels may be modulated to suit their functional requirements during these physiological processes. Authors' Contributions MC designed the quantitative RT-PCR techniques and carried out all experimental work. JJM recruited patients, organised the collection of tissues, and conceived of the study. TJS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.

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Anti-Plasmodium activity of ceramide analogs

Background Sphingolipids are key molecules regulating many essential functions in eukaryotic cells and ceramide plays a central role in sphingolipid metabolism. A sphingolipid metabolism occurs in the intraerythrocytic stages of Plasmodium falciparum and is associated with essential biological processes. It constitutes an attractive and potential target for the development of new antimalarial drugs. Methods The anti- Plasmodium activity of a series of ceramide analogs containing different linkages (amide, methylene or thiourea linkages) between the fatty acid part of ceramide and the sphingoid core was investigated in culture and compared to the sphingolipid analog PPMP (d,1-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). This analog is known to inhibit the parasite sphingomyelin synthase activity and block parasite development by preventing the formation of the tubovesicular network that extends from the parasitophorous vacuole to the red cell membrane and delivers essential extracellular nutrients to the parasite. Results Analogs containing methylene linkage showed a considerably higher anti- Plasmodium activity (IC 50 in the low nanomolar range) than PPMP and their counterparts with a natural amide linkage (IC 50 in the micromolar range). The methylene analogs blocked irreversibly P. falciparum development leading to parasite eradication in contrast to PPMP whose effect is cytostatic. A high sensitivity of action towards the parasite was observed when compared to their effect on the human MRC-5 cell growth. The toxicity towards parasites did not correlate with the inhibition by methylene analogs of the parasite sphingomyelin synthase activity and the tubovesicular network formation, indicating that this enzyme is not their primary target. Conclusions It has been shown that ceramide analogs were potent inhibitors of P. falciparum growth in culture. Interestingly, the nature of the linkage between the fatty acid part and the sphingoid core considerably influences the antiplasmodial activity and the selectivity of analogs when compared to their cytotoxicity on mammalian cells. By comparison with their inhibitory effect on cancer cell growth, the ceramide analogs might inhibit P. falciparum growth through modulation of the endogenous ceramide level.

Background Sphingolipids are essential components of eukaryotic cell membranes, predominantly found in the outer leaflet. Sphingosine and ceramide (Figure 1 ) are the two simplest molecules structurally, which belong to the sphingolipid family. Sphingosine represents the sphingoid backbone, and ceramide has a fatty acid linked in a amide bond to sphingosine. Sphingolipid species have two types of functional groups linked to the 1-position, i.e. sphingomyelin (SPM) (Figure 1 ) having a phosphorylcholine group, and a variety of glycolipids having either glucose, galactose, galactosyl-sulfate or oligo-glycosides linked to the sphingosine moiety of ceramide. Figure 1 structures of sphingolipids and analogs Until recently, sphingolipids were primarily considered to be structural components of membranes. However, data accumulated during the last decade have expanded the view of their biological functions. They are now also considered to be key molecules which regulate many functions essential to eukaryotic cells [ 1 - 5 ]. They are involved, for example, in the regulation of membrane fluidity and are part of discrete membrane microdomains or rafts implicated in signalling and trafficking in cells [ 4 , 6 - 8 ]. Interest in sphingolipids was strengthened by an increasing body of evidence demonstrating their role as secondary messengers for intracellular signal transduction pathways that regulate many cellular processes. For example, ceramide accumulates in response to several different inducers such as cytokines, cytotoxic agents or to stressful conditions, which lead to cell cycle arrest or to apoptosis [ 9 ]. Sphingosine is a protein kinase C inhibitor [ 10 ] that inhibits growth or stimulates proliferation, depending upon the cell type [ 11 , 12 ]. Ceramide plays a central role in sphingolipid metabolism [ 13 ]. It can be converted into SPM through transfer of the choline phosphate group from phosphatidylcholine or serves as a precursor for complex sphingolipids (cerebrosides which possess sugar residues and gangliosides which contain sialic acid residues in addition to the carbohydrate units). Moreover, ceramide can be phosphorylated by a distinct kinase and can also be produced by enzymatic hydrolysis of complex sphingolipids. In turn, ceramide can be hydrolyzed to sphingosine and fatty acid by ceramidases. In contrast to yeast and mammalian cells, the current understanding of sphingolipid metabolism and the biological role of sphingolipids in the development of Plasmodium falciparum , the causative agent of malaria, is still limited. Gerold et al. [ 14 ] provided evidence that de-novo synthesis of sphingolipids occurs in the intraerythrocytic stages of the human malaria parasite P. falciparum and can be inhibited by the well established inhibitors of de-novo ceramide biosynthesis, fumonisin B1, cyclo-serine and myriocin [ 15 , 16 ]. However, these compounds are weak inhibitors of parasite growth. Evidence was provided that another pathway for the synthesis of glycosylated sphingolipids exists in P. falciparum [ 14 , 17 ]. The importance of sphingolipid metabolism for parasite development was demonstrated by Haldar's work showing that: ( i ) The parasite contains two distinct forms of SPM synthase, one sensitive to sphingolipid analogs, d,1-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) or d,1-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) (Figure 1 ), known to inhibit the synthesis of glucosylceramide in mammalian cells [ 18 ], and the second insensitive to them [ 19 ]; ( ii ) These analogs blocked the parasite proliferation in culture by preventing the formation of the tubovesicular network (TVN) that extends from the parasitophorous vacuole to the red cell membrane and delivers essential extracellular nutrients to the parasite [ 20 - 22 ]. Neutral magnesium-dependent sphingomyelinase activity was also identified in P. falciparum [ 23 - 25 ], indicating that a sphingomyelin cycle (ceramide-SPM conversion) exists in Plasmodium . Recently, an increase in the intracellular ceramide content and an activation of parasite sphingomyelinase(s) were found to be associated with the parasite death process as induced by artemisinine and mefloquine [ 26 ]. Given the importance of sphingolipids in many cellular functions and the central role of ceramide in sphingolipid metabolism, the anti- Plasmodium activity of non-natural analogs of ceramides was investigated on the intraerythrocytic development of P. falciparum . Interestingly, a series of analogs containing a methylene (CH 2 -NH) linkage between the fatty acid and the sphingoid-analog core showed considerably higher anti- Plasmodium activity than their counterparts with a natural amide (CO-NH) linkage or than PPMP. The methylene analogs irreversibly blocked parasite development in contrast to PPMP whose effect is cytostatic. Their efficiency in inhibiting parasite growth did not correlate with their potential to inhibit parasite SPM synthase activity, indicating that SPM synthase is not their primary target. Possible mechanisms of action are discussed. Methods Materials D,1-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol-HCl (D,1-threo-PPMP) was purchased from Matreya (Pleasant Gap, PA). 6-((N- (7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino) hexanoyl sphingosine (NBD-C 6 -ceramide) and N- (4,4-difluoro-5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl) sphingosyl phosphocholine (BODIPY-FL-C 5 -ceramide) were obtained from Molecular Probes, Inc. (Eugene, OR). The compounds of Figure 3 and Figure 4 were synthesized according to the procedure described by Dagan et al [ 27 ], using specific starting materials for each analog. The compounds of Figure 2 were synthesized by linking specific fatty acids to the amino group of substituted 1,3-dihydroxy-2-aminophenyl derivatives. The full description of the synthesis of each specific analog will be described in a separate publication. Figure 2 Anti- P. falciparum activity of ceramide analogs having an amide linkage (series A). Figure 3 Anti- P. falciparum activity of ceramide analogs having a methylene or a thiourea linkage (series B). Figure 4 Anti- P. falciparum activity of selected derivatives P. falciparum culture and synchronization P. falciparum strains (FcB1/Colombia, K1/Thailand, F32/Tanzania, W2/Indochina) were maintained in continuous culture on human erythrocytes in RPMI medium containing 7% (v/v) heat-inactivated human serum under an atmosphere of 3% CO 2 , 6% O 2 , 91% N 2 , at 37°C, as described by Trager and Jensen [ 28 ]. Parasite synchronization was performed successively by treatment with 5% (w/v) sorbitol and by concentration in gelatin solution as previously described [ 29 ]. Anti- Plasmodium activity Drug susceptibility assays were performed using a modification of the semi automated microdilution technique of Desjardins et al . [ 30 ]. Stock solutions of test compounds were prepared in DMSO. Drug solutions were serially diluted twofold with 100 μl culture medium in 96-well plates. Asynchronous parasite cultures (100 μl, 1 % parasitemia and 1 % final hematocrite) were added to each well and incubated for 24 hours at 37°C prior to the addition of 0.5 μCi of [ 3 H] hypoxanthine (Amersham, France, 1 to 5 Ci.mmol/ml) per well. After a further incubation of 24 hour, plates were frozen and thawed. Cell lysates were then collected onto glass-filter papers and counted in a liquid scintillation spectrometer. The growth inhibition for each drug concentration was determined by comparison of the radioactivity incorporated in the treated culture with that in the control culture (having the same final % of DMSO) maintained on the same plate. The concentration causing 50% growth inhibition (IC 50 ) and 90% growth (IC 90 ) were obtained from the drug concentration-response curve and the results were expressed as the means ± the standard deviations determined from several independent experiments. The DMSO concentration never exceeded 0.1% (v/v) and did not inhibit the parasite growth. Cytotoxicity test upon human embryonic cells A human diploid embryonic lung cell line (MRC-5, Bio-Whittaker 72211D) was used to assess the cytotoxic effects towards eukaryotic host cells. MRC-5 cells were seeded at 5,000 cells per well in 100 μl. After 24 hours, the cells were washed and two-fold dilutions of the drug were added in 200 μl standard culture medium (RPMI medium + 5% fetal calf serum) and maintained for five days under 5% CO 2 atmosphere. The final DMSO concentration in the culture remained below 0.1%. Untreated cultures were included as controls. The cytotoxicity was determined using the colorimetric MTT assay according to the manufacturer's recommendations (Cell proliferation kit I, Roche Applied Science, France) and scored as a percentage of reduction in absorption at 540 nm of treated cultures versus untreated control cultures. IC 50 values were obtained from the drug concentration-response curve. The results were expressed as the mean ± the standard deviations determined from several independent experiments. The index of selectivity was defined as the ratio of the IC 50 value on MRC-5 to that of P. falciparum . Parasite stage-specific inhibitory effects and reversibility Synchronized cultures (1–2% parasitemia) at the ring stage (0–10 hours old parasites), the trophozoite stage (25–35 hours old parasites) and the schizonte stage (40–48 hours old parasites) were maintained in the presence of drug concentrations in the vicinity of IC 50 values. Aliquots were removed at the indicated times, washed three times with culture medium and maintained in culture in the absence or in the presence of a given drug. Parasite morphology was determined on Giemsa-stained smears defined according to the following criteria: the ring stage, when parasites exhibited a peripheral cytoplasm stained by Giemsa and a unstained intraparasitic vacuole; the trophozoite stage, when parasites showed a fully stained cytoplasm, haemozoin crystals and one nucleus; the schizont stage, when parasites presented several distinctive nuclei. Parasitaemias were determined by counting 3,000 cells for each sample. Controls consisted of parasites incubated with DMSO instead of drugs processed in the same way. Sphingomyelin synthase activity assays SPM synthase activity was measured as described by Haldar et al. [ 31 ]. Briefly, assays were performed on P. falciparum cultures at the trophozoite stage (20–30 h old parasites). 400 μl of culture (1 × 10 8 parasites) were incubated for 60 min at 37°C with 10 μM NBD-C 6 -ceramide and 0 to 500 μM PPMP or AD2646. Cells were then lysed by freezing and thawing of the culture. Lipids were extracted by a modification of the method of Bligh and Dyer [ 32 ]. To each sample, three volumes of a CH 3 OH/CHCl 3 mixture (1:2, v:v) were added and the mixture vortexed for one min. Organic and aqueous phases were separated by centrifugation (12,000 × g, five min) and the organic phase was dried. Lipids were dissolved in 15 μl ethanol and analysed by thin layer chromatography on HPTLC plates (Silica gel 60 F 254 , Merck, Darmstadt, Germany) in CH 3 OH/CH 3 Cl 3 /NH 4 OH (75:25:4, v:v:v). For qualitative analyses, the fluorescent lipids were detected under UV and for quantitative analyses, the fluorescent lipid spots were scraped, eluted in one ml methanol and quantified at an excitation of 470 nm and an emission of 530 nm in a spectrofluorometer. The percentage of SPM synthase activity for each drug concentration was determined by comparison of the fluorescence quantified in the analog-treated culture with that in the control culture (without drug). Labelling of infected red blood cells and fluorescence microscopy Infected erythrocytes treated with or without ceramide analogs were incubated for 30 min, at 37°C, in culture medium containing 10 μM BODIPY-FL-C 5 -ceramide, washed three times with culture medium without serum and fixed overnight, at 4°C, in 3.7% formaldehyde/0.05% glutaraldehyde. Cells were mounted on poly-L-lysine coated slides and viewed using a Nikon Eclipse TE 300 DV inverted microscope with an 100X oil objective mounted on a piezzo electric device using appropriate fluorescence emission filters. Image acquisition (z-series) was performed with a back illuminated cooled detector (CCD EEV: NTE/CCD-1024-EB, Roper Scientific, France) using a 0.2 μm step. Data acquisition and image deconvolution process were performed with Metamorph software (Universal Imaging Corporation, Roper Scientific, France). The images presented correspond to the maximum intensity projection of the deconvoluted z-series. Results and Discussion Anti-Plasmodium activity of non-natural ceramide analogs Non-natural analogs of ceramides were synthesized comprising two functional groups [ 27 ] : 1) A phenyl group substituted on carbon 3 of a sphingoid-like backbone; with the phenyl group replacing the sphingosine acyl chain [ 33 , 34 ] to which were linked nitro or amine groups, or carbon chains of varying lengths; and 2) a fatty acid with an amide (CO-NH) linkage (series A, Figure 2 ), a methylene (CH 2 -NH) or a thiourea (CS-NH) linkages (series B, Figure 3 ) on carbon 2. Analogs in which the alkyl group replaces the amide were investigated because the carbonyl group of ceramide was shown not to be necessary for triggering apoptosis in mammalian cells. In fact, replacement of the carbonyl group of ceramide by a methylene group substantially reduced the time required for cell death [ 35 ]. Only D / L - threo enantiomers were investigated on P. falciparum since reports demonstrated that D / L-erythro enantiomers of ceramide analogs were less efficient in inhibiting glucosylceramide synthase in mammalian cells [ 18 ] and did not inhibit SPM synthase activity in P. falciparum [ 19 ]. Figure 2 and Figure 3 show the IC 50 values obtained for the different compounds on the development of the chloroquine-resistant strain FcB1 of P. falciparum in culture (IC 50 value for chloroquine = 115 ± 25 nM, n = 3). Interestingly, the nature of the linkage considerably influences the anti- Plasmodium activity. Analogs with amide linkage were found to inhibit parasite growth with IC 50 values in the micromolar range (Figure 2 ). Best IC 50 values were similar to that obtained with the ceramide-related compound PPMP (IC 50 = 9.0 ± 1.7 μM, n = 3). However, this IC 50 value for PPMP differed from the previously reported value (IC 50 = 0.85 μM) [ 19 ]. The discrepancy may be due to drug susceptibility assay conditions which were performed on synchronized cultures at the ring stage for Lauer et al. [ 19 ] and on asynchronous cultures in the present study. Analogs with methylene linkages were more efficient than the amide analogs in killing parasites with IC 50 values in the nanomolar range (Figure 3 ). For the D-threo nitro phenyl analogs of series A, no particular increase of the inhibitory activity was observed with the increase of the N -acyl chain length (IC 50 values ranging from 10.8 to 40.4 μM, Figure 2 ). For the series B, best activities were observed for N -alkyl chain length of 12–16 carbons (IC 50 values ranging from 17 to 42 nM for the series B, Figure 3 ). In both series, substitution of the nitrophenyl group by an aminophenyl group instead of nitro group decreased the anti- Plasmodium activity significantly (compare compounds AD2495 and AD2623 of series A, Figure 2 ; and compounds AD2646 and AD2672 of series B, Figure 3 ). Increase of the analog hydrophobicity by substitution of the nitro group of the phenyl ring by alkyl chains seems to decrease the anti- Plasmodium activity of compounds of both series (compare compounds AD2583 and AD2603-7, Figure 2 and compounds AD2646 and AD2677-78-80, Figure 3 ). Surprisingly, in the B series, the anti- Plasmodium activity was restored in compounds with symmetrical alkyl chains of 6–8 carbon length (compounds AD2651 and AD2670, Figure 3 ). No systematic difference in anti- Plasmodium activity was observed between D-threo and L-threo enantiomer of a same analogue: e.g. the enantiomers AD2646 and AD2645 of the B series showed similar activity (Figure 3 ). It can also be noted that ceramide analogs containing a thiourea linkage also showed a significant anti- Plasmodium activity (Figure 3 , compounds AD2215-17) with, however, a less pronounced inhibitory effect than analogs with a methylene linkage. Inhibition of parasite growth by the methylene analog AD2646 was observed having similar IC 50 values on the P. falciparum strains K1 (IC 50 = 45 nM), F32 (IC 50 = 21 nM) and W2 (IC 50 = 28 nM), suggesting that the drug is not restricted to a specific strain and acts through a conserved mechanism in malarial parasites. Furthermore, analysis of drug combination with antimalarial drugs showed that AD2646 has a non-synergistic and non-antagonistic effect with CQ on the CQ-resistant strain K1, and with mefloquine and with artemether on the FcB1 strain (data not shown). Compound AD2646 (Figure 1 ) was selected to further investigate the biological effects of methylene analogs on parasite development. Structure-activity relationship around AD2646 showed that the presence of a nitro group linked to the phenyl is not essential for anti- Plasmodium activity (Figure 4 , compare IC 50 values of compounds AD2646 and AD2730) nor hydroxylation on carbon 1 (compare compounds AD2730 and AD2724). In contrast, hydroxylation of carbon 3 is important for anti- Plasmodium activity since removal of the hydroxyl group reduced the activity 13.5 times (compare compounds AD2730 and AD2729). Cytotoxicity on human cells MRC-5 of ceramide analogs in methylene linkage The cytotoxicity of methylene analogs upon human MRC-5 cells (diploid embryonic lung cell line) was evaluated (Table 1 ). Derivatives tested showed IC 50 values in the micromolar range, from 5 to 8 μM (except for AD2619), which are similar to the IC 50 value of PPMP. No major difference of toxicity was observed between D - and L-threo enantiomers (compare AD2646 and AD2645). In contrast to what was observed for P. falciparum , hydroxylation of the sphingosine carbon 3 does not seem important for cytotoxicity since similar IC 50 values were measured for AD2646 and AD2729, suggesting different mechanism(s) of action for AD2646 on MRC-5 cells and P. falciparum . AD2646 and 4 derivatives show high selectivity for P. falciparum as illustrated by the high index of selectivity of these compounds ranging from 160 to 624. The index of selectivity was defined as the ratio of the IC 50 value on MRC-5 cells to that on P. falciparum . It can be noted that no selectivity was observed for PPMP. A similar range of growth inhibition was measured on P. falciparum (Figure 2 ) and HL-60 cells [ 36 ] with ceramide analogs in amide linkage supporting a weak selectivity of these analogs for P. falciparum . Table 1 Cytotoxicity of methylene analogs and PPMP on human MRC-5 cells Compounds IC 50 (μM) IC 90 (μM) Index of selectivity AD2646 (-) 4.9 7.5 160 AD2645 (+) 6.1 10.3 161 AD2672 (-) 3.7 5.9 2 AD2730 (-) 6.1 9.9 322 AD2729 (-) 5.8 9.8 22 AD2619 (-) 26.7 42.3 624 PPMP 7.5 12.4 0.8 IC 50 and IC 90 values are the mean of three independent experiments. The S.E. were within 10% of the mean. (-): D-threo , (+): L-threo . Index of selectivity is defined by the ratio of the IC 50 value on MRC-5 cells to that on P. falciparum . It must be emphasized that the amide linkage of ceramide analogs is not required for activating apoptosis in cancer cells [ 35 ]. An increase of cytotoxicity of ceramide analogs in methylene linkage compared to their counterparts in amide linkage was also observed on the human histolytic lymphoma U937 [ 35 ] and the human leukaemia HL-60 cells [ 27 ] however, with higher IC 50 values than that observed with P. falciparum . Stage-specific inhibitory effects of AD2646 and reversibility To investigate the cytostatic or cytotoxic effects of AD2646 on the parasite development, cultures at the ring stage (0–10 hours), the trophozoite stage (25–35 hours) and the schizonte stage (40–48 hours) were incubated with 30, 100 or 250 nM of AD2646 for 24.5 hours for the ring stage, for 11 hours for the trophozoite stage, and for 14 hours for the schizonte stage. Aliquots were then taken, washed and incubated in the absence or the presence of drug for a further 13 hours to 24 hours depending upon the parasite stage tested (see Figure 5 ). Parasitaemia and parasite stages were determined on Giemsa-stained smears at time of aliquot removal and after the subsequent incubation. Figure 5 P. falciparum stage sensitivity to AD2646 . Parasites at the ring (A), trophozoite (B) and schizonte (C) stages were maintained in the presence of 30 nM (square) or 100 nM (triangle) AD2646 for 24 h30, 11 h and 14 h, respectively. Aliquots were then taken, washed and maintained in culture in the absence (open symbol) or in the presence (full symbol) of the same concentration of analog. Controls were cultures maintained in the absence of drug (full circle) and processed as the treated cultures. Parasitemia and parasite morphology were determined on Giemsa-stained smears at the indicated time. Each value is the mean of two independent experiments. Development of the ring stage was slightly affected by a continuous incubation with 30 nM AD2646. In contrast, when incubated with 100 and 250 nM, parasite growth was irreversibly blocked at the young trophozoite stage and the parasite degenerated. Drug removal after 24 hours of incubation did not allow a recovery of parasite growth (Figure 5A ). The trophozoite stages were more sensitive to AD2646 since a continuous incubation with 30 nM completely blocked development. Parasites did not enter into division and then degenerated. Only a partial recovery of parasite growth was observed when drug was removed after 11 hours of incubation. A more marked effect was observed with 100 nM AD2646 with degenerated parasites already observed after only 11 hours. No recovery of parasite growth was then observed after drug removal (Figure 5B ). The schizont stage appeared less sensitive than the trophozoite stage since a slight effect was only observed on the parasite development with 30 nM AD2646. However, parasite growth was irreversibly blocked by an incubation with 100 nM AD2646 and parasites degenerated (Figure 5C ). Similar results were observed for the methylene analogs AD2651 and AD2670, the trophozoite stage being the most sensitive with a complete inhibition of parasite development for 250 nM (data not shown). It can be noted that, in contrast to methylene analogs, addition of PPMP to parasite culture led to a preferential and reversible arrest of parasite development at the ring stage. The schizont stage (>30 hours old parasites) was insensitive to this concentration of drugs [ 14 , 19 ]. A cytostatic effect of PPMP on the ring-stage was effectively observed : rings blocked by a 24 hours incubation with 5 μM PPMP recovered to a normal growth after drug removal (data not shown). Blockage of parasite development was associated with the inhibition of a sensitive SPM synthase and TVN formation that delivers extracellular nutrients to the parasite [ 20 - 22 ]. Inhibition of sphingomyelin synthase activity and tubovesicular network formation of P. falciparum by compound AD2646 Figure 6 reproduces the inhibitory effects of PPMP and the methylene analogue AD2646 on the SM synthesis activity of young trophozoite (20–30 hours)-infected erythrocytes maintained in culture. As previously reported [ 19 ], no SPM synthase activity was measured in non-infected red blood cells and a biphasic inhibition curve was observed with PPMP in infected erythrocytes. Two pools of SPM synthase activity are present in parasites with respect to their inhibition by the ceramide analogue, one very sensitive to the drug and the second only inhibited by high concentrations of drug. The biphasic inhibition curve that superimposes on the PPMP inhibition curve was also recorded for AD2646 indicating that PPMP and AD2646 inhibit the SPM synthase activity of infected-red blood cells in a similar way. Figure 6 Inhibition of P. falciparum sphingomyelin synthase activity by AD2646 and PPMP . Trophozoite cultures (20–30 hours aged parasites) were incubated with 0–500 μM PPMP (full square) or AD2646 (open square) and 10 μM NBD-C 6 -ceramide for 60 min, at 37°C. SPM synthase activity was measured as described by Lauer et al. [19]. The percentage of SPM activity was determined by comparison of the activity measured in control cultures maintained without the analogs. Each value is the mean of triplicate experiments. In contrast, PPMP and AD2646 have completely different effects on the TVN formation for drug concentrations that block parasite growth. After 24 hours of incubation, ring development was totally inhibited by 5 μM PPMP and no TVN was observed as previously described [ 20 ] (Figure 7C ). As in controls maintained without drug (Figure 7A ), TVN was distinctly observed after 24 hours of incubation of rings with 60 nM AD2646 (Figure 7B ). This concentration blocks irreversibly the parasite development indicating that AD2646 has no major effect on TVN formation. Figure 7 Effects of AD2646 and PPMP on the formation of the tubovesicular network of P. falciparum . Infected erythrocytes at the ring stage were incubated for 24 hours in presence of 60 nM AD2646 (B) or 5 μM PPMP (C). TVN formation in treated cells and untreated cells (A) was evaluated by membrane staining using BODIPY-Fl-C5-ceramide. Arrow: TVN. Bar: 5 μm. These data do not support the hypothesis of parasite growth inhibition due to an inhibition of the parasite SPM synthase activity as was demonstrated for PPMP [ 19 - 22 ] : 1) The anti- Plasmodium activity of AD2646 does not correlate with its inhibitory activity on the SPM synthase. Although AD2646 and PPMP showed similar inhibitory activity on this enzymic activity in parasites in cultures, AD2646 is about 300 times more efficient in inhibiting parasite development than PPMP; 2) In contrast to PPMP which inhibits the parasite development preferentially and reversibly at the ring stage [ 19 ], AD2646 inhibited parasite development preferentially and irreversibly at the trophozoite stage (Figure 5 ); 3) Inhibition of the SPM synthase activity by PPMP is associated with an inhibition of the TVN formation [ 19 - 22 ]. This was not observed in the presence of AD2646 (Figure 7 ). What could be the mechanism(s) of action of ceramide analogs in methylene linkage on P. falciparum ? By their lipidic nature, these analogs might act through a detergent effect that could lead to lysis or modification of the integrity of infected-erythrocyte membranes. This apparently is not the case. No significant lysis of normal erythrocytes was observed after 48 hous of incubation with concentrations of analogs up to 10 μM (data not shown). Furthermore, no preferential lysis of infected-erythrocytes was observed on Giemsa-stained smears of infected cultures maintained 48 hours with 250 nM AD2646, a concentration inhibiting parasite growth totally. Interestingly, the absence of a fatty acyl carbonyl group (methylene linkage) in our ceramide analogs is a critical factor for the efficacy of their antiplasmodial activity. Sphingolipids preferentially interact with cholesterol in membranes, especially in detergent-resistant microdomains (DRMs or rafts). Rafts have been described in Plasmodium and are involved, at least, in the uptake of erythrocyte raft proteins and maintenance of the parasitophorous vacuole containing the parasite, inside the erythrocyte [ 37 ]. This interaction implies : 1) van der Waals interactions between the saturated acyl chain and sphingoid moiety of sphingolipids and the rigid planar tetracyclic rings of cholesterol [ 38 ] and 2) hydrogen bonds between the 3-β hydroxyl group of cholesterol and the fatty acyl carbonyl group resulting from the amide linkage with the sphingoid moiety [ 39 ]. The amide-linked fatty acid function seems to have a profound stabilizing effect on cholesterol-sphingolipid interactions [ 40 ]. It could be hypothesized that in a membrane context, methylene analogs might have a destabilizing effect on the cholesterol-sphingolipid interactions and, in consequence, modifications of membrane properties. Indeed, P. falciparum growth is characterized by a setting up of new permeabilities of the infected-erythrocyte membrane [ 41 ]. Although the biochemical nature of these new permeabilities is still unknown, they have been characterized from an electrophysiological point of view and involve a malaria-induced anion channel [ 42 , 43 ]. The effect of ceramide analogs was investigated on the properties of this channel. A 24 hours-incubation of infected-erythrocytes with 250 nM AD2646 or 10 μM PPMP did not modulate significantly the induced channel activity measured in the whole-cell configuration of the patch-clamp technique (S. Egee, unpublished data), suggesting that these ceramide analogs do not inhibit parasite growth through modifications of infected-erythrocyte membrane permeabilities. Ceramide is at the parting of different ways of sphingolipid metabolism. Analogs have the potential to inhibit different ceramide-metabolizing enzymes and then might have a pleiotropic effect. Ceramide analogs in amide linkage were described as potent inhibitors of alkaline ceramidase in HL60 human myeloid leukemic cells [ 44 , 45 ]. Methylene analogs inhibit the biosynthesis of SPM and glycosphingolipids in HL60 cells, and acid ceramidase in vitro [ 10 ]. When applied to cancer cells, such analogs induced an elevation of the endogenous level of ceramide with the consequent effects of growth suppression and cell death by apoptosis [ 44 , 45 ]. In contrast to what was observed for cancer cells [ 27 ], preliminary results suggest that the ceramide analog AD2646 induced non-apoptotic death of P. falciparum . Parasites exposed to 1 μM AD2646 for up to 36 hours failed to exhibit characteristic apoptosis, as determined by terminal deoxynucleotidyl transferase DNA fragmentation assay and DNA fragmentation using both gel electrophoresis and fluorescence microscopy methods, although the nucleus appeared highly condensed (M. Dellinger, unpublished data). Apoptosis in P . falciparum is still controversial although some characteristics of apoptosis has been described in Plasmodium [ 46 ]. Recently, an increase in the intracellular ceramide content and an activation of parasite sphingomyelinase(s) were found to be associated with a non-apoptotic parasite death process as induced by artemisinine and mefloquine [ 26 ]. The hypothesis that AD2646 induced parasite death through modulation of endogenous ceramide level, as observed for cancer cells, is under investigation. Authors's contribution ML and PG carried out the in vitro inhibition assays on P. falciparum and MRC-5 cells. MG and MD performed the fluorescence microscopy and apoptosis investigations on P. falciparum , respectively. SE and ST carried out electrophysiological studies on the malaria-induced anion channel. AD, CW and SG participated in the design and synthesis of ceramide analogs. All authors read and approved the final manuscript.

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Surveillance of antimicrobial resistance at a tertiary hospital in Tanzania

Background Antimicrobial resistance is particularly harmful to infectious disease management in low-income countries since expensive second-line drugs are not readily available. The objective of this study was to implement and evaluate a computerized system for surveillance of antimicrobial resistance at a tertiary hospital in Tanzania. Methods A computerized surveillance system for antimicrobial susceptibility (WHONET) was implemented at the national referral hospital in Tanzania in 1998. The antimicrobial susceptibilities of all clinical bacterial isolates received during an 18 months' period were recorded and analyzed. Results The surveillance system was successfully implemented at the hospital. This activity increased the focus on antimicrobial resistance issues and on laboratory quality assurance issues. The study identified specific nosocomial problems in the hospital and led to the initiation of other prospective studies on prevalence and antimicrobial susceptibility of bacterial infections. Furthermore, the study provided useful data on antimicrobial patterns in bacterial isolates from the hospital. Gram-negative bacteria displayed high rates of resistance to common inexpensive antibiotics such as ampicillin, tetracycline and trimethoprim-sulfamethoxazole, leaving fluoroquinolones as the only reliable oral drugs against common Gram-negative bacilli. Gentamicin and third generation cephalosporins remain useful for parenteral therapy. Conclusion The surveillance system is a low-cost tool to generate valuable information on antimicrobial resistance, which can be used to prepare locally applicable recommendations on antimicrobial use. The system pinpoints relevant nosocomial problems and can be used to efficiently plan further research. The surveillance system also functions as a quality assurance tool, bringing attention to methodological issues in identification and susceptibility testing.

Background Exaggerated and irrational use of drugs, availability of antibiotics without prescription, the use of pharmaceuticals of doubtful quality and the HIV epidemic may all contribute to the current worldwide surge in antimicrobial drug resistance. Emerging resistance to antimicrobial drugs increases morbidity and mortality by hampering the provision of effective chemotherapy, and makes treatment more costly [ 1 - 3 ]. The surge in antimicrobial resistance seen in many low-income countries is potentially disastrous because of the lack of resources for purchasing expensive second-line drugs [ 4 ]. It is widely held that surveillance of antimicrobial susceptibility is fundamental to combat the emergence of resistance [ 5 ]. Surveillance must be global since resistant bacteria can be transferred between countries, but it must also be local, since countries have very different resistance patterns and different treatment practices [ 6 ]. The primary task of a surveillance system is to provide locally applicable data to guide empiric therapy. Furthermore, surveillance may help assessing the magnitude of the resistance problem locally, nationally and internationally, monitoring changes in resistance rates and detecting the emergence and spread of new resistance traits. A well-functioning surveillance system is also necessary to measure the impact of any interventions. Surveillance systems also functions as a quality assurance tool and may help improving the quality of the susceptibility testing. This paper describes the experience with the implementation of a computerized surveillance system for antimicrobial drug susceptibility at Tanzania's major referral hospital, and its use to analyze the susceptibility patterns of 7621 consecutively recorded clinical bacterial isolates. Methods Setting The study was performed at Muhimbili National Hospital (MNH), Dar es Salaam, Tanzania. With more than 1000 beds, MNH is the largest hospital in the country and serves as a national referral and university teaching hospital, as well as a primary and referral hospital for a population of approximately 3.6 million in the Dar es Salaam area. The Department of Microbiology and Immunology at MNH examines specimens from inpatients and outpatients at MNH, and from a number of nearby hospitals. Bacteriological cultures are performed on more than 23,000 specimens per year. The surveillance system A free-of-charge software for the surveillance of antimicrobial resistance (WHONET, World Health Organization, Geneva, Switzerland) [ 7 ] was implemented at MNH in 1998. Currently a total of 880 microbiology laboratories in 76 countries use this software, however, among these are only 41 laboratories in four countries on the African continent (data from 2002, personal communication from John Stelling, author of the WHONET software). The software has three main parts, a laboratory configuration file which can be used to customize it to the particular laboratory, an interface for data entry and a part for analysis and reporting of resistance data. At our hospital, all bacterial isolates of clinical significance from specimens received during the period July 1 st 1998 to December 31 st 1999 were recorded and analyzed. The specimens examined included urine, pus/secretions (swabs from skin, surgical and traumatic wounds, burns, umbilical cords, throat, nose, eye and ear discharge and genital swabs), blood, cerebrospinal fluid, other body fluids, stools and other specimens. Mycobacteria and anaerobic bacteria were not included in the study. Apart from the WHONET software, we used Stata 8.0 for Macintosh (Stata Corporation, College Station, Texas, USA) to evaluate differences of proportions by Fisher's exact test (2-tailed, cut-off point for statistical significance at p-value of 0.05). Laboratory methods The specimens were cultured and the bacterial isolates identified using standard microbiological methods as described in Mackie & McCartney Practical Medical Microbiology [ 8 ]. Susceptibility testing was performed by Stokes' method [ 9 ] on Iso-Sensitest (Oxoid Limited, Basingstoke, UK) agar plates. This method, developed by Dr Joan Stokes half a century ago, was designed to monitor for both disc and agar quality in that both the clinical isolate and a control strain were tested on every plate. The clinical isolate is swabbed onto the middle of the agar plate and the control strain at the periphery. The antibiotic disk is placed precisely at the interface between the surface areas inoculated with the clinical isolate and the control strain. After overnight incubation, the relative size of the inhibition zones of the clinical isolate and the control strains are compared. The test results are classified as susceptible (S), intermediate (I) or resistant (R) by evaluation of the difference between the inhibition zones of the clinical isolate and the control strain. The control strains used in our lab are S. aureus NCTC 6571, E. coli NCTC 10418 or Pseudomonas aeruginosa NCTC 10662. The isolates showing intermediate resistance were few and were grouped together with sensitive isolates for the purpose of data analysis. Either methicillin or oxacillin disks were used to test for methicillin-resistance in S. aureus , the results being considered equivalent and interchangeable in the data analysis. ß-Lactamase testing was not routinely performed. The susceptibility of pneumococci to penicillin was examined by the use of penicillin 2 μg disks. Commercially produced antibiotic disks, mostly obtained from Oxoid Limited, were used, however, in some instances, antibiotic disks, prepared locally were used due to financial constraints. The Department of Microbiology and Immunology participates in an external quality assessment program in bacteriology led by the World Health Organization-collaborating centre, the National Institute for Communicable Diseases (NICD), Johannesburg, South Africa. The Department of Microbiology and Immunology at our hospital receives bacterial strains from NICD, performs species identification and antimicrobial susceptibility testing, and report the results back to NICD. Evaluation of the surveillance system We evaluated the strengths and shortcomings of the surveillance system in our setting, particularly in terms of how well it performed in its main application areas, providing locally applicable data to guide empiric therapy, monitoring antimicrobial susceptibility trends, detecting the emergence and spread of new resistance traits and as a tool for quality assurance. We also assessed the cost-implications of implementing the surveillance program in our setting. We considered direct costs, such as the purchase of equipment, and indirect costs, such as those related to the running of the laboratory, including human resources. We also comment on the benefits of the surveillance system related to both direct patient care and long-term implications of containing antimicrobial resistance. Results Bacterial isolates A total of 7617 bacterial isolates were registered during the study period, of which 67.4% (n = 5134) were Gram-negative and 32.6% (n = 2483) Gram-positive. Table 1 shows the most frequently encountered bacteria, overall and from various specimen types. The majority of the isolates were obtained from pus (44.3%), urine (43.5%) and blood cultures (10.1%). Cerebrospinal fluid accounted for 0.4% of the isolates. Among the 2034 blood cultures, 15.9% (n = 323) yielded growth of a total of 326 pathogenic bacterial isolates and 447 Coagulase-negative staphylococci (CoNS) as shown in Table 1 . CoNS are potential pathogens and are increasingly considered as a cause of blood-stream infections. However, in many cases they are merely contaminants, i.e. bacterial isolates present on the skin surface, which are introduced in the blood specimen and grow in the blood culture, but do not produce disease in the patient. For CoNS isolates to be considered a probable pathogen, it is commonly required that they are recovered from two separate blood cultures. Since multiple blood cultures were not routinely taken from the same patient in the hospital, the susceptibilities of these isolates were not evaluated further. CoNS and various other Gram-positive probable contaminants, mostly Bacillus spp. were recovered from 22.0% (n = 447) and 6.9% (n = 141) of the blood cultures, respectively. Furthermore, five Candida spp. isolates and one Cryptococcus neoformans were recovered. Among the 49 Salmonella isolates, two were identified as S . Typhi, 16 as S . Typhimurium, 16 as S . Paratyphi B and one each as S . Paratyphi C, S . Enteritidis and S . Arizonae. Twelve Salmonella isolates were not serotyped. Among the 41gonococcal isolates, 28 (68.3%) were from genital swabs. Eleven (26.8%) gonococcal isolates were obtained from the neonatal ward, out of which 4 were specified as from eye discharge. Table 1 Frequency of pathogenic a bacterial isolates from different specimen types at Muhimbili National Hospital, Tanzania Organism Blood (%) Spinal fluid (%) Urine (%) Pus b (%) Other (%) Overall (%) Gram-negative isolates E. coli 27 (3.5) 0 (0.0) 1466 (44.2) 417 (12.3) 26 (21.8) 1936 (25.4) Klebsiella spp. 91 (11.8) 8 (23.5) 1036 (31.3) 603 (17.9) 33 (27.7) 1771 (23.3) Pseudomonas spp. 10 (1.3) 2 (5.9) 52 (1.6) 531 (15.7) 9 (7.6) 604 (7.9) Proteus spp. 7 (0.9) 0 (0.0) 121 (3.7) 249 (7.4) 3 (2.5) 380 (5.0) Enterobacter spp. 4 (0.5) 0 (0.0) 97 (2.9) 1 (0.0) 0 (0.0) 102 (1.3) Salmonella spp. 37 (4.8) 2 (5.9) 6 (0.2) 0 (0.0) 4 (3.4) 49 (0.6) N. gonorrhoeae 0 (0.0) 0 (0.0) 0 (0.0) 41 (1.2) 0 (0.0) 41 (0.5) Haemophilus spp. 1 (0.1) 5 (14.7) 0 (0.0) 0 (0.0) 0 (0.0) 6 (0.1) Other GNR 32 (4.1) 7 (20.6) 12 (0.4) 184 (5.4) 10 (8.4) 245 (3.2) Subtotal, Gram-negative isolates 209 (27.0) 24 (70.6) 2790 (84.2) 2026 (60.0) 85 (71.4) 5134 (67.4) Gram-positive isolates Staphylococcus aureus 72 (9.3) 1 (2.9) 362 (10.9) 1120 (33.2) 12 (10.1) 1567 (20.6) Streptococcus pyogenes 1 (0.1) 0 (0.0) 0 (0.0) 160 (4.7) 2 (1.7) 163 (2.1) Other streptococci c 39 (5.0) 3 (8.8) 52 (1.6) 58 (1.7) 13 (10.9) 165 (2.2) Enterococci 3 (0.4) 0 (0.0) 64 (1.9) 3 (0.1) 1 (0.8) 71 (0.9) S. pneumoniae 2 (0.3) 6 (17.6) 0 (0.0) 11 (0.3) 6 (5.0) 25 (0.3) CoNS a 447 (57.8) ... 45 (1.4) ... ... 492 (6.5) Subtotal, Gram-positive isolates 564 (73.0) 10 (29.4) 523 (15.8) 1352 (40.0) 34 (28.6) 2483 (32.6) Total 773 (100.0) 34 (100.0) 3313 (100.0) 3378 (100.0) 119 (100.0) 7617 (100.0) GNR, Gram-negative rod-shaped bacteria, not further identified; CoNS, coagulase-negative staphylococci; "...", not applicable. a CoNS from blood and urine specimens are reported as possible pathogens, although many may be contaminants. CoNS from other specimen types are considered contaminants and not reported. b Pus includes swabs from skin, surgical and traumatic wounds, burns, umbilical cords, throat, nose, eye and ear discharge and genital swabs. c Streptococci other than S. pyogenes and S. pneumoniae , and streptococci not identified below genus level. Specimens from inpatients and outpatients contributed to 53.2% and 31.9% of the isolates, respectively. A further 6.0% were obtained from specimens from other hospitals in Dar es Salaam, while 8.8% were obtained from other or unknown locations. Among the isolates from inpatients, 36.5% were obtained from the Department of Pediatrics, 28.4% from the neonatal section and 8.1% from the other pediatric wards. The other isolates came from the Departments of Surgery (22.4%), Internal Medicine (16.6%), Obstetrics and Gynecology (9.8%), the Intensive Care Unit (4.9%) and other locations (9,8%). For 4900 isolates, the age or the estimated age group of the patient was known. Of these, 23.6% (n = 1155) were from neonates (≤ 1 month old), 6.8% (n = 335) from children aged one month to seven years, and 69.6% (n = 3410) from adults or children older than 8 years. Antimicrobial susceptibility Tables 2 and Table 3 show the antimicrobial susceptibility patterns of the most frequently isolated Gram-negative and Gram-positive bacteria, respectively. There were no clear-cut differences in the antimicrobial susceptibilities among the various serotypes of Salmonella isolates (data not shown). The majority of Pseudomonas aeruginosa isolates was susceptibility-tested to gentamicin only, to which 4.3% (15/350) were resistant. Among the isolates of Neisseria gonorrhoeae , 70.0% were resistant to penicillin, 45.2% to tetracycline, 59.3% to trimethoprim-sulfamethoxazole, 5.9% to erythromycin and none was resistant to spectinomycin, fluoroquinolones or amoxicillin-clavulanate (data not shown). Table 2 Percentage of Gram-negative bacterial isolates resistant to antimicrobial agents (number of tested isolates in brackets) Drug E. coli Klebsiella spp. Proteus spp. Enterobacter spp. Salmonella spp. GNR Ampicillin 80% (1761) 85% (1572) 60% (331) 72% (86) 70% (46) 56% (204) Amoxicillin- clavulanate 28% (1292) 32% (1153) 17% (247) 32% (78) 52% (23) 31% (124) Ceftazidime 5% (788) 6% (605) 2% (95) 10% (51) 0% (8) 14% (35) Tetracycline 77% (1223) 66% (1016) 77% (211) 72% (54) 42% (12) 45% (153) Gentamicin 8% (1634) 14% (1538) 7% (343) 15% (91) 9% (23) 8% (217) Trimethoprim- sulfamethoxazole 76% (1313) 69% (1174) 57% (224) 70% (56) 73% (44) 51% (172) Sulfonamides 84% (174) 84% (231) 74% (46) 100% (14) 95% (22) 62% (34) Nitrofurantoin 32% (929) 53% (652) 72% (71) 48% (48) ... ... Chloramphenicol 45% (250) 51% (372) 55% (132) ... 20% (41) 57% (138) Fluoroquinolones 13% (432) 6% (343) 3% (65) 6% (32) 0% (20) 15% (40) Nalidixic acid 28% (509) 16% (334) 18% (22) 31% (16) ... ... GNR, Gram negative rod-shaped bacteria, not further identified; "...", not tested. Table 3 Percentage of Gram-positive bacterial isolates resistant to antimicrobial agents (number of tested isolates in brackets) Drug S. aureus CoNS Enterococci S. pneumoniae S. pyogenes Other strept. a Penicillin 97% (1521) 93% (42) 67% (9) 4% (23) 0% (163) 23% (98) Ampicillin ... ... 6% (66) ... ... 13% (83) Methicillin/ cloxacillin 2% (1556) 21% (47) ... ... ... ... Tetracycline 49% (1042) 90% (39) 76% (51) 8% (13) 47% (131) 61% (90) Erythromycin 29% (1543) 69% (48) 26% (65) 6% (18) 7% (161) 26% (156) CoNS, Coagulase-negative staphylococci; "...", not tested. a Streptococci other than S. pyogenes and S. pneumoniae , and streptococci not identified below genus level. Comparison of resistance patterns of isolates obtained from inpatients and outpatients at MNH did not show large differences. However, ampicillin resistance was more frequent in urinary isolates of E. coli from inpatients than in those from outpatients as shown in Table 4 . Likewise, urinary isolates of Klebsiella spp. from inpatients were more frequently resistant to gentamicin and trimethoprim-sulfamethoxazole than isolates from outpatients. Table 4 Percentage of urinary E. coli and Klebsiella spp. isolates from inpatients and outpatients resistant to antimicrobial agents E. coli Klebsiella spp. Drug Inpatients Outpatients P a Inpatients Outpatients P a Ampicillin 87.2 82.7 0.036 a 92.2 91.1 0.624 Amoxicillin- clavulanate 31.4 28.3 0.344 37.7 33.9 0.327 Ceftazidime 4.9 5.6 0.731 7.6 6.0 0.577 Tetracycline 83.1 81.7 0.648 82.0 75.2 0.053 Gentamicin 8.6 7.7 0.572 14.9 5.4 <0.001 a Trimethoprim- sulfamethoxazole 86.0 81.3 0.067 82.7 74.2 0.012 a Sulfonamides 92.1 87.8 0.510 95.2 100.0 0.553 Nitrofurantoin 33.7 33.1 0.881 52.1 58.0 0.157 Fluoroquinolones 17.8 12.7 0.217 7.2 6.7 1.000 Nalidixic acid 29.0 28.2 0.913 14.0 18.8 0.334 a P < 0.05 (Fisher's exact test, 2-tailed) indicates statistical significance of the differences in resistance rates. Comparison of resistance patterns in isolates blood cultures with those from other specimen types showed apparent great differences for some drugs, however, in most cases the number of blood culture isolates were few and did not show statistically significant differences. However, as shown in Table 5 , blood culture isolates of Klebsiella spp. were indeed more frequently resistant to gentamicin than those from other specimen types. A significantly greater proportion of blood culture isolates of S. aureus were resistant to tetracycline than among those from other specimen types, whereas for penicillin the isolates from blood cultures were resistant in a lower proportion than the others. Table 5 Percentage of bacterial isolates from different specimen types resistant to antimicrobial agents E. coli Klebsiella spp. S. aureus Drug Blood Other P a Blood Other P a Blood Other P a Penicillin ... ... ... ... ... ... 91.5 96.9 0.028 a Ampicillin 84.0 79.4 0.803 84.3 85.3 0.759 ... ... ... Amoxicillin- clavulanate 40.0 27.8 0.383 29.8 31.9 0.873 ... ... ... Methicillin ... ... ... ... ... ... 1.4 2.2 1.000 Ceftazidime 0.0 5.3 1.000 5.7 6.0 1.000 ... ... ... Tetracycline 54.5 77.3 0.139 66.7 66.4 1.000 84.6 48.3 <0.001 a Erythromycin ... ... ... ... ... ... 21.1 29.0 0.179 Gentamicin 13.0 7.7 0.416 41.3 12.3 <0.001 a ... ... ... Trimethoprim- sulfamethoxazole 72.0 76.3 0.636 63.0 69.1 0.297 ... ... ... Sulfonamides 83.3 84.0 1.000 86.8 83.4 0.809 ... ... ... Chloramphenicol 58.3 43.8 0.199 57.9 49.3 0.200 ... ... ... Fluoroquinolones 40.0 13.1 0.136 0.0 6.5 0.381 ... ... ... "...", not applicable. a P < 0.05 (Fisher's exact test, 2-tailed) indicates statistical significance of the differences in resistance rates. Evaluation of the surveillance system A great number of bacterial isolates were recorded in the system. All age groups and both inpatients and outpatients were represented in the study. More than a third of the isolates were from outpatient populations from the Dar es Salaam area, however we cannot exclude the possibility of a selection bias in favour of patients with infections caused by resistant organisms, since many patients get treatment at primary health facilities before reaching MNH. We do not know how well the rural population is represented in this material, but we assume that the outpatients in the study are mostly from the Dar es Salaam area. Ten percent of the isolates represented systemic infections, i.e. isolates from blood cultures and spinal fluid. The susceptibility test results were recorded as interpreted values (i.e. "R" (resistance), "I" (Intermediate) or "S" (susceptible)) and not as inhibition zone diameters. In this study, no molecular techniques were available for the detection of resistance genotypes and evaluation of genetic relatedness of bacterial isolates. The direct cost of implementing the surveillance system was limited to the purchase of a computer at approximately 1000 Euro. However, less expensive second-hand computers would be sufficient. The software was downloaded free of charge from the WHO website. The indirect costs of running this surveillance program are related to human sources for operating the software, including data entry and analysis, and the costs of the susceptibility testing activities. It is difficult to separate these indirect costs from the costs of running the daily laboratory activities. In our setting, a laboratory technologist from the department took on the task of operating the software in addition to her regular duties. In our experience, for a hospital of our size, it is recommendable to allocate approximately 50% of a laboratory technologist position to operating the surveillance software. In our setting, this would translate into a monthly cost of approximately 100 Euro for the department. The surveillance system is dependent on susceptibility testing of acceptable quality. The susceptibility testing incurs costs related to human resources and the purchase of laboratory reagents including antimicrobial disks and agar media. Implementing a surveillance system may increase these costs by focusing on the importance of quality reagents. However, since the susceptibility testing activities are an integral activity of the department, which would have been performed regardless of the surveillance system, we choose not to attribute their costs to the surveillance system in this context. The benefits of a surveillance system are difficult to quantify, but are of potentially great magnitude. Foremost, surveillance data may improve empiric therapy for infections and thus save lives and reduce suffering. It may reduce treatment costs by enabling the use of the least expensive effective drugs. Additionally, surveillance systems may contribute to containing or reducing antimicrobial resistance, which in the long term perspective may have great benefits in reducing morbidity and mortality, and diminish the need for expensive second-line antimicrobial agents. The strengths and weaknesses are elaborated on in the Discussion part. Discussion Resistance patterns and implications for therapy Experience from the World Health Organization's External quality assurance system for antimicrobial susceptibility testing has shown that disk diffusion testing is suitable for routine surveillance [ 10 ]. However, disk diffusion is not optimal for testing of certain important resistance traits, such as penicillin-resistance in pneumococci. The lack of international standardization of methods and interpretive criteria causes concern, but there are indications that routine susceptibility testing data are suitable for surveillance even if obtained with different methods [ 11 ]. Consistent with observations from a number of other countries in the region [ 12 - 15 ] and elsewhere [ 16 ], Gram-negative bacilli displayed high rates of resistance to common inexpensive antibiotics. Reasonably priced antibiotics such as ampicillin, tetracycline, trimethoprim-sulfamethoxazole and sulfonamides are now of limited benefit in the treatment of infections caused by important Gram-negative bacteria such as E. coli , Klebsiella spp., Proteus spp. and Salmonella . Chloramphenicol may fail to cure as much as a quarter of infections caused by Salmonella and half or more of infections caused by E. coli , Klebsiella spp. and Proteus spp. Fluoroquinolones appear to be the only reliable drugs for oral treatment of infections caused by common Gram-negative bacilli, whereas gentamicin and third-generation cephalosporins remain useful for parenteral therapy. The study showed a very low prevalence of methicillin-resistant S. aureus , consistent with previous data from the same hospital [ 17 , 18 ]. While the results should be interpreted with some caution since confirmatory nucleic acid based techniques were not available, the data support the current use of isoxazolyl penicillins, such as cloxacillin for the treatment of staphylococcal infections at the hospital. There were few isolates of enterococci compared to studies from high-income countries [ 19 ]. It is reassuring that the current study showed a low rate of ampicillin-resistant enterococci, indicating that nosocomial infections caused by these micro-organisms is a minor problem compared with many high-income countries. Low consumption of broad-spectrum antibiotics such as third-generation cephalosporins, fluoroquinolones, imipenem and vancomycin may explain this finding [ 19 - 21 ]. While other countries in the region have been affected by penicillin-resistant pneumococci [ 22 , 23 ], the current study indicates that pneumococcal disease in Dar es Salaam can safely be treated with penicillin or erythromycin. However, the results should be interpreted with some caution since the number of isolates tested was small. More than a quarter of the gonococcal isolates (11/41) were obtained from the neonatal ward, and most or all of these isolates probably represent gonococcal conjunctivitis. Amoxicillin-clavulanate, spectinomycin, fluoroquinolones and erythromycin appear to be good alternatives for the treatment of gonococcal infections. An apparent increase in resistance to trimethoprim-sulfamethoxazole (from 18% to 59%) is noted since the study by Mbwana [ 24 ] from 1993 to 1995, however, this may be due to the use of different methodology for susceptibility testing. Applicability of data to guide treatment of serious infections Recommendations for antibiotic treatment of serious bacterial infections such as bloodstream infections and meningitis should preferably be based on knowledge of the prevalence and antimicrobial susceptibility patterns of pathogens isolated from blood and spinal fluid. While a fair number of bacterial isolates were tested in the current study, the number of blood culture isolates was limited (n = 329, excluding the CoNS isolates). As shown in Table 5 , there appears to be differences in resistance between isolates obtained from blood cultures and those from other specimen types, but these are difficult to assess because of the low number of blood culture isolates. Thus, the data from the current surveillance should be interpreted with caution with regards to the treatment of serious infections. The CoNS isolates obtained from blood were recorded in the WHONET database, since they may represent clinically important infections such as bacteremia in patients with compromised immunity, patients with indwelling intravascular devices and the newborn [ 25 ]. The study showed that a high proportion (21.9%) of blood culture bottles yielded CoNS isolates. However, the conventional way to distinguish pathogenic isolates of CoNS from contaminants, by requiring growth of a similar CoNS isolate in a separate blood culture, could rarely be used, since follow-up cultures were seldom available. Consequently, the susceptibilities of these isolates were not evaluated further. Relevance of data for outpatient and rural populations It is important to specify for which population the surveillance data are valid. At our hospital, specimens from both inpatients and outpatients were examined. The hospital is to a great extent used as a primary hospital for the population in the Dar es Salaam area. However, among the cases coming to the hospital, there may be a degree of selection of patients with infections caused by resistant microbes, since many patients rely on health centers and pharmacies to cure simple ailments, and only come to the hospital when primary treatment fails. The study found that a few resistance traits, such as ampicillin resistance in E. coli and gentamicin and trimethoprim-sulfamethoxazole resistance in Klebsiella spp. were more frequent in urinary isolates from inpatients than from outpatients. Apart from that, there were no dramatic differences between isolates from inpatients and outpatients. The data from the study should be representative for both the hospital setting and to some degree the population in Dar es Salaam. However, the majority of the population of Tanzania lives in rural areas, where resistance patterns may be substantially different. Thus one should be cautious to extrapolate the results of the current study to be valid for populations in the countryside. Ability to monitor trends of antimicrobial susceptibility Certain trends in antimicrobial susceptibility could be identified by comparison with data from other studies. While resistance to ampicillin, tetracycline and sulfonamides in Gram-negative bacteria was frequent already in the seventies [ 26 , 27 ], it is worrying that resistance to trimethoprim-sulfamethoxazole, chloramphenicol, nitrofurantoin, nalidixic acid and amoxicillin-clavulanate appear to have increased compared to previous studies [ 27 , 28 ]. The extensive use of chloramphenicol for the treatment of presumed cases of typhoid fever and the use of trimethoprim-sulfamethoxazole for the ambulatory treatment of chest infections, malaria and other infections, may have contributed to the high prevalence of resistance to these two drugs. Although still low, it is of concern that the rate of gentamicin-resistance in E. coli has increased from zero in 1978–79 [ 27 ] to 2% in 1995 [ 28 ] and 8% in the current study. In neighboring Kenya, the rate of gentamicin-resistance in E. coli has increased from 2% in the late seventies [ 29 ] to 20% and above in recent studies [ 12 ]. Resistance to gentamicin is common in Gram-negative bacteria with extended-spectrum beta-lactamases (ESBL), sometimes in as much as 96% of isolates [ 30 ]. Such an association cannot be investigated in the current study, since less than half of the isolates of E. coli and Klebsiella spp. were tested for susceptibility to third-generation cephalosporins and other methods for detection of ESBL (double disk synergy test, Etest, PCR) were not available. Also in P. aeruginosa the rate of gentamicin-resistance has increased, from zero in the seventies [ 27 ] to 4% in the current study. Resistance to penicillin and erythromycin was common among S. aureus isolates in this study. However, the rate of tetracycline resistance (49%) was lower than reported from the same hospital in 1979 (57%) and 1982 (74%) [ 17 ]. In the late seventies, tetracycline was used in great quantities in Tanzania to prevent and treat cholera; as much as 1788 kilograms of the drug were used during a period of only 5 months [ 31 ]. Due to the rapid emergence of tetracycline-resistant Vibrio cholerae , the use of the drug was subsequently greatly reduced, and this may have contributed to a concurrent decline in the rate of tetracycline-resistance in an unrelated species such as S. aureus . For meaningful comparison of data from different studies, whether from the same or different laboratories, the same method of susceptibility testing should preferably be employed. In our laboratory, the same method has been used for a number of years. The WHONET software features a number of sophisticated ways to analyze susceptibility information based on the measurements of inhibition zone diameters. Recording the diameter of the inhibition zones in disk diffusion testing is generally recommended [ 32 ], and may increase the accuracy of results and enable the detection of gradual shifts in antibiotic susceptibility over time. It also makes the data independent of the current breakpoints. With the WHONET software, data can easily and rapidly be re-analyzed with reference to new breakpoints. However, the Stokes' method for susceptibility testing [ 9 ], which is used in our laboratory, is based on visual interpretation of the difference in inhibition zones between the clinical isolate and the control strain. The interpretation is recorded as interpreted values, i.e. either susceptible "S", intermediate susceptible "I" or resistant "R". The WHONET software also accepts susceptibility data to be entered and analyzed as "interpreted values", i.e. "S", "I" and "R". The use of such interpreted values enables most of the analysis features of WHONET, but not all. Foremost, analyzing data based on zone diameters (or MIC values) is superior for the early detection of subtle shifts in antimicrobial resistance over time, which may alert clinicians about emerging resistance trends at an early stage. However, one asset of the Stokes' method, particularly under tropical conditions, is that unsuspected poor antibiotic disk quality will be discovered quickly since a control strain is tested on every plate. Furthermore, variations over time in the battery of antibiotics tested makes comparison of data less useful. Laboratories in low-income countries are sometimes vulnerable to this because of unreliable supplies of antibiotic discs. Ability to detect emerging resistance traits Disk diffusion testing may give indications of emerging resistance traits such as methicillin-resistance in S. aureus and ESBL in Gram-negative bacteria. The current surveillance indicated that methicillin-resistance is rare in S. aureus at the hospital. Ideally this should be confirmed with PCR-based methods to detect the mec A gene. Likewise, the disk diffusion testing showed the presence of resistance to ceftazidime in Gram-negative isolates, albeit at a low rate, which calls for further investigation with regard to the possible presence of ESBL. Our laboratory did not employ molecular methods for detection of resistance genes on a routine basis, but a recent study showed low prevalence of methicillin-resistant S. aureus (MRSA) [ 18 ]. Resistance surveillance should be coupled with awareness of signs of various resistance traits and, preferably, the possibility of using molecular methods to verify emerging resistance traits. Ability to detect nosocomial problems The WHONET software is well suited to analyze antibiograms in order to detect suspicious nosocomial outbreaks. These functions too are dependant on the use of a consistent battery of test drugs, and also works better when results are entered as actual values for MIC or zone diameters, as opposed to the interpreted value ("S", "I" or "R"). In our hospital, comparison of resistance rates did not show dramatic variation between isolates from inpatients and outpatients. The exception was a trend for more frequent gentamicin-resistance in inpatient isolates of Gram-negative bacteria, particularly Klebsiella spp., which may suggest possible nosocomial spread. The analysis of antibiograms did not produce convincing evidence of clonal patterns spread of bacterial isolates, possibly partly due to the variations in the battery of antibiotics tested. Molecular methods for the evaluation of the genetic relatedness of bacteria were not available in this study. Suitability for international comparison of resistance data In 2002 a total of 880 laboratories in 76 countries across the world used the software, including 41 laboratories in 4 African countries. The WHONET system has been implemented at MNH since 1998. Unfortunately, there is no international consensus on a recommended method for antimicrobial susceptibility testing. Worldwide at least twelve different in vitro methods are followed, and only in Europe the number is at least ten [ 5 ]. Furthermore, there are ongoing changes in the interpretive criteria for susceptibility testing [ 10 ]. In addition to this, there is an abundance of molecular methods to describe various genetic markers of resistance. In vivo clinical assessment is of great importance in understanding bacterial drug resistance and the gold standard for evaluating resistance in malaria parasite. The multitude of methods employed for antimicrobial susceptibility testing has to some extent hampered the meaningful sharing and comparison of resistance data among countries. Recently, much work has been done in Europe to harmonize resistance surveillance efforts across country borders [ 33 , 34 ]. While many laboratories record inhibition zones for disk diffusion results, interpretation is usually according to national guidelines. Thus, susceptibility patterns from different countries must be compared prudently. The lack of standardization in methods is a problem that must be addressed at an international level. The surveillance system as a quality assurance tool The implementation of the surveillance system brought focus on methodological issues, including microbial identification and susceptibility. The WHONET software has built-in functions to alert the operator if isolates with unexpected resistance patterns are entered. During the surveillance exercise in our laboratory, it was discovered that four isolates of Streptococcus pyogenes were reported as resistant to penicillin. This was subsequently double-checked, and consulting the laboratory bench-book we found that clerical errors were the explanation for this. The use of the surveillance software enabled the easy detection, investigation and correction of such errors, and consequently may contribute to increase the attention to quality issues and generally improve the performance of the lab. The current surveillance project highlighted some methodological issues, most of which were caused by budgetary limitations, such as the occasional use of locally made antibiotic disks and limitations in the identification of organisms due to lack of reagents. Impetus for further research Routine surveillance makes use of available large data sets at little additional cost and may be representative for a greater part of the population. However, often it is necessary to supplement the routine surveillance with ad hoc studies aimed at investigating particular problems. While ad hoc studies generally are more expensive to conduct, they allow for the use of more advanced and expensive laboratory methods and are better at targeting particular populations of interest. The current surveillance study identified a need for more data from bloodstream infections in order to provide reliable guidance for the treatment of serious bacterial infections. As a consequence of this, we started a study of bloodstream infections with the pediatric department at the hospital. Another laboratory-based research was started to ascertain the finding that methicillin-resistance in staphylococci is still relatively infrequent at this hospital. Influencing popular opinion on antimicrobial resistance issues Resistance surveillance is a platform from which to promote focus on antimicrobial resistance issues, both within the hospital and the medical community, but also among the general population. In conjunction with the surveillance exercise, we have highlighted issues regarding antimicrobials and resistance in local newspaper letters [ 35 ], and there is ongoing work to establish a chapter of APUA (Alliance for the prudent use of antibiotics, ) in Tanzania. Cost considerations and human resources The study suggests that laboratories, which perform susceptibility testing, can gain useful information on antimicrobial susceptibility with a minimal budget. As appropriate software can be obtained free-of-charge, the main cost of the surveillance system is associated with purchasing a computer. However, there are other, indirect costs, which may be attributed to the surveillance program depending on the situation of the laboratory, such as running costs for microbiologic procedures, including susceptibility testing. Particularly, it is important to ensure availability of antimicrobial discs of satisfactory quality. A susceptibility surveillance system also implies the need for some additional human resources for data entry and analysis. In our experience, it is recommendable to allocate approximately 50% of a laboratory technologist position to this task. While the WHONET program is excellent for entry, analysis and reporting of resistance data, the software is not intended to function as a complete patient management system for the laboratory. Data can be transferred from other databases into WHONET by the use of a complementary software called BacLink (also free-of-charge). However, in laboratories such as ours, where the management of patients' laboratory tests (i.e. receipt of specimens and laboratory forms, inscription in registers, return of test results, etcetera) is handled manually via register-books, the data must be punched into WHONET by hand. Since the WHONET database is not used directly for patient management, the surveillance activity tends to become less integrated in the clinical routine work than it should. Thus, although the program performs its task very well, in a long-term perspective, a surveillance system that is integrated with a patient management system might be more sustainable. It is difficult to quantify the potential benefits of a well-functioning surveillance system. However, we are fully convinced that the modest costs of the surveillance program are highly justified since the data generated may improve empiric therapy, help contain or prevent the further emergence of antimicrobial resistance, decrease the need for expensive second-line antimicrobial drugs and, ultimately, save lives and reduce suffering. Conclusions It is imperative to preserve the effectiveness of common antibiotics by promoting rational use of antibiotics based on sound knowledge of local resistance patterns. In a hospital with bacteriology services, the implementation of a computerized surveillance system is a low-cost tool to make use of available resistance data. In our hospital, the resistance surveillance system has generated information on resistance patterns that is useful as guidance for empiric therapy of infections. It can help alert clinicians of trends of antimicrobial resistance, guide drug-policy decisions and facilitate rational use of drugs to prevent the further emergence of antimicrobial resistance. The surveillance system has also served as a quality assurance tool and led to increased focus on antimicrobial resistance and prudent use of drugs. There is need for more data from blood cultures for reliable guidance for the treatment of severe, systemic bacterial infections. For antibiotic policy recommendations to be applicable for the general population, more information is needed from outpatients and rural areas. There is limited information on antimicrobial resistance trends on the African continent. Only four African countries use the WHONET system for antimicrobial resistance surveillance, although some countries may use other similar software. Recently much work has been done to establish consensus and a more standardized approach to resistance surveillance in Europe [ 34 ]. Susceptibility data based on recorded zone diameters, instead of interpreted values ("S", "I" and "R"), would make the surveillance system more effective in detecting subtle changes in antimicrobial resistance. We believe there is a need for a standardized approach to antimicrobial resistance surveillance also in the African region, as well as globally. This would facilitate liaisons and sharing of information among countries. Competing interests The authors declare that they have no competing interests. Authors' contributions BB was the principal investigator, participated in the planning and execution of the study, performed data entry and data analysis, and was the main responsible author. DSMM, WU, SYM and AD participated in the planning of the study and contributed to the writing process. MM contributed to designing the WHONET database, performed data entry and microbiological work, and contributed to the writing process. SH participated in the writing. NL was the project coordinator and participated in planning, data analysis and writing. Pre-publication history The pre-publication history for this paper can be accessed here:

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549038

Cross-talk between phosphatidic acid and ceramide during ethanol-induced apoptosis in astrocytes

Background Ethanol inhibits proliferation in astrocytes, an effect that was recently linked to the suppression of phosphatidic acid (PA) formation by phospholipase D (PLD). The present study investigates ethanol's effect on the induction of apoptosis in astrocytes and the formation of ceramide, an apoptotic signal. Evidence is presented that the formation of PA and ceramide may be reciprocally linked during ethanol exposure. Results In cultured rat cortical astrocytes, ethanol (0.3–1 %, v/v) induced nuclear fragmentation and DNA laddering indicative of apoptosis. Concomitantly, in cells prelabeled with [ 3 H]-serine, ethanol caused a dose-dependent, biphasic increase of the [ 3 H]-ceramide/ [ 3 H]-sphingomyelin ratio after 1 and 18 hours of incubation. As primary alcohols such as ethanol and 1-butanol were shown to inhibit the phospholipase D (PLD)-mediated formation of PA, a mitogenic lipid messenger, we tested their effects on ceramide formation. In astrocytes prelabeled with [ 3 H]-serine, ethanol and 1-butanol, in contrast to t-butanol, significantly increased the formation of [ 3 H]-ceramide. Moreover, exogenous PA, added to transiently permeabilized astrocytes, suppressed ethanol-induced [ 3 H]-ceramide formation. Vice versa, addition of C 2 -ceramide to astrocytes inhibited PLD activity induced by serum or phorbol ester. Conclusion We propose that the formation of ceramide in ethanol-exposed astrocytes is secondary to the disruption of phospholipase D signaling. Ethanol reduces the PA:ceramide ratio in fetal astrocytes, a mechanism which likely participates in ethanol-induced glial apoptosis during brain development.

Background The proliferation of astrocytes is stimulated by polypeptide growth factors such as PDGF, EGF, bFGF and IGF-1 acting on cellular signaling pathways which involve tyrosine kinases, protein kinase C, and the Ras-Raf-MAP kinase pathway [ 1 , 2 ]. Astroglial proliferation is also stimulated by neurotransmitters such as acetylcholine and glutamate [ 3 , 4 ], by direct stimulation of protein kinase C with phorbol ester [ 5 , 6 ], and by peptides such as endothelin and prolactin [ 7 , 8 ]. Astroglial proliferation is prominently inhibited by ethanol both in vivo and in vitro [ 9 - 11 ], and this interference likely contributes to the development of the fetal alcohol syndrome (alcoholic embryopathy) (reviewed in [ 12 ]). Ethanol has been shown to potently antagonize proliferative effects of several individual astroglial mitogens including PDGF, IGF-1, acetylcholine and prolactin [ 8 , 13 - 15 ]. The molecular target of ethanol's antimitogenic actions in astroyctes is not known with certainty, but inhibitory interactions of ethanol with lipid signaling pathways have been implicated [ 15 ]. Our group has recently reported strong evidence that the growth-inhibitory effect of ethanol in astrocytes is caused by the disruption of the phospholipase D (PLD) signaling pathway [ 16 , 17 ]. Under physiological conditions, PLD catalyzes the hydrolysis of phosphatidylcholine (PC) to yield phosphatidic acid (PA) and choline. In the presence of ethanol, however, PLD forms phosphatidylethanol (PEth), a non-physiological phospholipid, at the expense of PA. This PLD-specific phenomenon of transphosphatidylation is the reason why downstream events mediated by PLD activation and PA formation are dose-dependently inhibited in the presence of ethanol (or other primary alcohols such as 1-butanol). In our previous work, we have found that astroglial PLD is activated by mitogenic factors including fetal calf serum (FCS), PDGF, and phorbol ester, and we observed that ethanol reduced both astroglial proliferation and PA formation in a parallel manner. 1-butanol reduced PA formation and DNA synthesis with the same potency while t-butanol was inactive for both effects [ 16 ]. More recently, we demonstrated that exogenous PLD as well as PA, when introduced into the cytosol by transient permeabilization, stimulated astroglial cell proliferation. Importantly, the action of PLD was suppressed in the presence of ethanol (0.3 %, v/v) while the mitogenic effect of PA was not affected [ 17 ]. Thus, disruption of the PLD signaling pathway by ethanol is sufficient to suppress astroglial cell proliferation. Recent findings from other groups are also compatible with a central role for the PLD signaling pathway in ethanol toxicity in astrocytes. First, several mitogenic factors including those that are known to be particularly sensitive to ethanol activate PLD activity in astrocytes. This holds true for PDGF [ 16 ], acetylcholine [ 5 , 18 ], glutamate [ 19 ], phorbol esters [ 5 , 6 ], endothelin [ 20 , 21 ], and prolactin [ 22 ]. In fact, disruption of PLD signaling by ethanol was recently found to be responsible for ethanol's inhibitory effect on astroglial DNA synthesis induced by muscarinic agonist [ 23 ]. Second, PLD is activated via the mitogenic Ras-Ral pathway in many cell types [ 24 ], and PA, the immediate product of PLD activity, interacts with and activates proteins such as Raf kinase, protein kinase Cζ, and mTOR which are known to be central to mitogenic signaling (reviewed in [ 25 , 26 ]). In addition, PA is a precursor of diacylglycerol (DAG), the endogenous activator of classical PKC's, and of lyso -PA, a potent mitogen in many cell types [ 25 , 26 ]. Taken together, current evidence suggests that intact PLD signaling is a prerequisite for the proliferative effects of several mitogens, and that disruption of the PLD pathway by ethanol may be a common theme in ethanol-induced inhibition of astroglial proliferation. The present study in fetal astrocytes was motivated by recent reports that ethanol induces apoptosis in astrocytes, an effect that was accompanied by activation of the sphingomyelinase pathway and formation of ceramide [ 27 , 28 ]. Apoptosis denotes an active cellular program causing cellular death upon contact with toxicants. Apoptotic cell death in the CNS has been under intensive study in recent years and involves several intracellular reaction cascades linked by the activation of caspases (reviewed in [ 29 ]). Apoptosis is almost universally accompanied by the formation of ceramide which may occur through de novo -synthesis, inhibition of ceramide breakdown, or activation of (acidic and/or neutral) sphingomyelinase (SMase), an enzyme which catalyzes the hydrolysis of sphingomyelin to ceramide and phosphocholine (see [ 30 ] for review). Ceramide has emerged as a second messenger for apoptotic pathways targeting kinases and phosphatases which are required for the execution of apoptotic cell death (reviewed in [ 31 , 32 ]). In cerebellar astrocytes and in glioma cells, ceramide levels were found to be reciprocally related to cell proliferation [ 33 , 34 ]. For the present study, we developed the hypothesis that the formation of ceramide may be secondary to the inhibition of PLD signaling which we had described earlier (see above). We now report that ethanol-induced ceramide formation in astrocytes is mimicked by 1-butanol, but not by t-butanol, and that PA, the product of PLD activity, antagonizes ethanol-induced formation of ceramide. We also found that ceramide is a potent inhibitor of stimulated PLD activity. Thus, we obtained evidence of a cross-talk between PA and ceramide, two lipid messengers with opposite effects on cellular proliferation. Results Markers of apoptosis When primary astrocyte cultures were exposed to ethanol (0.3–1%, v/v), staining of the cells with Hoechst 33258, a dye intercalating into DNA, revealed condensation and fragmentation of the nuclei which was visible after 16 hrs; the maximum effect was observed after 21 hours (Fig. 1A ). Higher magnification demonstrated the presence of "apoptotic bodies" in the nuclei (Fig. 1B ). A similar effect was observed after treatment of the cells with the well-known apoptogen, staurosporine (1 μM), or with C 2 -ceramide (50 μM) but not with t-butanol (1%, v/v) (not illustrated). In parallel experiments, ethanol caused fragmentation of nuclear DNA in serum-starved astrocytes which is reflected by "DNA laddering" on agarose electrophoresis (Fig. 2 ). Serum withdrawal alone was not effective while incubation with C 2 -ceramide (50 μM) mimicked the effect of ethanol. Ethanol at 0.3% (v/v) was almost as effective as 1 % (Fig. 2 ). Figure 1 Nuclear fragmentation of astrocytes after treatment with ethanol. In this experiment, astrocytes were incubated with ethanol (1 %, v/v) for 21 hours, fixed in methanol/acetic acid (3:1) and stained with bisbenzimide (Hoechst 33258, 1 μg/ml). The characteristic condensation and fragmentation of nuclei indicates apoptosis. Enlargement: left picture, 400 fold; right picture, 1,000 fold. The experiment was repeated three times with similar results. Figure 2 DNA fragmentation in astrocytes after treatment with ethanol. Astrocytes were incubated with the compounds given: lane 1, control (serum-free medium); lane 2, serum-containing medium; lane 3, C 2 -ceramide (50 μM) in serum-free medium; lanes 4–7, ethanol in serum-free medium (concentrations and times as given); lane 8, size markers. After incubation, cells were lysed, DNA was purified, separated on a 3 % agarose gel and stained with ethidium bromide. The experiment was repeated three times with identical results. Effects of ethanol on sphingomyelin hydrolysis Formation of [ 3 H]-ceramide was measured after labeling sphingomyelin with [ 3 H]serine. Under basal conditions, the ratio of [ 3 H]-ceramide to [ 3 H]-sphingomyelin ("C/S ratio") was approximately 1:30. This ratio was not significantly changed during serum withdrawal (Figs. 3 and 4 ). The incubation of astrocytes with ethanol (1 %, v/v) in serum-free medium caused an increase of [ 3 H]-ceramide (cpm per dish) but did not significantly change the total labeling of the large pool of [ 3 H]-sphingomyelin by [ 3 H]-serine (data not shown). As the total incorporation of [ 3 H]-serine into [ 3 H]-sphingomyelin was somewhat variable between individual preparations, we used the C/S ratio to calculate ethanol-induced changes. As shown in Figs. 3 and 4 , ethanol caused a significant increase of the astroglial C/S ratio in a biphasic and dose-dependent manner. Figure 3 Formation of ceramide in ethanol-treated astrocytes: time course. Astrocytes were labeled with [ 3 H]-serine for 72 hours, washed and treated with ethanol (0.3 %, v/v) in serum-free medium. At the indicated time points, the cells were extracted with methanol/chloroform (2:1), lipid extracts were separated by TLC, and radioactivity associated with [ 3 H]-ceramide and [ 3 H]-sphingomyelin was determined by liquid scintillation counting. Data (N = 3–7) are means ± S.E.M. and are expressed as [%] ceramide/sphingomyelin. Statistics: ANOVA, F 1,53 = 2.28, p = 0.02. *, p < 0.05 vs. control at time zero (Dunnett's post test). Figure 4 Formation of ceramide in ethanol-treated astrocytes: concentration dependence. Astrocytes were labeled with 3 H-serine for 72 hours, washed and treated with ethanol (0.1–1 %, v/v). After (A) 1 hour and (B) 18 hours, the cells were extracted with methanol/chloroform (2:1), phospholipids were separated by TLC, and the radioactivity associated with ceramide and sphingomyelin was determined by liquid scintillation counting. Data (N = 5–6) are means ± S.E.M. and are expressed as [%] ceramide/sphingomyelin. Statistics: one-way ANOVA for repeated measurements, (A) F 3,19 = 3.98, p = 0.03; (B) F 3,23 = 4.88, p = 0.02. *, p < 0.05 vs. controls (Dunnett's post test). The rapid and transient phase of ceramide formation occurred within 15 min and reached a maximum at 1 hour after addition of ethanol (1 h value without ethanol: 2.93 ± 0.35%; 1 h value with 0.3% ethanol: 3.92 ± 0.60%; p = 0.02). A second increase gradually developed after 4 hours and reached a maximum at 18 hours of ethanol exposure (18 h value without ethanol: 3.46 ± 0.41 %; 18 h value with ethanol: 4.18 ± 0.51 %; p = 0.005). At this later time point, staurosporine (1 μM) caused an increase of the C/S ratio to 7.97 ± 1.78 % (p < 0.01; not illustrated). Inhibition of ceramide formation by PLD activity and phosphatidic acid To investigate whether ceramide formation is secondary to a disruption of PLD signaling, we used the isomeric alcohols, 1-butanol and t-butanol. 1-Butanol – but not t-butanol – is a substrate of PLD for transphosphatidylation and leads to the formation of phosphatidyl-1-butanol at the expense of PA. The correlations between butanol exposures and disruption of PLD signaling (i.e., suppression of PA formation) in astrocytes were documented in detail in our previous work [ 16 ]. In the present experiments we measured the differential effects of 1- and t-butanol on the formation of ceramide (C/S ratio) in astrocytes. As shown in Fig. 5 , there was a tendency for an increased level of the C/S ratio after addition of 1-butanol at 0.1% while highly significant increases were observed with 0.3%. In contrast, t-butanol (0.1 and 0.3%) had no effect. This pattern was identical at the 1 hour and 18 hours time points (Fig. 5A and 5B ). This pattern suggested a change of the C/S ratio that was secondary to the disruption of the PLD pathway. Figure 5 Effects of isomeric butanols on ceramide formation in astrocytes. Astrocytes were prelabeled with [ 3 H]-serine for 72 hours, washed and treated with 1-butanol ("1-But") or t-butanol ("t-But"). After (A) 1 hour and (B) 18 hours, the cells were extracted with methanol/chloroform (2:1), phospholipids were separated by TLC, and the radioactivity associated with ceramide and sphingomyelin was determined by liquid scintillation counting. Data (N = 8–10) are means ± S.E.M. and are expressed as [%] ceramide/sphingomyelin. Statistics: Repeated measures ANOVA, (A) F 4,49 = 7.2, p = 0.0002; (B) F 4,39 = 25.0, p < 0.0001. *, p < 0.05; **, p < 0.01 vs. controls ("Ctr"). #, p < 0.05; ##, p < 0.01 vs. effect of 1-butanol (Tukey-Kramer multiple comparisons test). To obtain more direct evidence for this hypothesis, we tested the influence of exogenous PA, the product of PLD, on the C/S ratio. For this purpose, we used a permeabilization procedure which makes use of an oxygen-insensitive mutant (C530A) of streptolysin-O (SL-O) to introduce the membrane-impermeable PA into the astroglial cytosol [ 17 ]. In preliminary experiments, we found that transient permeabilization with SL-O for 15 min by itself did not affect [ 3 H]-ceramide levels (data not shown). Basal ceramide levels were also unchanged if exogenous PA was added to the astrocytes in the absence (data not shown) or presence of SL-O (Fig. 6 ). However, the ceramide formation evoked by ethanol (0.3 %) was significantly reduced in the presence of PA (Fig. 6 ). At the 1 h timepoint, the ethanol-induced effect (C/S ratio: 5.14 ± 0.53 %) was reduced by PA pretreatment to 3.98 ± 0.33 %, a relative reduction by 71 percent (Fig. 6A ). At the 18 h timepoint, PA pretreatment reduced ethanol-induced ceramide formation by 61 percent (Fig. 6B ). Figure 6 Effects of phosphatidic acid on ceramide formation in astrocytes. Astrocytes were prelabeled with [ 3 H]-serine for 72 hours, washed and treated with PA (200 μM) or ethanol (EtOH, 0.3 % v/v) during transient permeabilization with streptolysin-O (144 ng/ml) in calcium-free medium. After 15 min, the cultures were washed and re-exposed to calcium-containing medium to initiate pore repair. After (A) 1 hour and (B) 18 hours, the cells were extracted with methanol/chloroform (2:1), phospholipids were separated by TLC, and the radioactivity associated with ceramide and sphingomyelin was determined by liquid scintillation counting. During the experiments, PA was only present for 15 min during cell permeabilization whereas ethanol was present throughout the incubation period. Data (N = 9) are means ± S.E.M. and are expressed as [%] ceramide/sphingomyelin. Statistics: Repeated measures ANOVA, (A) F 3,35 = 5.52, p = 0.005; (B) F 3,35 = 14.5, p < 0.0001. **, p < 0.01 vs. controls ("Ctr"). #, p < 0.05; ##, p < 0.01 vs. effect of ethanol (Tukey-Kramer multiple comparisons test). Inhibition of phospholipase D activity by ceramide As the previous experiments indicated an inhibitory effect of the PLD pathway on ceramide formation, the following experiment tested a possible effect of ceramide on PLD activity measured by the transphosphatidylation assay. In these experiments, the membrane-permeable C 2 -ceramide (50 μM) slightly but non-significantly reduced basal PLD activity by 30% (Fig. 7 ). However, when PLD activity was stimulated by addition of serum (Fig. 7A ) or by PDB, a phorbol ester and stimulator of protein kinase C (Fig. 7B ), C 2 -ceramide at both 10 and 50 μM strongly and significantly reduced PLD activity. Interestingly, serum-induced PLD stimulation was more sensitive to ceramide than PDB-induced PLD activity; the relative inhibitions for 10 and 50 μM C 2 -ceramide were (for serum stimulation) 84 and 93 % and (for PDB stimulation) 53 and 64 %, respectively. Figure 7 Inhibition of phospholipase D activity by ceramide. PLD activity in serum-starved, [ 3 H]-glycerol-labeled astrocytes was determined by the transphosphatidylation assay; in the presence of ethanol, PLD converts [ 3 H]-phosphatidylcholine (PC) into [ 3 H]-phosphatidylethanol (PEth) which reflects PLD activity. In (A), PLD activity was stimulated by addition of medium containing 10% fetal calf serum (FCS) for 5 min. In (B), PLD activity was stimulated by 4β-phorbol-12β,13α-dibutyrate (PDB; 1 μM) for 30 min. C 2 -ceramide ("C 2 ") was added to the cultures in concentrations of 10 and 50 μM 45 min before the addition of FCS and PDB, respectively. Data are means ± S.E.M. of 7–8 experiments and are expressed as [%] PEth/PC. Statistics: ANOVA, (A) F 4,36 = 14.0, p < 0.0001; (B) F 4,37 = 75.2, p < 0.0001. **, p < 0.01 vs. controls. ##, p < 0.01 vs. stimulated PLD activity (Tukey-Kramer multiple comparisons test). Discussion We have previously documented that ethanol suppresses signaling through the mitogenic phospholipase D (PLD) pathway, and we and others have provided evidence that this effect may be responsible for the antiproliferative actions of ethanol in astrocytes [ 16 , 17 , 23 ]. The present study was motivated by findings that ethanol induces astroglial apoptosis via activation of the sphingomyelinase pathway [ 27 , 28 ]. We confirm these earlier reports by showing the induction of apoptotic markers and ceramide formation in ethanol-treated astrocytes. The novel finding of our study is that ethanol-induced formation of ceramide is reciprocally regulated by phosphatidic acid (PA) and the phospholipase D pathway which is itself inhibited by ceramide. We used three different approaches to demonstrate that apoptotic cell death in astrocytes can be induced by exposure to ethanol. First, we report that ethanol can induce nuclear condensation and degradation (Fig. 1 ). Second, application of ethanol to serum-starved astroglial cultures caused "DNA laddering", a typical hallmark of apoptotic degradation of nuclear DNA (Fig. 2 ). The effect of ethanol was mimicked by a cell-permeable ceramide, C 2 -ceramide (Fig. 2 ), and by staurosporine (not illustrated). Third, ethanol induced an increase of ceramide in astroglial cultures (Figs. 3 and 4 ). Our findings clearly confirm that ethanol can induce apoptosis and ceramide formation in astrocytes, a finding which is in agreement with some [ 28 ] but not all [ 35 ] previous studies. We performed a time course of [ 3 H]-ceramide formation after ethanol exposure and found that it was maximal at 1 and 18 hours. Biphasic formations of ceramide such as those observed here have been described previously in a range of peripheral cell types although their significance is unclear; they may reflect different modes of ceramide formation, or different pools of ceramide [ 36 ]. At both time points of maximum ceramide formation (1 hour and 18 hours), we observed the same ethanol-evoked enhancement of ceramide formation. Importantly, apoptotic cell death and ceramide formation were induced by ethanol levels as low as 65 mM which corresponds to blood alcohol levels (0.3%) which are found in heavy drinkers. It should be noted that the present experiments do not unequivocally identify the mechanism of ceramide formation. We used [ 3 H]-serine to pre-label sphingomyelin for 72 hours, removed the precursor, and measured formation of ceramide as an increase of the [ 3 H]-ceramide/ [ 3 H]-sphingomyelin ratio during incubations with ethanol. This ratio most likely reflects the activity of sphingomyelinase(s), and sphingomyelinase activity was actually shown to be responsible for ethanol-induced ceramide formation in a recent study [ 28 ]. However, our present data do not exclude alternative pathways of increased ceramide formation such as de novo -synthesis of ceramide or inhibition of ceramidase. The important findings of this study relate to the interaction between lipid second messenger pathways. It was known from previous work (see Introduction) that PA, the product of phospholipase D (PLD), mediates mitogenic stimulation in astrocytes whereas formation of ceramide by sphingomyelinase activation accompanies apoptosis. We now tested the hypothesis that ethanol causes astroglial apoptosis by inhibiting PLD and, as a consequence, stimulates the sphingomyelinase pathway. The results shown in Figs. 5 and 6 are evidence of a direct inhibitory influence of the PLD pathway on ceramide formation. First, we observed that the addition of 1-butanol, a primary alcohol which suppresses PLD signaling, caused an increase of ceramide levels (Fig. 5 ). This effect was not seen with the inactive isomer t-butanol which does not interfere with PLD signaling in astrocytes (see our previous study [ 16 ]). Second, we used transient permeabilization of astrocytes by streptolysin-O to introduce PA, the product of PLD, into the astroglial cytosol [ 17 , 37 ]. We found that exogenous PA almost completely prevented the ethanol-induced increase of ceramide at early (1 hr) and delayed (18 hrs) phases of ceramide formation (Fig. 6 ). The fact that ethanol and 1-butanol, but not t-butanol increase ceramide formation, whereas PA antagonized this effect, gives strong evidence that PLD-mediated formation of PA keeps ceramide levels low under basal conditions (Fig. 5 ), and that PLD activity antagonizes ceramide formation under the influence of toxicants (Fig. 6 ). Unfortunately, we could not determine the effect of exogenous PA on astroglial apoptosis because the permeabilization procedure was found to induce a delayed apopototic response in astrocytes (not shown). We also probed the reciprocal effects of ceramide signaling on PLD activity. The results shown in Fig. 7 demonstrate that C 2 -ceramide inhibits PLD activity at a concentration as low as 10 μM. Basal PLD activity was only slightly inhibited, but the increases of PLD activity induced by addition of serum or phorbol ester were strongly antagonized. This finding in astrocytes corroborates previous reports that ceramide can inhibit PLD signaling in peripheral cell types [ 38 , 39 ]. It remains a matter of speculation why serum-induced PLD was somewhat more susceptible to inhibition by ceramide than phorbol ester-stimulated activity. Growth factors in serum and phorbol ester may affect different signaling pathways leading to PLD activation, and we previously presented inhibition data with bacterial toxins which supported this idea for astroglial PLD [ 40 ]. Interestingly, ethanol was also observed to inhibit serum- and growth factor-mediated astroglial proliferation more effectively than phorbol ester-induced proliferation [ 6 , 16 ]. At this time, we cannot distinguish which isoform of PLD is responsible for PA formation; previous attempts to selectively down-regulate astroglial PLDs failed due to the long biological half-lives of the proteins [ 41 ]. The molecular target of ceramide for inhibiting PLD also remains to be identified; previous work has implicated direct inhibition of PLD by ceramide as well as upstream molecules such as protein kinase C which activate PLD [ 42 , 43 ]. Conclusions In summary, the present results give evidence of a cross-talk between lipid-signaling pathways in astrocytes such that the product of PLD, namely PA, inhibits ceramide formation whereas ceramide inhibits PLD activation (Fig. 8 ). The experimental evidence suggests that the ratio "PA:ceramide" contributes to the decision whether astrocytes proliferate or undergo apoptosis. Our data suggest that ethanol induces astroglial apoptosis during brain development by disrupting PLD signaling, thereby reducing PA and increasing ceramide formation. This effect likely contributes to the microencephaly and delay of brain development observed in fetal alcohol syndrome. Figure 8 Hypothetical cross-talk between phospholipase D and sphingomyelin pathways in astrocytes, and effect of ethanol. Phosphatidic acid (PA), the product of phosphatidylcholine (PC) hydrolysis by phospholipase D (PLD), inhibits hydrolysis of sphingomyelin (SM) by sphingomyelinase (SMase). Vice versa, ceramide (Cer), the product of SM hydrolysis by SMase, inhibits activation of PLD. Ethanol induces apoptosis by disrupting the mitogenic PLD signaling pathway thereby decreasing the PA:Cer ratio and disinhibiting the pro-apoptotic SMase pathway. Methods Materials [ 3 H]-Serine and [ 3 H]-glycerol were from Biotrend (Köln, Germany). Ceramide (from bovine brain), C 2 -ceramide (N-acetyl-D- erythro -sphingosine), and staurosporine were from Alexis (Lausen, Switzerland). Hoechst 33258, L-α-phosphatidic acid (sodium salt, from egg yolk) and 4β-phorbol-12β,13α-dibutyrate (PDB) were from Sigma (Deisenhofen, Germany); most other chemicals and TLC plates were obtained from Merck (Darmstadt, Germany) or Roth (Karlsruhe, Germany) at the highest purity available. Fetal calf serum (South America) was from Invitrogen, cell culture materials were from Sarstedt (Nürnbrecht, Germany). Phosphatidylethanol (PEth) standard was synthesized as described [ 16 ]. Recombinant, oxygen-insensitive streptolysin-O (C530A) was prepared as described [ 44 ]. Cell culture Astrocyte-rich cultures were prepared from newborn rat pups. Cerebral hemispheres were collected, meninges and blood vessels were removed, the brain tissue was dissociated by trituration, passed through a 50 μm nylon mesh, and the cells were seeded onto plastic culture dishes (14,000 cells per cm 2 ). The growth medium was DMEM containing 10% fetal calf serum (FCS), 2 g/l NaHCO 3 , 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were incubated at 37°C in a 95/5% mixture of air and carbon dioxide. For the experiments, astrocytes were grown for two weeks in culture and were used when they reached confluency. As judged by GFAP immunostaining, these cultures contained >90% astrocytes. Fluorescence microscopy Cells were seeded on microplates (5,000 cells per 200 μl) and incubated with different apoptogens (ethanol 0.3 and 1 %, C 2 -ceramide 50 μM or staurosporine 1 μM) for 24 hrs in serum-free medium. Subsequently, cells were washed and fixed with ice-cold methanol/acetic acid (3:1). Dried and re-hydrated cells were stained with bisbenzimide (Hoechst 33258, 1 μg/ml) solution for 10 min, washed and sealed in gelatin. Photographs were obatined using a Leica Leitz DMRB fluorescence microscope and a Nikon Digital Camera DXM. DNA fragmentation The test was carried out as described [ 45 ] with some modifications. Briefly, cells were incubated with apoptogens (Ethanol 0,3%, 1%; C2-Ceramide 50 μM) in serum-free medium. Then, the cells were transferred and resuspended in Tris-EDTA buffer containing 0.5% Igepal CA 630. Further lysis was performed in buffer containing RNAse A (100 μg/μl), proteinase K (0,5 μg/ml) and SDS (1.2 %). After 5 minutes, the clear solution was mixed with 3 M CsCl in acetate buffer. Precipitated debris and chromosomal DNA was removed by centrifugation, and the supernatant was loaded onto a QIAprep column (twice), centrifuged and eluted by hypotonic Tris-EDTA buffer. The eluate was analyzed by electrophoresis on a 3 % agarose gel and visualized with ethidium bromide. Measurement of [ 3 H]-ceramide formation Phospholipids were labeled by addition of [ 3 H]-serine (1 μCi/ml) to astrocytes kept in growth medium (DMEM plus FCS) for 72 h. After washing, cells were incubated in serum-free medium with ethanol, butanol, C 2 -ceramide or staurosporine (see Results). C 2 -ceramide or staurosporine were added in DMSO; the final DMSO concentration was < 0.1 %. At the end of the incubation, cells were fixed with methanol, transferred and extracted first with with chloroform: methanol (1:1), then with chloroform: methanol: water (10:10:9) to separate water and lipid phases. After addition of ceramide and sphingomyelin standards, the lower (lipid) phase was evaporated, taken up in chloroform:methanol (3:2) and separated by thin-layer chromatography (TLC). For the determination of [ 3 H]-ceramide, we used one-dimensional TLC (HP-TLC plates Merck 11845; eluent: chloroform/acetic acid 9:1). For the determination of [ 3 H]-sphingomyelin, we used two-dimensional TLC (TLC plates Merck 1.05721); solvent I was chloroform/methanol/25% aqueous ammonia (13:7:1), solvent II was chloroform/ methanol/water/acetic acid (30:30:2:5). 3 H-lipids were visualized by using iodine vapor, spots were scraped and suspended in scintillation cocktail (Lumasafe Plus), and radioactivity was measured by liquid scintillation counting (Packard 1600 CA). Formation of [ 3 H]-ceramide was calculated as percentage of radioactivity found in [ 3 H]-sphingomyelin. Cell permeabilization For the introduction of PA, astrocytes were transiently permeabilized with streptolysin-O [ 37 ]. Briefly, astrocytes were washed and exposed to an oxygen-insensitive mutant of streptolysin-O (C530A) in calcium-free HBSS buffer (prepared by dissolving 8 g NaCl, 0.4 g KCl, 60 mg KH 2 PO 4 , 60 mg Na 2 HPO 4 × 2 H 2 O, 100 mg glucose in 1 L of sterile water). PA (sodium salt; 200 μM) was added as a suspension in buffer prepared by sonication. After 15 min, the cells were washed and incubated in serum-free, calcium-containing DMEM. Formation of [ 3 H]-ceramide was determined as described above. Ethanol, if present, was present throughout the incubation period. Using 144 ng/ml of streptolysin-O, this procedure yielded transient permeabilization of > 80% of astrocytes followed by repair of the pore in > 80% of permeabilized cells. The procedure was previously found to allow the entry of approx. 10 6 molecules of PA per cell [ 17 ]. Determination of phospholipase D activity Phospholipase D activity was determined using the transphosphatidylation assay [ 46 ]. For this purpose, astrocytes were kept in serum-free medium containing [ 3 H]-glycerol (1 μCi/ml) for 24 h in order to label phospholipids. More than 60% of the phospholipid label was associated with phosphatidylcholine (not illustrated). Subsequently, the cells were washed and re-exposed to medium containing ethanol (2 %) and PDB (1.0 μM) or FCS (10 %, v/v) as stimulators. PDB and C 2 -ceramide, when used, were dissolved in DMSO (end concentration of DMSO < 0.1 %). After 30 min of reaction time, the cells were washed in cold phosphate-buffered saline and extracted as described above for ceramide determinations. After addition of phosphatidylethanol (PEth) and PA standards, aliquots of the lipid phase were separated by two-dimensional TLC (TLC plate Merck 1.05721) using chloroform/methanol/25 % aqueous ammonia (13:7:1) for the first run and the upper phase of ethylacetate/isooctane/ acetic acid/water (13:2:3:10) for the second run. Individual phospholipids were stained by iodine, and the spots corresponding to PEth, PA and phosphatidylcholine (PC) were isolated and counted for radioactivity in a scintillation counter. To determine PLD activity, formation of [ 3 H]- PEth was calculated as percentage of [ 3 H]-PC. Statistics Data are shown as means ± SEM of N experiments whereby N refers to the number of different astroglial preparations from different animals. Results were obtained from two replicate dishes which were pooled to represent a single experiment. Statistical calculations were performed by GraphPad InStat 3.0 program package, using analysis of variance (ANOVA) of paired or unpaired data as indicated in text and figure legends. Abbreviations Cer, ceramide; FAS, fetal alcohol syndrome; FCS, fetal calf serum; PA, phosphatidic acid; PC, phosphatidylcholine; PDB, 4β-phorbol-12β,13α-dibutyrate; PEth, phosphatidylethanol; PKC, protein kinase C; PLD, phospholipase D; SL-O, streptolysin-O; SM, sphingomyelin. Author's contributions B.S. carried out the cell culture experiments and phospholipid measurements and participated in the experiments concerning apoptotic markers. S.J. contributed substantially to the apoptotic marker experiments. K.L. participated in the design of the study and the final draft of the manuscript. J.K. conceived and supervised the study, performed the statistical analyses and drafted the manuscript.

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549212

In vitro bioassay as a predictor of in vivo response

Background There is a substantial discrepancy between in vitro and in vivo experiments. The purpose of the present work was development of a theoretical framework to enable improved prediction of in vivo response from in vitro bioassay results. Results For dose-response curve reaches a plateau in vitro we demonstrated that the in vivo response has only one maximum. For biphasic patterns of biological response in vitro both the bimodal and biphasic in vivo responses might be observed. Conclusion As the main result of this work we have demonstrated that in vivo responses might be predicted from dose-effect curves measured in vitro .

Background In vitro bioassay is very useful in biomedical experiments. It has the potential to yield very important data about molecular mechanism of action of any biologically active compounds. However, the major challenge for such experiments is extrapolation to in vivo responses. Unfortunately, there is a substantial discrepancy between in vitro and in vivo experiments, and there is a paucity of work directed to prediction of in vivo response from in vitro bioassay. So, the purpose of the present work was development of a theoretical framework to enable improved prediction of in vivo response from in vitro bioassay results. Results A survey of literature revealed that most cases of dose-effect curves for in vitro experiments fall into three classes. They are: • monophasic response; • biphasic pattern; • bimodal or polymodal dose-effect curve. MONOPHASIC RESPONSE is the form most commonly reported in articles on in vitro bioassay. In these cases, with increasing dose of biologically active substance (BAS), the cellular response increases to a maximum (dose-response curve reaches a plateau). The most general schemes exhibiting this class of response can be classified as 3 classes: (I) BAS regulation of enzyme activity, (II) Ligand interaction with one type of receptor, and (III) Ligand interaction with negatively cooperative receptors. We will consider these three classes: (I): BAS might regulate enzyme activity. It might be: • substrate: E+S ←→ ES → E+P → cell response ,     (scheme 1) where E is enzyme, S is substrate, ES is enzyme-substrate complex, P is product. Cellular response is suggested to be proportional to product concentration. Scheme (2) approximates the classic Michaelis scheme [ 1 ]. • enzyme activator ( A ) E+S ←→ ES → E+P → cell response E+A ←→ EA (scheme 2) EA+S ←→ EAS → EA+P → cell response increasing , Scheme (3) is characteristic of many BAS. The majority of these groups are vitamins and minerals, which are known to be enzyme cofactors and serve to increase enzyme activity. • enzyme inhibitor ( I ) E+S ←→ ES → E+P → cell response E+I ←→ EI → no cell response ,     (scheme 3) For example, there is the large class of drugs, whose action can be described with the help of scheme (4). This class is called "inhibitors of angiotensin-converting enzyme". These drugs are commonly used for hypertension treatment and prevention [ 2 ]. (II) Ligand interaction with one type of receptors: L+R ←→ LR → cell response (scheme 4) where L is ligand (BAS), R is receptor, LR is ligand-receptor complex. Scheme (4) is "classic" receptor theory as described by Clark (1937) [ 3 ]. For example, kinetic schemes of such type were proved in the case of estrogen regulation of gene expression [ 4 ], apolipoprotein AI, CII, B and E synthesis [ 5 ]. (III) Ligand interaction with negative cooperative receptors L+R ←→ LR L+LR ←→ L 2 R → cell response (5) where L 2 R is complex ligand-receptor complexes. Scheme (5) is characteristic for insulin receptors [ 6 ]. Kinetic equations for schemes (1)–(5) are well known [ 7 ]. They include "classic" Michaelis [ 1 ] and Clark [ 3 ] equations. It can be shown, due to the first order Taylor series, equations for the schemes (1)–(4) can be re-formulated from particle counter theory as: y = B * x /( 1 + A * x )     (6) and for scheme (5): y = B * x 2 /( 1 + A * x 2 )     (7) where x is incoming signal ( x is BAS concentration). For scheme (1) x is substrate concentration, for scheme (2) it is activator concentration, for scheme (3) it is inhibitor concentration, for schemes (4) and (5) it is ligand concentration. y is cellular response for the in vitro system. A and B are scaling coefficients. The BAS concentration in the whole organism changes as a function of time according to equation (14) (see Methods.) i.e. x(t) = C(t) = C 0 [exp(-k el γt)-exp(-k 1 t)] (8) We used equation (8) as the incoming signal, substituted this into equations (6) and (7) and solved analytically using Math Cad 8 graphing software (MathSoft Inc., Cambridge, MA, USA) to predict in vivo responses for monomodal in vitro dose-effect curves for schemes (1)–(5). We used illustrative values from works [ 8 , 9 ] and demonstrated that for such in vitro dose-effect curves, the in vivo response has only one maximum (fig. 1 ). Figure 1 In vivo response for monophasic dose-effect curves measured in vitro . B = 1. a) equation (6), b) equation (7). k el = 0.0714 1/min, k 1 = 0.0277 1/min, C 0 = 1 nM, γ = β. Illustrative values for fig. 1, 2, 4 taken from Veldhuis et al., (1993) [8] and similar to those measured by Baumann et al., (1987) 9 for the clearance of growth hormone (GH). We define β (degree of conjugation) as the proportion of BAS that is free of binding proteins and is available to interact with cognate receptors. The larger is β, the larger the proportion of "free" BAS (see Methods). For equation (6) the value of this maximum is increasing as β increases; for equation (7) this value is maximum for mid-range β values. BIPHASIC PATTERNS OF BIOLOGICAL RESPONSE In this case, in in vitro experiments the low doses of BAS stimulate cellular response, and the high doses inhibit it. So, a maximum is observed on the dose-response curve. The most common kinetic schemes for such response are: • Negative back loop (substrate and product inhibition): a) E+S ←→ ES → E+P → cellular response ES+S ←→ ES 2 (9) b) E+S ←→ ES → E+P → cellular response ES + P ↔ ESP Such schemes are characteristic of glucose metabolism [ 1 ]. • Presence of two receptor types: one type stimulates cellular response, another type inhibits it. L+R ←→ LR → "positive" cellular response L+R' ←→ LR' → "negative" cellular response (10) where R are receptors of the first type, R' are receptors of the second type, LR , LR ' are ligand-receptor complexes with different receptor types. This mechanism has been proven for estrogen regulation of nitric oxide synthase (activity in the rat aorta [ 10 ]; protein pS2 expression in hormone-dependent tumors [ 11 ] and so on. • Desensitization of cellular receptors L+R ←→ LR → positive cellular response LR → decrease in receptor number (11) It has been suggested, that mechanism (11) is basic for drug tolerance [ 7 ]. For example, this mechanism was described for uretal cell stimulation by 17-β-estradiol. Before estradiol treatment, expression of estrogen receptors mRNA in cells was much higher then after 12-days estradiol administration [ 12 ]. It is well known that endogenous opioid receptors become down regulated after chronic exposure to exogenous opioids [ 13 ] and receptor down-regulation has often been observed to follow acute exposure to hormones including growth hormone [ 14 ]. • Change of effector's molecule conformation: "Active" conformation + ligand suplus ←→ "Passive" conformation (12) Scheme (12) was suggested by Bootman and Lipp (1999) [ 15 ] for Ca ++ regulation of 1,4,5-trisphosphate activity. The authors suggested that Ca ++ surplus induces a change in Ca ++ -channel conformation from "open" or "active" to "closed" or "passive" [ 15 ]. For schemes (9)–(12), due to the first order Taylor series, this kinetic equation can be derived: y = A*x*exp(-B*x) (13) Using equation (13), we obtained a prediction of in vivo biphasic dose-effects curves (fig. 2 ). As is apparent from the figure, the magnitude and the analytical appearance of in vivo response is affected by the dose of BAS and its degree of conjugation (β). Both the bimodal and biphasic in vivo responses might be observed for biphasic dose-effect curves. Changes of dose of BAS concentration or its conjugation with blood proteins (or their concentration) might dramatically change the form of in vivo response. For the simulations shown in Figure 2 we used values for k el and k 1 and blood volume (4.9 liters) based on measurements by Baumann et al. (1987) [ 9 ] and Veldhuis et al. (1993) [ 8 ] for growth hormone secretion, clearance and pulsatility. Polymodal biological responses are commonly observed in biological systems. It has been demonstrated, that in some experimental systems, administration of a single, bolus dose of hormone produces a polymodal response [ 16 ]. Figure 2 In vivo response for biphasic dose-effect curves measured in vitro . B = 1. a) variation of β, C 0 = 1 nM, b) variation of C 0 , β = .388. k el = 0.0714 1/min, k 1 = 0.0277 1/min, γ = β. Bimodal dose-effect curves are usually observed for BAS with regulatory activity [ 17 , 18 ]. The mechanism of their formation is still unclear. From our point of view, bimodal dose-response curve might be described by superposition of two biphasic dose-effect curves with different B value. This might be observed in cascade system of signal transduction and amplification. If x regulate intermediate z formation in biphasic way with B 1 , and z has biphasic response on y formation with B 2 , then if B 1 < B 2 , summary dose-effect curve ( y concentration from x ) is bimodal (fig. 3 ). Differences in B 1 and B 2 value define the maximum points. For example, with B 2 increasing, the interpeak distance will also increase. Figure 3 Possible mechanism of bimodal dose-effect curve formation for in vitro systems. a) intermediate z formation as function of x concentration, B 1 = 1, b) final product y formation as function of z concentration, B 2 = 5, c)summary dose-response curve. See comments in the text of the article. For systems, which have bimodal dose-effect curve in vitro , the polymodal response in vivo is observed (fig. 4 ). The form of this response might be change to "seems constant" due to BAS concentration of β value. The differences of maximum values are observed, this differences is time-dependent: the highest maximum is observed with the longest observation. It might be demonstrated, that with change of B 2 value to 20, only bimodal in vivo response will be observed. So, the form and the value of maximums are dependent from the dose of BAS and degree of conjugation. Figure 4 In vivo response for bimodal dose-effect curves measured in vitro . B 1 = 1 , B 2 = 5 . a) variation of β, C 0 = 1 nM, b) variation of C 0 , β = .388. k el = 0.0714 1/min, k 1 = 0.0277 1/min, γ = β. Discussion Analogues of hormones are commonly used in medicine for hormone replacement therapy (for example in post-menopausal women), for oral contraception, as anabolic drugs, for asthma therapy and so on [ 2 ]. But engineered modifications of hormones, growth factors or their analogs are likely to differ from the native analogues in their affinity for binding proteins. In view of this, an important practical consequence of our simulations results are that the testing of newly designed hormones in in vivo systems (with endogenous binding proteins) will require measurements of acute biological response at multiple concentration and time points. For longer-term responses requiring protein synthesis (such as a secretion of body mass or longitudinal bone growth), it could be argued that such multiple time point studies would not be as important. However, in so far as long term biological responses are the consequence of critical initial events which may require threshold concentrations of free hormone, or repeated patterns of hormone exposure over prolonged periods [ 16 , 19 ], this assumption may not be justified. Another application of our work may be the study of hormone functions in glandular tumour disorders. With these disorders, there is usually serious metabolic or hormonal dysfunction. From our point of view, it may be not only due to gland biosynthesis of abnormal hormone. Tumour-produced hormones may not differ structurally from their normal analogues. The dysfunctional occurs due to abnormal concentrations of hormones, which are synthesised by tumours. As it follows from our results, changes in concentrations can dramatically change the form and value of biological response. On the other hand, in many tumour disorders the concentrations of binding proteins are changed. For example, in ovarian carcinoma the changes of sex binding protein and ratio free/bound sex hormones (β) are observed [ 20 ]. As follows from our results, this can dramatically change the biological response to such hormones, i.e. apparent biological functions. So with testing in vitro such hormones seems to be normal (and they may be normal), but in vivo they may have abnormal effects due to changes of their binding protein concentration, or ratio free/bound hormone. Conclusion So, as a result of this work we have demonstrated that in vivo responses might be predicted from dose-effect curves measured in vitro . For monophasic curves, in vivo response is proportional to BAS concentration. For the most complex in vitro curves, the value and the form of in vivo response depends in a predictable way on the dose of BAS and its degree of conjugation. Methods To obtain the discussed results we used linear pharmacokinetics model: where: m 1 (t) mass of biologically active substance (BAS) in the place of infusion, m 2 (t) mass of BAS in compartment (blood), k 1 , k el constants of hormone diffusion from place of infusion to blood and excretion form blood (accordingly). Many of biologically active substances are conjugate into complexes with blood proteins (for example: GH, nerves growth factor, IGF-1): B+P ⇔ K HP (15) where B is BAS, P is blood protein, BP is BAS-protein complex, K is dissociation constant. For many BAS, concentration of free (not bound with blood proteins) BAS is equal to: [B] ≈β [B 0 ] (16) where β is constant ("degree of conjugation"), [ B ] is concentration of free BAS, [ B 0 ] is initial concentration of BAS. If β = 1 then BAS dose not conjugate with protein. If β = 0 then all BAS is in conjugate form. It may be that only conjugate BAS (for example, bilirubin), or only unconjugated BAS can be excreted form the blood (for example, sex hormones). This means that for scheme (14) the law of mass action will be written in the next way: dm 1 /dt = - k 1 m 1 , m 1 (0) = M dm 2 /dt = k 1 m 1 - γ k el m 2 , m 2 (0) = 0 (17) where γ is a constant. γ = 1-β if only conjugate form of BAS can be excreted and γ = β if only unconjugated form is excreted. But γ is a constant with respect to t : γ = const(t) . This means that solution of system (17) is: C(t) = C 0 [exp(-k elγ t)-exp(-k 1 t)] (18) where C(t) is BAS concentration in the blood compartment ( C = m 2 / V , V = const (about 5 liters) is blood volume), C 0 is seems initial BAS concentration ( C 0 = M/V ).

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549212.xml

509408

Amplification of Trial-to-Trial Response Variability by Neurons in Visual Cortex

The visual cortex responds to repeated presentations of the same stimulus with high variability. Because the firing mechanism is remarkably noiseless, the source of this variability is thought to lie in the membrane potential fluctuations that result from summated synaptic input. Here this hypothesis is tested through measurements of membrane potential during visual stimulation. Surprisingly, trial-to-trial variability of membrane potential is found to be low. The ratio of variance to mean is much lower for membrane potential than for firing rate. The high variability of firing rate is explained by the threshold present in the function that converts inputs into firing rates. Given an input with small, constant noise, this function produces a firing rate with a large variance that grows with the mean. This model is validated on responses recorded both intracellularly and extracellularly. In neurons of visual cortex, thus, a simple deterministic mechanism amplifies the low variability of summated synaptic inputs into the large variability of firing rate. The computational advantages provided by this amplification are not known.

Introduction In the primary visual cortex (V1), different trials of presentation of an identical stimulus yield highly variable firing rates ( Heggelund and Albus 1978 ). This trial-to-trial variability is not inherited from subcortical inputs, as these respond in a much more consistent fashion ( Kara et al. 2000 ). Instead, variability has been related to spontaneous variations in cortical state ( Arieli et al. 1996 ; Buracas et al. 1998 ; Tsodyks et al. 1999 ; Kenet et al. 2003 ). These variations may reflect the perceptual effects associated with a stimulus, rather than the presence of the stimulus itself ( Ress and Heeger 2003 ). A key property of trial-to-trial variability is that it depends on the strength of the stimulus: Response variance across trials is approximately proportional to response mean ( Tolhurst et al. 1981 ). An example of this effect can be seen in the responses of a cell in cat V1 to drifting gratings ( Figure 1 A– 1 C). Different trials of an identical stimulus elicit firing rates that vary greatly ( Figure 1 A). As a result, the standard deviation of the firing rates is roughly comparable to their mean amplitude ( Figure 1 B and 1 C). The ratio of variance to mean is close to the value predicted for a Poisson process ( Figure 2 A, dashed line). For a Poisson process, the variance of the spike counts is equal to the mean. Once spike counts are converted to firing rate by binning in 10-ms windows (i.e., at 100 Hz), the ratio of variance to mean becomes 100. The Poisson-like behavior of firing rates is well known, although reports differ on the exact value of the ratio of variance to mean ( Tolhurst et al. 1981 ; Bradley et al. 1987 ; Vogels et al. 1989 ; Geisler and Albrecht 1997 ; Gur et al. 1997 ; Reich et al. 1997 ; Buracas et al. 1998 ; Kara et al. 2000 ). Figure 1 Variability in the Responses of a Simple Cell (A) Firing rate in response to a cycle of an optimal drifting grating. Three trials are shown. (B) Firing rate averaged over seven trials. Shaded area indicates 2 s.d. (C) Same, for three other stimuli: a grating drifting in the orthogonal direction (top), a grating drifting in the opposite direction (middle), and a blank stimulus (bottom). (D) Membrane potential trace measured for the first cycle. Dashed line is resting potential V rest . Dotted line is firing threshold V thresh (from [H]). (E–G) As in (A–C), for coarse potential. (H) Relation between firing rate and coarse potential. Curve is fit of rectification equation. (I–K) As in (A–C), for predictions of rectification model. Figure 2 Relation between Response Variance and Mean for Three Cells (A) Variance versus mean for firing rate of the simple cell in Figure 1 measured with 13 stimuli (the four in Figure 1 plus nine additional orientations). Line is linear regression. Diagonal line is prediction for a Poisson process. (B) Variance versus mean for coarse potential. Error bars are 2 s.d. Curve is linear fit to standard deviation versus mean. Dashed line is resting potential V rest . Dotted line is firing threshold V thresh . (C) Variance versus mean for firing rate predicted by the rectification model. Details as in (A). (D–F) As in (A–C) for a complex cell. (G–I) As in (A–C) for a third neuron, whose behavior is intermediate between those of simple cells and complex cells. Because the production of firing rates within a neuron introduces remarkably little noise ( Calvin and Stevens 1968 ; Mainen and Sejnowski 1995 ; Carandini et al. 1996 ), trial-to-trial variability is thought to arise from the membrane potential fluctuations that result from summated synaptic input ( Calvin and Stevens 1968 ; Stevens and Zador 1998 ). I have tested this hypothesis by considering membrane potential responses recorded intracellularly in vivo. Results From traces of membrane potential obtained at high temporal resolution ( Figure 1 D), I obtained an estimate of overall synaptic drive by removing the action potentials and low-pass filtering the resulting traces ( Carandini and Ferster 2000 ; Volgushev et al. 2000 ). The outcome of this procedure ( Figure 1 E) is a coarse potential (or “generator potential”; Lankheet et al. 1989 ) that approximates the synaptic current ( Anderson et al. 2000a ). This technique allows one to estimate synaptic currents while concurrently recording firing rates. Variability of Coarse Potential during Visual Stimulation We can now consider the mean and variance across trials for coarse potential. The mean, V mean , is the “signal” reflecting the stimulus-driven synaptic input to the neuron ( Figure 1 F and 1 G, traces). The variance, instead, is the “noise” reflecting the synaptic input's trial-to-trial variability ( Figure 1 F and 1 G, shaded areas). The variability of potential depended only slightly on stimulus strength. Variance was slightly higher when the stimuli depolarized the cell than when they hyperpolarized it ( Figure 1 F and 1 G). For the example simple cell in Figure 1 , standard deviation of potential was 2.8 ± 1.2 mV (s.d.) for V mean between –70 and –65 mV, and 4.0 ± 1.7 mV for V mean between –55 and –50 mV. The relation between standard deviation of potential and V mean can be described by a regression line ( r = 0.27 ± 0.04, s.d., bootstrap) whose slope is 0.08 ± 0.01 and whose intercept at V rest = −60.4 mV is 3.3 ± 0.1 mV. Similar values were obtained in the rest of the population (e.g., Figure 2 E and 2 H): correlation coefficient was r = 0.40 ± 0.19 (s.d., N = 22), intercept at V rest was 3.3 ± 1.4 mV, and mean slope was a shallow 0.14 ± 0.09. In occasional cells (such as that of Figure 2 H), the standard deviation of potential did not grow monotonically with V mean . The ratios of variance to mean seen in membrane potentials were negligible when compared to those seen in firing rate. For the example simple cell, over the entire range of mean potentials the variance of potential grew by less than a factor of four ( Figure 2 B). By contrast, over the entire range of firing rates the variance of firing rate grew by a factor of almost 100 ( Figure 2 A). Similar results were obtained in the remaining cells, such as the complex cell of Figure 2 D and 2 E and the intermediate cell of Figure 2 G and 2 H. In the last cell, the difference between potential and firing rate was particularly striking, as the former shows a downward slope that is clearly absent in the latter. These differences in variability are meaningful because potential and firing rate were recorded from the same responses to the same set of stimuli. They are not simply due to differences in time scale ( Buracas et al. 1998 ; Kara et al. 2000 ) because firing rate and potential were sampled at the same resolution (100 Hz). Accounting for the Variability of Firing Rate The origin of the large variability in firing rate lies not in an unforeseen source of noise, but rather in a deterministic mechanism, the nonlinear transformation of potentials into firing rates. This transformation ( Figure 1 H) can be fitted by a simple rectification model ( Granit et al. 1963 ) describing how firing rate R grows with potential V once this potential is above a threshold V thresh . As expected ( Anderson et al. 2000b ; Carandini and Ferster 2000 ), this rectification model captures the relation between potential and firing rate ( Figure 1 H, curve) and can be used to predict the rough features of firing rate both in individual trials (compare Figure 1 A and 1 I) and in averages across trials (compare curves in Figure 1 B and 1 C with those in Figure 1 J and 1 K). Of course, rectification is not a full account of the transformation between synaptic inputs and firing rates. Indeed, the relationship between firing rate and potential exhibits substantial error bars ( Figure 1 H). These error bars do not denote noise involved in generating spikes, which is negligible ( Calvin and Stevens 1968 ; Mainen and Sejnowski 1995 ; Carandini et al. 1996 ). They simply indicate that (as evident in the Hodgkin–Huxley equations) instantaneous potential is only one of the determinants of firing rate; additional determinants include the membrane potential's recent history ( Azouz and Gray 1999 ) and frequency content ( Carandini et al. 1996 ; Volgushev et al. 2002 ). Despite its simplicity, the rectification model is sufficient to predict the large variability of firing rate, and the increase of firing-rate variance with firing-rate mean. The predicted standard deviation resembles the measured one both in amplitude and in time course (compare shaded areas in Figure 1 B and 1 C with those in Figure 1 J and 1 K). Indeed, a plot of variance versus mean for the predicted firing rate ( Figure 2 C) indicates almost as much variability as that seen for the actual firing rate ( Figure 2 A). Similar results were obtained in the other example cells (compare Figure 2 D to 2 F, and 2 G to 2 I) and in the rest of the population ( Figure 3 A and 3 B). While the rectification model often underestimated the vertical intercept of the line relating mean and variance ( Figure 3 A), it generally captured the line's slope ( Figure 3 B). The model, therefore, accounts for the growth of firing-rate variance with the mean. Figure 3 Performance of the Rectification and Gaussian–Rectification Models in Predicting Firing-Rate Variability Distributions of firing-rate variance versus firing-rate mean were fitted with a line in logarithmic scale, corresponding to the equation variance = a mean b , where a is the intercept of the line and b is the slope of the line. Fitting was performed on the measured distributions (e.g., Figure 2 A), on the distributions predicted by the rectification model (e.g., Figure 2 C), and on those predicted by the Gaussian–rectification model (e.g., Figure 6 B). Dashed lines indicate predictions for a Poisson process. (A) Comparison of measured intercept versus predicted intercept. Diagonal line indicates equality between measured and predicted values. (B) Same, for the slope. (C and D) Same as in (A) and (B), for the predictions of the Gaussian–rectification model. The reason why the rectification model explains the large variability of firing rate is rather intuitive. Trial-to-trial fluctuations in potential are critical to obtain spikes, because many visual stimuli (such as the 210° grating in Figure 1 ) elicit a mean potential that barely reaches the firing threshold ( Anderson et al. 2000b ). Therefore, small fluctuations in membrane potential make the difference between a trial with few or no spikes and one with plenty of spikes. In other words, the firing threshold amplifies small fluctuations in potential into large fluctuations in firing rate. Perhaps less intuitive is the reason why the rectification model explains the growth of firing-rate variance with firing-rate mean. One may think that a necessary condition for this effect is the growth in potential variance observed with increasing mean potential ( Figure 2 B). This is not the case: The variance of potential could stay constant or even decrease (as it does for the cell in Figure 2 H), and the variance of firing rate would still grow with the mean ( Figure 2 G). Predicting the Variability of Firing Rate An intuition and a quantitative account for these properties can be obtained by applying the rectification model to an idealized random distribution of potentials, which we take to be Gaussian. Such a Gaussian–rectification model has been used to explain the dependence of mean firing rate on mean synaptic input ( Anderson et al. 2000b ; Hansel and van Vreeswijk 2002 ; Miller and Troyer 2002 ). It resembles a model proposed by Abeles (1982 , 1991 ) to study neuronal integration time. In the Gaussian–rectification model, the stimulus determines the mean of the Gaussian ( Figure 4 B), and the portion of Gaussian that crosses threshold determines the distribution of firing rates ( Figure 4 A). The mean of the Gaussian is the average potential V mean evoked by the stimulus at that instant ( Figure 4 B). The rectification function ( Figure 1 H) operates on this distribution and determines the distribution of firing rates ( Figure 4 A): Each potential contributes a firing rate given by the rectification function, with a probability given by the value of the Gaussian at that potential. When mean potential V mean is low, the Gaussian lies mostly below the threshold V thresh , so the predicted firing rate is mostly zero ( Figure 4 A, a ). When V mean is higher, however, the tail of the Gaussian that lies above threshold becomes substantially larger, and the distribution of firing rates reaches higher rates ( Figure 4 A, e ). The large peak at 0 spikes/s corresponds to the area of the Gaussian that lies below V thresh . Figure 4 The Gaussian–Rectification Model (A and B) Distributions across trials of model potential V (B) and of model firing rate R (A) for five values of the mean potential V mean . Firing rate is obtained from potential by applying the rectification model ( Figure 1 H). The value for R = 0 is shown at 1/3 of veridical height. (C and D) Mean (data points) and standard deviation (error bars) for the distributions in (A) and (B) as a function of mean potential V mean . Curve and shaded area indicate model predictions for the full range of mean potentials. Arrows indicate the five mean potentials (± 2 mV) used in (A) and (B). Throughout, dashed lines indicate resting potential V rest and dotted lines indicate firing threshold V thresh . Such a simple model is sufficient to predict that the variance of firing rate should increase with mean firing rate. As mean potential V mean increases, the distribution of firing rate becomes broader ( Figure 4 A), increasing not only in mean but also in standard deviation ( Figure 4 C). This phenomenon occurs even though in the model the standard deviation of potential is the same at all mean potentials ( Figure 4 D). The main assumption of the model, that of a Gaussian distribution of potentials, is generally borne out by the data. In most cells, the distribution of potential is close to a Gaussian, especially at the lowest values of mean potential, where spiking seldom occurs ( Figure 5 B). For the example simple cell, the distribution of z -scores (the difference between potential and mean potential, normalized by the standard deviation at that potential) appears remarkably Gaussian ( Figure 6 A). Similar results were obtained in the other cells (e.g., Figure 6 C and 6 E), although in some cells the tails of the distributions exceeded those of a Gaussian, and a large skewness clearly favored the more depolarized tails (not shown). A Gaussian distribution of potentials is commonly predicted in the theoretical literature (e.g., Svirskis and Rinzel 2000 ; Amemori and Ishii 2001 ; Rudolph and Destexhe 2003 ). It would be expected in a passive membrane summating many independent, high-rate presynaptic spike trains ( Rice 1944 ; Tuckwell 1988 ). Figure 5 Application of the Gaussian–Rectification Model to the Data from the Example Simple Cell (A and B) Distributions across trials of potential V (B) and of firing rate R (A) for five values of the mean potential V mean . Curves are best-fitting Gaussians (B) and predicted distributions of firing rate (A). Bin for R = 0 is shown at 1/3 of veridical height (and is three times wider than the others so that area is veridical). (C and D) Mean (data points) and standard deviation (error bars) for the distributions in (A) and (B), as a function of mean potential V mean . Curve and shaded area indicate model predictions for the full range of mean potentials. Arrows indicate the five mean potentials (± 2 mV) used in (A) and (B). Even a reduced model with constant standard deviation of potential (D, shaded area) predicts a growing standard deviation (A, shaded area). Figure 6 Variability of Potential and Predictions of the Gaussian–Rectification Model for Three Cells (A) Distribution of normalized deviations from the mean ( z -scores) for the potential of the simple cell in Figure 1 and Figure 2 A– 2 C. These were computed by subtracting from each potential the corresponding mean potential V mean (the abscissa in Figure 2 B) and dividing by the standard deviation (the square root of the ordinate in Figure 2 B). The results were cumulated. The curve is a normal Gaussian. (B) Variance versus mean for firing rate for the same cell and its prediction by the Gaussian–rectification model. Data points are same as Figure 2 A. Red curve: prediction of Gaussian–rectification model Shaded area: region where the Gaussian–rectification model predicts the occurrence of 75% of the points. Line is linear regression. (C and D) Same as (A) and (B) for the complex cell in Figure 2 D– 2 F. (E and F) Same, for the intermediate cell in Figure 2 G– 2 I. The Gaussian–rectification model has four parameters. Three of these parameters describe the rectification stage and are thus fully constrained by the measured relationship between potential and firing rate ( Figure 1 H). The remaining parameter, the standard deviation of the Gaussian, σ, was obtained from maximum likelihood estimation, i.e., by searching for the standard deviation that maximized the probability of observing the distributions of firing rate ( Figure 5 A). The result, σ = 4.6 mV, slightly overestimates the standard deviation observed for low mean potentials, but correctly estimates it at higher mean potentials ( Figure 5 D, compare shaded area to error bars). The model predicts the main features of the distributions of firing rate ( Figure 5 A). It predicts that when mean potential is low (e.g., V mean = −64 mV; Figure 5 A, a ), the firing rate is always zero, whereas larger mean potentials yield a distribution of firing rates that spans values from 0 to 300 spikes/s (e.g., V mean = −54 mV; Figure 5 A, d ). Deviations from the predictions are largest where they are least significant, i.e., at high firing rates for the high values of V mean (e.g., V mean = −50 mV; Figure 5 A, e ). These high values were achieved seldom; for example, only 21 data points were obtained at V mean = −50 mV ( Figure 5 A, e ), compared to 273 at V mean = −54 mV ( Figure 5 A, d ) and 1,575 at V mean = −64 mV ( Figure 5 A, a ). In fact, the model closely predicts both the firing rate's mean and standard deviation ( Figure 5 C). It predicts the two key effects of increasing mean potential: (1) an increase in the firing rate's mean (as a power law; Anderson et al. 2000b ; Hansel and van Vreeswijk 2002 ; Miller and Troyer 2002 ), and (2) an increase in the firing rate's standard deviation. Crucially, the model closely predicts how firing-rate variance depends on firing-rate mean ( Figure 6 B, red curve). Because of noise in the estimation of variance from a limited number of measurements (in this experiment, seven trials), the data are not expected to fall exactly on the model's prediction; Monte Carlo simulations with a matched number of trials determined the area in which 75% of the observations are predicted to fall ( Figure 6 B, gray area). Similar results were obtained in the remaining cells of the population, except that the model has a mild tendency to underestimate the intercept and overestimate the slope of the relation between variance and mean ( Figure 3 C and 3 D). Overall, the Gaussian–rectification model applied to the trace of mean potential performed as well as the rectification model applied to the individual traces of potential. Both models underestimated the intercept of the lines fitted to the relationship between firing-rate variance and mean: the rectification model by 25 ± 42% ( Figure 3 A), and the Gaussian–rectification model by 44 ± 26% ( Figure 3 B). Both models correctly estimated the slope of the line (the growth in variance with increasing mean), with insignificant errors of 0.10 ± 0.25 for the rectification model ( Figure 3 C), and −0.01 ± 0.22 for the Gaussian–rectification model ( Figure 3 D). This performance is remarkable, given that the Gaussian–rectification model replaces detailed knowledge of potential in individual trials with just one free parameter, the standard deviation σ of potential. These results illustrate how the key element in producing the steep growth in firing-rate variance observed with growing stimulus strength is the nonlinear transformation between potential and firing rate ( Figure 1 H). Indeed, the model was intentionally implemented with the constraint that the standard deviation of potential, σ, be constant. This constraint serves to demonstrate that a mild growth in variance of potential ( Figure 5 D, error bars) is not necessary to produce the steep growth in firing-rate variance ( Figure 5 C, error bars). Variability of Responses to Current Injection The predictions of the Gaussian–rectification model apply to any neuron that meets minimal criteria: a relationship between synaptic input and firing rate that is monotonic and includes a threshold, and noise in the input that has a Gaussian distribution. As an example, let us consider a neuron that is closer to biological reality than the Gaussian–rectification model, one that receives currents (not potentials) in its input and produces individual spikes (not continuous firing rates). In particular, consider an enhanced integrate-and-fire neuron, where each spike is accompanied by a temporary increase in spike threshold and by the entry of calcium, which in turn determines an after-hyperpolarization potassium current (see Materials and Methods ). To ensure realism, I fitted the model parameters to responses to injected currents of a regular spiking neuron. This neuron was recorded in vitro in the visual cortex of the guinea pig, in the near absence of synaptic inputs ( Carandini et al. 1996 ). The injected currents include sinusoids ( Figure 7 A, top four panels) and approximately Gaussian-distributed noise ( Figure 7 A, bottom panels). Once its parameters are appropriately tailored, the enhanced integrate-and-fire model accurately predicts the cell's responses, both in the subthreshold membrane potential waveforms and in the timing of individual spikes ( Figure 7 B and 7 C). Figure 7 Responses of a Regular-Spiking Neuron in the Visual Cortex to Current Injection, and Predictions by an Enhanced Integrate-and-Fire Model Neuron (A) Injected currents were sinusoids or noise waveforms. Noise was obtained by summing eight sinusoids with incommensurate frequencies. (B) Membrane potential responses of a regular-spiking neuron (cell 19s2, experiment 4) recorded with sharp electrodes in a study of guinea pig visual cortex in vitro ( Carandini et al. 1996 ). (C) Predictions of an enhanced integrate-and-fire neuron model fine-tuned to resemble the responses of the cell. Just as predicted, this spiking neuron responds to noisy injected currents with a firing rate whose variance grows with the mean ( Figure 8 ). To simulate the synaptic drive to a simple cell recorded in vivo ( Figure 1 A– 1 D) I injected sinusoidal currents, to which I added Gaussian noise. The model responses ( Figure 8 A– 8 D) resemble those seen in vivo ( Figure 1 A– 1 D). The firing rate is highly variable ( Figure 8 B), with a standard deviation that is roughly comparable to the mean ( Figure 8 C and 8 D), even though the standard deviation of the injected current is constant ( Figure 8 H and 8 I). In fact, for firing rate the variance grows proportionally to the mean ( Figure 8 E), even though for injected current the variance is constant ( Figure 8 J). Figure 8 Variability in the Responses of the Spiking Model Neuron (A) Response of the model neuron to a 0.6-nA sinusoidal current in the presence of Gaussian noise (s.d. 0.25 nA). (B) Corresponding firing rate. Three trials are shown. (C) Firing rate averaged over 16 trials. Shaded area indicates 2 s.d. (D) Same, for three other stimuli: a 0.4-nA sinusoid (top), a 0.2-nA sinusoid (middle), and noise alone (bottom). (E) Variance versus mean for firing rate. Diagonal line is prediction for a Poisson process. Red curve: prediction of Gaussian–rectification model, with no parameters allowed to vary to fit the data. Shaded area: region where the Gaussian–rectification model predicts the occurrence of 75% of the points. (F) Relation between firing rate and injected current. Curve is fit of rectification equation. (G–I) As in (B–D), for injected current. (J) Variance versus mean for injected current. The Gaussian–rectification model captures the essence of this behavior. Once it is given the standard deviation of the noise and the relationship between injected current and firing rate ( Figure 8 F), the Gaussian–rectification model makes a parameter-free prediction of the relationship between variance and mean ( Figure 8 E, curve). This prediction is not perfect (it consistently underestimates firing-rate variance), but it does capture the most important behavior: that variance grows with the mean for firing rate ( Figure 8 E, curve) but not for injected current ( Figure 8 J, horizontal line). Similar results were obtained when the stimulus parameters were changed to simulate synaptic inputs to a complex cell, or when the parameters of the spiking neuron were changed to simulate other cells measured in vitro, or even chosen randomly within reasonable bounds. As predicted, as long as the relationship between synaptic input and firing rate involved a threshold and the input noise was Gaussian, the variance grew with the mean for firing rate but not for injected current. Role of Firing-Rate Encoder Having validated the Gaussian–rectification model, we can now investigate the role of its parameters in determining the curves relating firing-rate variance and mean ( Figure 9 ). The model has four parameters (see Materials and Methods ): (1) the standard deviation σ of potential, (2) the firing threshold, V thresh , (3) the gain k of the relationship between firing rate and potential above threshold, and (4) the exponent n of this relationship. For the purpose of studying the model, we can assume, without loss of generality, that potential is unitless and has standard deviation σ = 1. Then, because V thresh can only determine the range of firing rates that is achieved, only k and n control the shape of the variance versus mean curves ( Figure 9 ). Figure 9 Role of Parameters of Gaussian–Rectification Model The standard deviation of potential was set to σ = 1, so that the shape of the curves relating firing-rate variance to firing-rate mean depends entirely on the gain k and the exponent n of the curves relating firing rate to membrane potential. The effects of these two parameters are explored: varying n (columns) and varying k (rows). Red curves: predictions of the Gaussian–rectification model; shaded areas: regions where the model predicts the occurrence of 75% of the points. Insets illustrate the corresponding curves relating firing rate to membrane potential. The gain k controls curve position, and the exponent n controls curve shape ( Figure 9 ). Increasing the gain k lifts the curves upward by twice as much as it shifts them rightward ( Figure 9 , rows). These shifts occur because variance grows with k 2 and mean grows with k. Decreasing the exponent n causes the curves to saturate ( Figure 9 , columns): The variance saturates to a plateau if n = 1 ( Figure 9 , middle), and it reaches a maximal value and then decreases if n < 1 (as in Figure 9 , left). Saturation occurs because when potential goes well above threshold, increases in mean potential cease to reveal ever larger portions of the Gaussian. If the curves relating firing rate to potential saturate ( n < 1), variations in potential are compressed into proportionally ever smaller variations in firing rate; the opposite occurs if the curves expand ( n > 1). This analysis predicts that it should be fairly common for the firing-rate variance to saturate at high firing rates, possibly showing a plateau or even a decrease. Indeed, in the sample of V1 neurons recorded intracellularly, exponents are typically close to unity ( n = 1.1 ± 0.6). Accordingly, a mild form of saturation is common in the plots of firing-rate variance and mean ( Figure 6 B). To quantify the saturation, however, one needs reliable estimates of firing-rate variance. These estimates are not very reliable in the intracellular sample, which typically involves only a few hundred spikes per cell, leading to large clouds of points in the scatters of variance versus mean ( Figure 6 B). Variability of Extracellularly Recorded Firing Rates To test the model's prediction rigorously, I considered a set of V1 responses obtained with extracellular recordings. Thanks to the large number of spikes (commonly >4,000 per cell), measurements in this dataset yield more precise estimates of firing-rate variance over a wider range of firing rates than are available in the intracellular sample. An analysis of firing-rate variance versus mean for these extracellularly recorded cells supports the predictions of the model ( Figure 10 ). Extracellular data do not afford independent estimates of gain k and exponent n of the transformation of potential into firing rate. I thus first computed the model predictions for a variety of combinations of k and n (such as those shown in Figure 9 ). I then made Bayesian estimations of the values of k and n that maximize the likelihood of the data, while imposing a broad prior for n = 1.1, the median value measured intracellularly. The quality of these two-parameter fits was excellent ( Figure 10 ), of higher quality than could be obtained by fitting a line, the two-parameter “model” commonly used to describe data of this kind ( Figure 2 A). Moreover, a number of cells exhibited the saturation in variance predicted by the model. The eight representative cells shown in Figure 10 are arranged in order of increasing exponent n. The first three ( n = 0.9 to 1.0) show evident saturation in firing-rate variance as mean firing rate increases. The remaining five show a milder saturation, as expected from their higher exponents ( n = 1.1 to 1.2). Saturation was common, as the median n was 1.06, with n < 1 in 13/37 cells. Yet to my knowledge, except for an anecdotal account ( Mechler 1997 ), this common property had not been previously reported. It constitutes further support for the usefulness of the Gaussian–rectification model. Figure 10 Relationship between Variance and Mean for Eight Cells Recorded Extracellularly in Cat V1, and Fits by the Gaussian–Rectification Model For each mean firing rate, data point and error bars indicate mean ± 1 s.d. of the observed variance. Red curves and shaded areas are predictions of the model. Values of exponent n and gain k are reported next to each graph. Cells are arranged in order of increasing exponent n. Discussion We have seen that a large amplification takes place between the trial-to-trial variability of synaptic input and that of firing rate: The variance of synaptic input is small compared to the dynamic range, and it is roughly constant. The amplification of variability arises from the threshold in the transformation of synaptic input into firing rate. A Gaussian–rectification model attributes this amplification to very simple causes: approximately constant Gaussian noise in the input, and rectification due to threshold in the output. It indicates that firing-rate variance would grow with the mean even if the variance of synaptic input were constant. Both of the assumptions of the model, constant Gaussian noise and rectification, are borne out by the data. These assumptions are rather minimal, so they are naturally satisfied by more realistic models. For example, a realistic integrate-and-fire model behaves as predicted: Once it is given constant Gaussian noise in the input, it produces a firing-rate variance that grows with the firing-rate mean. Further support for the Gaussian–rectification model comes from its novel, and correct, prediction that firing-rate variance should saturate at high firing rates. In confirming this prediction I showed that the model can be used to account for variability in firing rate without knowledge of cellular properties. The extension to extracellular data is important because extracellular methods constitute the norm in visual neurophysiology, especially in awake animals, and are the ones used in previous studies of firing-rate variability. These results further lengthen a list of properties of V1 neurons that are simply explained by the firing threshold. In addition to the amplification of trial-to-trial variability demonstrated here, these include the sharpening of tuning for stimulus direction and orientation ( Jagadeesh et al. 1993 ; Carandini and Ferster 2000 ; Volgushev et al. 2000 ), the power-law behavior of firing rate at low contrast ( Heeger 1992 ; Anderson et al. 2000b ; Hansel and van Vreeswijk 2002 ; Miller and Troyer 2002 ), and even the establishment of the dichotomy between simple and complex cells ( Carandini and Ferster 2000 ; Mechler and Ringach 2002 ; Priebe et al. 2004 ). It is remarkable that a mechanism as simple as the firing threshold can determine phenomena that might prima facie require more complex explanations at the level of the network. Limitations of the Approach One limitation of this study lies in the use of coarse potential. Coarse potential is not completely independent of firing rate: Even when spikes are removed and the traces smoothed, there is still a likely contribution of active conductances that has not been removed. Fortunately, this limitation strengthens my observation that coarse potential is not nearly as variable as firing rate: Any unwanted remaining echo of the spikes would make coarse potential more similar to firing rate and, thus, more variable. Therefore, in reality the variance of the actual synaptic input might be even less dependent on the mean than appears, for example, in Figure 2 B, 2 E, and 2 H. A partial control for these effects would be to perform some of the measurements while blocking spikes. However, blocking spikes would prevent the key measurements of this study, which require concurrent measurement of firing rate and estimation of synaptic input. Another limitation of the approach is that I have mostly considered firing rates, not individual spikes. Unlike firing rates, individual spikes can occur only in integer numbers and are separated by refractory periods. These properties can become relevant to response variability, for example, if firing rates become so high that refractory period becomes a limiting factor ( Kara et al. 2000 ). Such concerns are assuaged by the realistic integrate-and-fire model ( Figure 7 ), which shows an increase of firing-rate variance with the mean similar to that predicted by the Gaussian–rectification model. As to the saturation in firing-rate variance that was observed in some neurons, it invariably occurred at firing rates much lower than predicted from the refractory period. A more serious limitation of coarse potentials and firing rates is that they make sense only in a limited range of time windows. The windows should be long enough to be able to contain more than one spike, and short enough that mean potential is approximately constant within the window. An informal analysis of the effect of time window indicates that a range of 5–20 ms is satisfactory. This range, however, might be appropriate only for V1 neurons; further investigations are required before applying these methods elsewhere. Finally, a broader limitation of this work is that it concentrates on variability across trials, with little bearing on another form of variability, the one observed within trials in the irregularity of spike trains ( Softky and Koch 1993 ; de Ruyter van Steveninck et al. 1997 ; Reich et al. 1997 ; Troyer and Miller 1997 ; Buracas et al. 1998 ; Shadlen and Newsome 1998 ; Stevens and Zador 1998 ). Thanks to recent advances, however, the cellular origins of this form of variability have been largely explained ( Reich et al. 1997 ; Stevens and Zador 1998 ). In particular, it is now clear that high variability within trials is to be expected if neurons receive synaptic inputs with slow temporal correlation ( Svirskis and Rinzel 2000 ). In fact, variability within trials is most evident with visual stimuli that provide a roughly stationary response, being greatly diminished with richer stimuli, which elicit highly precise responses ( Bair and Koch 1996 ; Reich et al. 1997 ; Buracas et al. 1998 ). Conversely, trial-to-trial variability is endemic, being present regardless of type of visual stimulus ( Reich et al. 1997 ; Buracas et al. 1998 ). Implications for Cortical Processing What computational advantage might cortical neurons derive by amplifying the variability that they receive in their input? Why reduce the signal/noise ratio? To answer these questions, it might help to clarify the sources of “signal” and “noise.” The main source of variability in synaptic inputs to a V1 neuron is likely to be intracortical because thalamic responses are half as variable ( Kara et al. 2000 ). Variability thus results largely from ongoing cortical activity ( Arieli et al. 1996 ; Buracas et al. 1998 ; Tsodyks et al. 1999 ; Kenet et al. 2003 ). It appears to us as noise simply because it is not synchronized with stimulus onset. By contrast, the mean across trials of potential or firing rate constitutes a signal that is driven entirely by the stimulus. The results of this study suggest that threshold affects the interaction between stimulus-driven activity and ongoing activity, turning it from additive to multiplicative. At the level of firing rates, this interaction is largely multiplicative because the variance of firing rate grows proportionally to the stimulus-driven mean firing rate. At the level of synaptic inputs, instead, this interaction is nearly additive because the variance of potential barely depends on the stimulus-driven mean potential. Indeed, additivity has been seen between local field potentials and ongoing voltage-sensitive dye signals ( Arieli et al. 1996 ). We have seen that the rectification due to firing threshold is single-handedly responsible for the variability of firing rate and is, thus, responsible for turning a largely additive interaction into a multiplicative interaction. It is thus conceivable that the computational role of firing threshold is to multiply stimulus-driven responses by ongoing cortical activity, i.e., to multiply what we call “signal” by what we call “noise.” What may appear as lowering the signal/noise ratio can in fact be seen as a useful process, one that progressively amplifies the ongoing activity that ultimately guides our actions. Materials and Methods Data acquisition in vivo Measurements in vivo were obtained in paralyzed, anesthetized cats. Methods for animal preparation and maintenance have appeared elsewhere ( Carandini and Ferster 2000 ) and were approved by the Animal Care and Use Committees at Northwestern University and at the Smith-Kettlewell Eye Research Institute. The 22 cells recorded intracellularly belong to a sample that has been analyzed in two previous studies by Carandini and Ferster (2000) and by Anderson et al. (2000a) . These studies describe in detail the recording methods, which involved the whole-cell patch technique. The electrical noise associated with this technique is commonly <0.1 mV (as judged from records obtained after losing the patch). From the sample I excluded a few cells that produced less than ten spikes per block of stimuli, or that failed to satisfy other minimal requirements (firing rate >2 spikes/s, spike height >10 mV). Stimuli were optimal gratings drifting in 12 directions in 30° intervals, and a blank screen of uniform gray. The resting potential V rest was taken as the mean potential measured with the blank screen. Coarse potential traces were obtained from traces of membrane potential sampled at 4 kHz by removing spikes ( Lankheet et al. 1989 ) and by applying a low-pass filter with a cutoff of 50 Hz ( Carandini and Ferster 2000 ; Volgushev et al. 2000 ). The same low-pass filter was applied to spike trains sampled at 4 kHz to yield firing rate. Both coarse potential and firing rate were subsampled at 100 Hz. The 37 neurons recorded extracellularly are part of a study of the organization of receptive fields and suppressive surrounds in area V1 ( Bonin et al. 2003 ). This dataset was chosen because it involved lengthy experiments that yielded many thousands of spikes per cell at a variety of firing rates. Recordings were made with quartz-coated platinum/tungsten microelectrodes; methods for data acquisition and animal maintenance have been described by Freeman et al. (2002) . Stimuli were drifting gratings presented at the optimal orientation, spatial frequency, and temporal frequency, and enclosed in one of 66 possible windows. The windows were stationary square gratings with variable period and orientation. Stimuli typically lasted 2 s, and each block of stimuli was typically repeated three to six times. Firing rates were extracted from the spike train by low-pass filtering at 50 Hz and were subsampled at 100 Hz. Data acquisition in vitro Measurements in vitro were made with sharp intracellular electrodes from slices of guinea pig visual cortex. Methods for this preparation were approved by the Animal Care and Use Committee at New York University. The cells are part of the dataset presented by Carandini et al. (1996) ; the cell in Figure 7 is the one whose responses are extensively illustrated in that study (cell 19s2). Rectification model The relation between potential V and firing rate R (e.g., Figure 1 H) was fitted with an extension of the rectification model ( Mechler and Ringach 2002 ), where R ( V ) = k [ V − V thresh ] n + , with [.] + indicating rectification, k a proportionality factor, and n an exponent. Fitted parameters were V thresh = −55.3 mV, k = 16.7, and n = 1.2 for the simple cell in Figure 1 , and V thresh = −46.6 ± 10.5 mV, k = 12.4 ± 7.9, and n = 1.1 ± 0.6 for the whole intracellular population ( N = 22). The distance between V thresh and V rest was 5.1 mV for the simple cell in Figure 1 , and 8.0 ± 4.2 mV for the population. Gaussian–rectification model The mean potential V mean in response to a stimulus was defined as the mean across trials of coarse potential. In the Gaussian–rectification model, the probability of observing a firing rate r ( Figure 4 A) given a mean potential V mean is where R ( V ) is the relation between firing rate and potential V ( Figure 1 H), and N [ V mean , σ ] is the probability distribution of potential ( Figure 5 B), a Gaussian with mean V mean and standard deviation σ. The value of p ( r ) depends on whether r is zero or positive: for r > 0, and for r = 0. The first expression is simply the value of the Gaussian for V = R −1 ( r ). The second expression is the area of the portion of Gaussian that is below threshold ( erf is the error function). These expressions allow maximum likelihood estimation of model parameters from measured firing rates. When parameters of the relation between firing rate and potential R ( V ) are obtained independently (in intracellular recordings; Figure 1 H), the only free parameter was the standard deviation σ of potential. Across the intracellular population, the average value of σ obtained by the fits was 5.4 ± 2.0 mV (s.d., N = 22). The required σ was always larger (by 2.1 ± 1.5 mV) than the standard deviations observed when V mean = V rest , but it was comparable (larger by only 0.9 ± 1.6 mV) to the standard deviations observed when V mean = V thresh . Statistics Let V mean ( t ) be the mean potential at time t from stimulus onset. Because the sample rate is 100 Hz, each time sample corresponds to a 10-ms interval. Of course, V mean ( t ) depends on the stimulus. To simplify the notation, however, consider the case of a single stimulus. Distributions for potential at a given mean potential ( Figure 5 B) were computed as follows: (1) Select a value of interest, v (e.g., v = −55 mV; Figure 5 B, a ); (2) find the times ( t k ) when the mean potential V mean ( t k ) is within 2 mV of v; (3) pooling across trials j, look at the distribution of potential [ V j ( t k )] (e.g., Figure 5 B, a ). Distributions for z -scores (normalized deviations from the mean) of potential ( Figure 6 A, 6 C, and 6 E) were computed as follows: (1) Divide the range of values of V mean in 1-mV intervals, with centers ( v i ); (2) for each interval i, find the set of times ( t ik ) when the mean potential is in the i -th bin; (3) pooling across trials j, compute σ i , the standard deviation of V j ( t ik ); (4) transform each V j ( t ik ) into a z -score: z ijk = [ V j ( t ik ) − v i ]/ σ i ; (5) look at the distribution of (z ijk ) (e.g., Figure 6 A). Enhanced integrate-and-fire model The enhanced integrate-and-fire model was derived in collaboration with Davide Boino (2000) by simplifying a model by Wang (1998) . The model neuron has a single compartment with membrane equation where the currents are: with t spike the time of the last spike. Ca ( t ) is the (unitless) calcium concentration: where the sum extends over all spikes with t spike < t. Spikes result from stereotyped conductances g Na ( t ) and g K ( t ) derived from Hodgkin–Huxley equations and are scaled to approximate the spikes from the recorded neuron. They occur when V m exceeds a threshold, which depends on the time since the last spike: The reversal potentials for sodium and potassium were set to V k = −80 mV and V Na = 55 mV. Passive parameters of the membrane ( C m = 120 pF, g leak = 12.4 nS, V leak = −60.3 mV) were obtained by fitting the membrane potential responses to sinusoids. The remaining parameters ( g AHP = 23.0 nS, Ca 50 = 10, τ Ca = 200 ms, V thresh = −43.5 mV, τ thresh = 36 ms) were obtained by a search algorithm aimed at maximizing the quality of the predictions for firing rate.

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499543

Structural characterization of genomes by large scale sequence-structure threading: application of reliability analysis in structural genomics

Background We establish that the occurrence of protein folds among genomes can be accurately described with a Weibull function. Systems which exhibit Weibull character can be interpreted with reliability theory commonly used in engineering analysis. For instance, Weibull distributions are widely used in reliability, maintainability and safety work to model time-to-failure of mechanical devices, mechanisms, building constructions and equipment. Results We have found that the Weibull function describes protein fold distribution within and among genomes more accurately than conventional power functions which have been used in a number of structural genomic studies reported to date. It has also been found that the Weibull reliability parameter β for protein fold distributions varies between genomes and may reflect differences in rates of gene duplication in evolutionary history of organisms. Conclusions The results of this work demonstrate that reliability analysis can provide useful insights and testable predictions in the fields of comparative and structural genomics.

Background Recent advances in networks theory have demonstrated a key role of uneven distributions occurring in many natural processes. It has been found that seemingly unrelated systems such as economic, professional, sexual and social networks, airline routing, power lines connections, language networks and internet hyperlinks all exhibit a power law decay of the cumulative distribution P x ≈ x - γ , where x is the number of links connected to each network node and γ is the value of the exponent typically varying in the range of 2–3 [ 1 ]. The heterogeneous architecture of scale-free networks imparts a robustness and error-tolerance from random perturbation and is often viewed as a possible common blueprint for naturally occurring large-scale networks. The critical role of the power law distribution has also been acknowledged in many areas of life sciences: metabolic and other cellular networks, proteins interaction maps, brain cellular organization, food and ecological webs all have been described as scale-free systems. It would be fair to say that the advances in the scale free network studies have revitalized the original Pareto's inequality law introduced more then a century ago [ 2 ]. The applicability of the scale free networks has been examined in numerous structural genomics studies. It has been proposed that the genomic occurrence of protein families, superfamilies and folds can follows an asymptotic power law: SDF(GO) = aGO - b (1) , where SDF(GO) is survival distribution function of genomic occurrence GO of a certain protein family, superfamily and fold. These findings have laid the foundation for characterizing the evolution of the protein universe in terms of a growing scale-free system in which individual genes are represented as nodes of a propagating network [ 3 - 7 ]. In our previous work [ 9 ], we have used the large-scale sequence-structure threading to assign protein folds to 33 genomes from all three superkingdoms of life. It has been found that more then 60% of the studied eukaryotic, 68% of archaeal and 70% of bacterial proteomes could be assigned to defined protein folds by threading. The estimated results have been used to analyze the distribution of protein architectures, topologies and domains (or homologous superfamilies according to the CATH classification [ 8 ]). Thus, we have found that the frequencies of genomic occurrence of assigned protein domains (homologous superfamilies) and topologies can be described by a power function (1) with moderate accuracy. According to the formalism of network theory, such a power law representation of the cumulative distribution of node connections governs a scale-free character of the system [ 10 ]. At the same time we have noted that the values of the power exponent b estimated in the study generally fall below the 2–3 range typical for scale-free systems (analogous observations could also be noted in a number of similar investigations [ 3 - 5 ]). Table 1 (see Additional file 1 ) features the estimated parameters a and b along with the corresponding correlation coefficients r 2 reflecting the goodness of fit of experimental data with the logarithmic linear plots (1) (Table 1 also reflects the total number of the analyzed ORF-s in each genome and the corresponding number of proteins for which the THREADER has confidently assigned certain fold). The established lowered values of the power exponent and modest accuracy of the power law dependences (1) encouraged us to seek alternative approaches to more accurately describe protein folds distributions. Results Weibull (reliability) analysis The Weibull distribution is a general-purpose statistical function defined within Extreme Value Theory [ 11 ] and widely used in reliability engineering to model material strength, durability of electronic and mechanical components or equipments. In the most common case the probability density distribution is described by a two-parameter Weibull distribution , where α is a scaling factor and β is a shape parameter also known as the slope [ 12 ]. The Weibull analysis operates on life data, i.e it utilizes time-to-failure (or time under the testing stress) to assess the reliability of a system and to forecast its stability through parameters of the characteristic life span α and shape β . A typical Weibull experiment is based on application of disruptive stress to multiple samples representative of the population until the tested objects achieve a state of failure and produce time-to-failure numbers. The corresponding time-to-failure values form heterogeneous Weibull distributions described by (2). Application of Weibull function to genomic analysis The distribution of protein folds in a genome can be viewed much like the behavior of a mechanical system under disruptive testing. It is feasible to stipulate that the increase of genomic abundance of any protein fold occurs under evolutionary pressure. Some folds are able to expand their genomic occurrence over a course of evolution others have higher probability to be lost through genetic drift and other random events, i.e. to fail. Considering these analogies, we anticipated that the Weibull logistic can provide some natural explanations for highly heterogeneous abundance of protein folds in genomes. To test this hypothesis we used two independent approaches to examine whether the genomic occurrence of protein topologies and domains can indeed be adequately described by the Weibull function. First of all, we employed the maximum likelihood (ML) method [ 13 ] to fit the survival distribution function SDF(x) of the genomic occurrences of protein topologies and homologous superfamilies into the Weibull dependence (2). The corresponding Weibull shape parameters have been established by solving the equation while the scaling factors have been calculated as . The ML method allowed very accurate description of the distribution of protein folds among the genomes. Figures 1a and 1b feature the survival distributions of CATH topologies and homologous superfamilies among all the studied genomes in combined (these experimental (observed) data curves are marked in red). On the same graphs we have plotted the SDF(GO) parameters reproduced within equation (2) through α and β values estimated by the ML approach. It is obvious that these computed blue curves labeled as 'Weibull analytical' resemble the experimental distributions (marked in red) very precisely. The corresponding α and β values estimated by the ML approach have been collected into Table 2 (see Additional file 2 ). The second way of examining applicability of the Weibull function (2) was based on notion that the double logarithmic transformation of the SDF(x) leads to the equation of a straight line: log(- log( SDF ( x )) = β log( x ) - β log( α )     (3) We performed the transformation (3) on the experimental SDF(GO) data to estimate the Weibull coefficients α and β and squared correlation coefficients r 2 which all have also been collected into Table 2 (marked as 'Weibull by plotting'). The values of the estimated squared correlation coefficients r 2 from Table 2 demonstrate very high accuracy of the linear dependences (3) established from the survival distributions of CATH folds in the studied genomes. These parameters also allow comparing the accuracy of double logarithmic dependences (3) with accuracy of simple logarithmic dependences derived from the power law model (1): log ( SDF ) = a - b * log ( GO )     (4) As it has been mentioned earlier, the dependences (4) have been estimated for the SDF(GO) functions for individual genomes, superkingdoms and for the combined set of proteins. The comparison of r 2 values from Table 2 and Table 1 established from the linear functions (3) and (4) respectively, reveals that for all studies cases (individual genomes, superkingdoms, total dependences) the statistical quality of Weibull dependences (3) is much better than of power law function (4). Figures 1a and 1b feature the Weibull distributions estimated by plotting (double logarithmic transformation) which reproduce the experimental SDF(x) curves with remarkable accuracy. Apparently, the distributions calculated from (3) (labeled as 'Weibull by plotting') are much closer to the experimental distributions than the power law curves (labeled as 'power law') computed within the conventional power function (1). Apparently, that the Weibull distributions established by the double logarithmic representations (4) (marked on Figure 1 'Weibull by plotting') are very close to those calculated by the ML method ('Weibull analytical'). It should be mentioned, however, that despite close resemblance between the Weibull distributions established by the analytical ML method and the 'double logarithmic' approach, the corresponding values of α and β parameters from Table 2 differ (due to the different data fitting algorithms employed by two methods) and the preference should, perhaps, be given to more stringent ML-derived data. Characteristic conditions for the Weibull distribution Although the estimated statistical criteria clearly demonstrate the suitability and superiority of a Weibull function over a power function in describing protein fold distributions, we decided to examine several additional criteria characteristic of the Weibull distribution. As it has been suggested by Romeu [ 14 ] there are four such characteristic properties immanent for the Weibull function. The double logarithmic plot of life data (also called 'a Weibull paper') should be linear As it can be seen from Table 1 the estimated r 2 values from the columns marked as 'Weibull by plotting' are all contained within the range ~ 0.95 – 0.98 what demonstrates that the 'Weibull papers' do indeed describe protein folds distribution in the studied genomes with high accuracy. Figures 2a,2b feature the 'Weibull papers' for the distribution of protein topologies and domains among all the studied species and illustrate that deviations from linearity are very insignificant. The slope of the 'Weibull paper' is an alternative estimator of β The data from Table 2 demonstrate that the estimated slopes of the 'Weibull papers' are very close to the values of β derived by analytical maximum likelihood approach. The x β transformation should yield an exponential distribution with mean α β The genomic occurrences of protein topologies and domains in the genomes and superkingdoms have been transformed into GO β distributions through the power factors β . The exponential character of the resulting distribution has been examined by several statistical tests and in all cases has been confirmed. The observed medians of the exponential distributions GO β accumulated in Table 3 (see Additional file 3 ) demonstrate strong correlations with the calculated α β values. Characteristic life α of the Weibull distribution lies approximately at the 63% of the population The values of the Weibull characteristic life at 63% of distributions have been calculated and collected in Table 3. It is obvious that these parameters closely match values α defined by plotting. Thus, all four specific criteria studied indicate that the genomic occurrence of protein topologies and domains can be characterized as true Weibull distributions. To support this notion further we have also considered another important property of the he Weibull distribution – the dependence of its median ( MDN ) from shape and scale parameters [ 13 ]: To assess the applicability of this condition, we calculated Weibull medians using sets of α and β parameters – estimated by graphical (double logarithmic transformation) and analytical (ML) approaches. The corresponding ' MDN Calctd ' values have been collected into Table 3 along with the observed medians of the Weibull distributions (marked ' MDN Obsrvd '). The estimated high quality linear dependences between the theoretical and observed medians are present on figures 3a and 3b for topologies and domains distributions respectively. The graphics demonstrate that calculated and observed median values are virtually the same what unanimously confirms validity of the condition (5). Thus, multiple independent tests have demonstrated that occurrence of protein folds in genomes obey the Weibull distribution and therefore can be interpreted in terms of the reliability theory what can provide additional insight into folds evolution. Discussion Interpretation of the Weibull parameters The very fact that we were able to assign the Weibull character to the distributions of the CATH protein topologies and homologous superfamilies within genomes ultimately implies that parameters of genomic occurrence can be classified as extreme values. According to the Extreme Values Theory the Weibull distribution will successfully model life systems for which many competing similar failure processes are "racing" to failure and the first to reach it produces the observed failure time [ 15 ]. In regard to genomic occurrence this may suggest that protein folds increase their genomic occurrence in a competitive manner and that those folds having a greater potential to duplicate, will continue to duplicate at the cost of less abundant folds which may ultimately disappear from genome. On another hand, according to reliability theory a Weibull distribution with β > 1 characterizes a life system that increasingly deteriorates. If the shape parameter is smaller then unity ( β <1), there is a reliability growth as the failure rate of the system decreases with time [ 14 ]. It is not clear at the moment, whether a reliability criterion is directly applicable to protein folds distributions. However, β does indeed describe the "skewdness" of the fold distribution, for example Caenorhabditis elegans has the lowest calculated value β among the studied organisms, whilst this genome has also been characterized for its recent expansion and duplication of several gene families [ 16 ]. Presumably, many of these folds are present at lower abundances in other genomes. It could be proposed that such a low β (according to the reliability theory characterizing the genome of C. elegans as the most stable amongst the studied) may reflect the fact that chances of loosing some lower abundant fold families are lower for C. elegans (considering that >70% of the translated ORFs C. elegans genome have been covered by the sequence-structure threading we have assumed that the recently duplicate genes are accordingly represented in the results). In this context, the reliability of a proteome can be viewed as its ability to maintain and expand its composition without loss of protein folds. We can speculate that life systems that enjoy evolutionary success will tend to minimize β <1 i.e. to have more balanced (less heterogeneous) folds representation in their genomes. The fact that most β values presented in Table 2 fall below the unity threshold demonstrates that, in general, the reliability of genome fold composition increases with time, i.e. less protein folds reach the failure state (termination of multiplication and, likely, following evolutionary extinction) as an organism evolves. Interestingly, little difference is observed has been found between the β shape parameters for topologies distributions across the three superkingdoms. All three linear dependences ln(- ln( SDF ( GO ))) ~ ln( GO ) for Bacteria, Eukaryote and Archaea presented on Figures 4a,4b appear very similar. As it has been already mentioned above, it is difficult to decide at this point whether the observed Weibull character of protein folds distribution can be placed in a larger context. We can only speculate that protein folding preferences may lead to a greater abundance of favorable protein configurations and to extinction of those folds which are less favorable. Such selection may represent a mechanism of evolutionary quest for searching for better protein folds. In any case, the observed phenomenon illustrates the act of natural selection in determination of the protein fold repertoires and that the propagation of protein folds in a genome occurs in a competitive manner, i.e. more abundant folds tend to expand their genomic presence even further causing lesser abundant folds to extinct. It also remains to be seen whether some other properties of genomes and proteomes can also be described by the Weibull statistics. In our studies we plan to use the Weibull approach to examine other distributions such as genomic occurrence of transcriptional promoters and regulatory elements, levels of gene expression and occurrence of protein domains per gene, among others. Another possible development for the reliability analysis in structural genomics might be to investigate whether the standard libraries of proteins folds themselves can be adequately described by the Weibull function. As it has been stipulated, in the study we have used the CATH standard library of protein folds, which is one of the most accepted and used protein folds classifications. Ii is not unfeasible, that the representation of protein architectures, topologies, homologous superfamilies, etc in the CATH can be adequately described by the Weibull law. Thus, it has been previously demonstrated that another widely used folds library – SCOP does indeed obey the power low [ 4 ]. Such observations would not necessarily contradict the uneven character of the fold distributions in individual proteomes or superkingdoms as a given protein fold library should reflect the proportion of protein folds occurrence in nature. At the same time, we anticipate that the analysis of the standard fold libraries in terms of the Weibull distributions may bring an additional insight into the field and will be carried out in the near future. To summarize the current work, it is possible to conclude that the use of the Weibull distribution allows more accurate description of protein topologies and domains distributions within and among genomes than power function used in conventional structural genomic studies. In addition, we were able to establish the Extreme Values relationships for protein folds distributions to demonstrate that the protein fold repertoire of an organism most likely occurs as a result of the competition amongst folds. This may reflect a mechanism of natural selection searching for an optimal protein structures when more evolutionary favorable folds tend to populate the entire genomic space and cause the extinction of lesser favorable protein configurations. Conclusions Use of a Weibull function allows describing cumulative distribution of protein topologies and domains within individual genomes and superkingdoms with higher accuracy compared to the conventional power function used in the related studies. The developed approach may be applied to quantification of the distribution of different properties of genomes and can be particularly useful for assessing and comparing fold distributions between different organisms and possible impact of the "reliability" of organisms due to a higher redundancy in their fold composition. In general, the results of investigation demonstrate the feasibility and importance of using the reliability analysis to improve the bioinformatics analysis of proteomes. Methods Assignment of protein folds The prediction of the protein folds has been conducted using the THREADER2 program [ 17 ]. The CATH homologues superfamily representative has been assigned to a given protein sequence if the THREADER2 produced an output above 2.9 for the Z score for the threading energy. After a certain CATH entry has been assigned to a protein sequence, it has also been associated with the corresponding higher level CATH representations: class, architecture and topology. The translated protein sequences for 33 complete genomes downloaded from the NCBI and ENSEMBL databases have been processed in this manner. The threading computations have been paralleled for processing on a Beowulf cluster consisting of 52 dual processor blades (2 × 1 GHz, 1 G RAM). The automated control was implemented by our own PVM-supporting Perl scripts enabling to distribute and query the individual threading processes over multiple computer servers. Survival distribution calculation After the occurrences of distinct classes, architectures, topologies and homologue family representatives have been established within the individual genomes, superkingdoms and in total, the corresponding survival distributions have been computed. First of all, we have established the counts of protein architectures, topologies and domains (homologues families) with a given genomic occurrence GO . At the next step these numbers have been converted into the fractional values. After that the survival distribution functions SDF(x) have been computed for genomes, superfamilies and for the combined set of proteins. The SDF(GO) numbers have been calculated for each integer GO value in the range from 0 to the maximal GO estimated within the set (genome/superfamily/total). Statistical analysis The fitting of the SDF ( GO )~ GO functions has been conducted by the SAS 9.0 statistical package (SAS Inc.). The power law dependences SDF(GO) = aGO - b have been analyzed as a logarithmic transforms Log( SDF(GO) = a - b * GO where the fitting has been conducted for the linear function. The Weibull – like dependences SDF(GO) = exp(-aGO b ) have been fitted using both non-linear approximation (by maximum likelihood method) and by the linear fitting of the double logarithmic transform: log(- log( SDF ( x )) = β log( x ) - β log( α ) The calculation of median valued for the survival distribution has been done by the 'R-project' open source statistical package. Supplementary Material Additional File 1 Parameters of power – law dependences for the survival distribution of genomic occurrences SDF(GO) = a GO b . Click here for file Additional File 2 Parameters α , β and medians (calculated and observed) of Weibull distribution of survival functions of genome occurrences established by maximum likelihood and plotting methods. Click here for file Additional File 3 Statistical parameters for 'Weibull papers' for genomic occurrences of protein topologies and domains. Click here for file

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The lack of public health research output from India

Background Systematic assessment of recent health research output from India, and its relation with the estimated disease burden, is not available. This information would help understand the areas in health research that need improvement in India to enhance the health of India's population. Methods The health research output from India during 2002, which was accessible in the public domain, was assessed by searching PubMed and other internet health literature databases, and was related to the disease burden suggested by the Global Burden of Disease Study. The main outcome measures were number of health papers with abstracts in basic, clinical and public health sciences; quality-adjusted research output based on the impact factors of journals in which the papers were published; classification of papers in disease/condition categories and comparison of research output with the estimated disease burden in each category. Comparison of the health papers from India during 2002 included in PubMed was done with those from Australia during one quarter of 2002. Results Of the 4876 health papers from India in 2002 in PubMed, 48.4%, 47.1% and 4.4% were in basic, clinical and public health sciences, respectively. Of the 4495 papers based on original research, only 3.3% were in public health. Quality-adjusted original research output was highest for non-communicable diseases (62% of total). Of the total quality-adjusted original research output, the proportions in injuries (0.7%), cardiovascular diseases (3.6%), respiratory infections (0.2%), diarrhoeal diseases (1.9%), perinatal conditions (0.4%), childhood cluster diseases (0.5%), unipolar major depression (0%), and HIV/AIDS (1.5%) were substantially lower than their proportional contribution to the disease burden in India. Human resources, health policy, health economics, and impact assessment of interventions were particularly poorly represented in public health research. The Australia-India ratio for quality-adjusted health research output per unit gross domestic product was 20 and for public health research output was 31. Conclusions Good-quality public health research output from India is grossly inadequate, and strategic planning to improve it is necessary if substantial enhancement of population health were to be made possible. There is inordinately low relative research output in several diseases/conditions that cause major disease burden in India.

Background India suffers a large proportion of the disease burden of the world, which has been estimated to be more than its 16.8% share of the world's population [ 1 , 2 ]. One of the vital elements in improving this situation is the need for a comprehensive and relevant evidence base that would equip India to take informed actions. A systematic assessment of recent health research output from India is not available. Without objective information about the current deficiencies and strengths in the health research output from India, it is difficult to plan substantial improvements in health research output that could enhance India's health status. We analysed the health research output from India in 2002 and related it with the estimated disease burden to identify areas that require particular attention to facilitate effective action to reduce disease burden in this world's second most populous country. Methods Health research output was defined as tangible research information related to human health that was readily accessible in the public domain. PubMed [ 3 , 4 ] (which includes MEDLINE) of the US National Library of Medicine, the most widely used online health literature search database in the world, and websites of major academic institutions in India, international agencies, and publishing houses, were searched to ascertain the health research output from India in the year 2002. PubMed was searched for papers published from India in 2002 using "India" in the author affiliation option in PubMed for all journals, and also by searching the Indian journals in PubMed as several papers in these journals mention only city and state but not India in the author affiliation. Only papers with abstracts were included, as the aim was to review all abstracts and classify the papers in various categories, including type of research, type of paper, disease/condition covered, allopathic or traditional system of medicine, and type and location of first author's institution. PubMed gives institutional affiliation and its location only for the first author. Papers that showed the first author affiliation with an Indian institution were considered as research output from India. Definitions were used to classify the Indian papers located in PubMed. Health research was defined as research that could be related to health. Basic research was considered either pure or applied , pure being experimental or theoretical work to advance health knowledge without a defined specific application and applied having such an application. Clinical research was categorised as patient series / management if the paper was about clinical cases or issues in management of patients, laboratory if it dealt mainly with laboratory analysis of patient specimens, clinical trial if it was a trial in the clinical setting, and clinical epidemiology if it was about distribution and determinants of disease assessed in the clinical setting. Public health research was categorised into epidemiology , environment/ social , and health systems / policy . Epidemiology included population epidemiology that dealt with study of distribution and determinants of disease and health in the population, and biostatistics / methods that dealt with methodological issues in epidemiology. Environment/social included environmental sciences that dealt with environmental influences on health, and social aspects that dealt with social dimensions of health. Health system/policy included health services that dealt with aspects of health service provision, and health policy that dealt with concepts and frameworks related to the health system. A paper was classified as original research if it had original data collection and its analysis, and review / viewpoint if it was not based on original data. An attempt was made to classify each paper under the disease/condition that it covered, according to the listing used in the Global Burden of Disease Study [ 2 ]. If a paper covered generic issue(s) which could not be classified under a particular disease/condition, it was considered unclassifiable for disease/condition. The 2002 impact factor of the journal, in which each paper was published, was used as a measure of the quality of each paper [ 5 , 6 ]. The proportion of papers and the quality-adjusted output for the diseases/conditions were related to the proportion of burden caused by each disease/condition in India as estimated for 2000 by the Global Burden of Disease Study [ 2 ]. The publications of 2002 were related to the disease burden of 2000, as research initiation to publication may take on an average a couple of years. Percent quality-adjusted research output was calculated for papers in the categories of several classifications as follows: IndMED [ 7 ], an online database of the Indian Medlars Centre, which covers several Indian biomedical journals was also searched. However, this database could not be included in the study, as the abstracts/papers for all the months of 2002 were not included in this database with substantial portions missing. As reports on commissioned research in public health may be available on the websites of agencies/organisations, the websites of several international agencies (DFID, European Commission, UNAIDS, USAID, WHO, World Bank), twelve academic institutions of India involved with public health, and sixteen publishing houses, were searched to locate research on public health reported in the public domain from India in 2002. For comparison of the Indian health research output with a developed country, a PubMed search was also done for papers published from Australia during the April–June 2002 quarter, using "Australia" OR "names/abbreviations of the states and territories of Australia [ 8 ]" in the author affiliation option in PubMed for all journals as several papers in Australian journals mentioned only city and state but not Australia in the author affiliation. Data were entered in an MS Access database and analysed using SPSS software. Results Of the 5718 papers with abstracts located on PubMed that were published from India in 2002, 842 (14.7%) papers were considered as non-health papers as they were on pure botany, chemistry, physics or zoology that could not be related to human health, and the other 4876 were health papers. The distribution of the types of research and the types of papers for the health papers is shown in Table 1 . The basic and clinical science papers predominated, with public health papers comprising a very small fraction (4.4% of the total). The proportion of papers based on original research was substantially lower for public health (68.5%) than for basic sciences (94.4%) and clinical sciences (92.3%); of the total 4495 original research papers, public health made up only 3.3%. 4700 (96.4%) of the total health papers were on the allopathic system of medicine and 176 (3.6%) on the traditional systems of medicine in which the majority were on ayurveda (144 [81.8%]). Table 1 Distribution of the types of health research and papers from India in 2002 included in PubMed Type of research No. (%)* of papers Type of paper Original research No. [%]† (%)‡ Review / Viewpoint No. [%]† (%)‡ Basic science 2358 (48.4) 2227 [49.6] (94.4) 131 [34.3] (5.6) Pure 525 (10.8) 518 [11.5] (98.7) 7 [1.8] (1.3) Applied 1833 (37.6) 1709 [38.0] (93.2) 124 [32.5] (6.8) Clinical science 2296 (47.1) 2119 [47.2] (92.3) 177 [46.3] (7.7) Patient series / management 1805 (37.0) 1639 [36.5] (90.8) 166 [43.5] (9.2) Laboratory 283 (5.8) 277 [6.2] (97.9) 6 [1.6] (2.1) Clinical trials 155 (3.2) 153 [3.4] (98.7) 2 [0.5] (1.3) Clinical epidemiology 53 (1.1) 50 [1.1] (94.3) 3 [0.8] (5.7) Public health 216 (4.4) 148 [3.3] (68.5) 68 [17.8] (31.5) Epidemiology 85 (1.7) 72 [1.6] (84.7) 13 [3.4] (15.3) Social / environmental 38 (0.8) 31 [0.7] (81.6) 7 [1.8] (18.4) Health systems / policy 93 (1.9) 45 [1.0] (48.4) 48 [12.6] (51.6) Other§ 6 (0.1) 0 [0.0] (0.0) 6 [1.6] (100.0) Total 4876 (100) 4494 [100.0] (92.2) 382 [100.0] (7.8) *Percent of the total 4876 papers † Percent of total in each type of paper ‡ Percent of total in each type of research §Papers that could not be classified in the above categories of type of research; these mostly consisted of biographies of persons or organizations Table 2 shows the distribution of the diseases/conditions covered by the original research papers from India as compared with the estimated disease burden. A large proportion of the basic science papers (49%) were not classifiable into specific disease/condition categories, as they were generic in nature, as compared with 2.9% papers in clinical science and 13% in public health. Overall, the relative proportion of quality-adjusted original research output for non-communicable diseases was higher than their relative contribution to the disease burden, and this was most marked for clinical sciences. However, some major categories/sub-categories within non-communicable diseases were not covered adequately, as a fairly large proportion of research output was on conditions or issues that were not contributing as much to the disease burden. For example, cardiovascular diseases with a disease burden of 11.4% of the total in 2000 had a relatively low quality-adjusted research output of 3.6% of the total. The estimated disease burden due to neuro-psychiatric conditions was 9.6% of the total and the quality adjusted original research output in this category was relatively fair at 8.8%, but the two major sub-categories of unipolar major depression and biopolar disorder that made up 5.2% of the total disease burden had only 0.2% of the total quality-adjusted original research output. A similar mismatch was seen for infectious & parasitic diseases and respiratory infections that had 33.3% of the total quality-adjusted original research output for 33.9% of the total disease burden, but the six major sub-categories under this group contributing 30.1% of the total disease burden had only 11.8% of the total quality-adjusted original research output (Table 2 ). Table 2 Distribution of original research health papers from India as compared with the estimated disease burden Disease / Condition* % DALY loss in 2000* % DALY loss in 2010* No. (%) of original research health papers† % quality-adjusted output for original research health papers‡ No. (%) of original research basic science papers§ % quality-adjusted output for original research basic science papers¶ No. (%) of original research clinical science papers# % quality-adjusted output for original research clinical science papers** No. (%) of original research public health papers†† % quality-adjusted output for original research public health papers‡‡ Communicable, Maternal, Perinatal and Nutritional Conditions 44.2 34.1 950 (28.6) 37.4 397 (34.9) 42.9 484 (23.5) 29.1 69 (53.9) 59.4 Infectious & parasitic diseases 25.9 22.7 762 (22.9) 33.1 358 (31.5) 40.2 362 (17.6) 23.6 42 (32.8) 48.6 Tuberculosis 6.8 7.0 143 (4.3) 7.4 49 (4.3) 7.2 87 (4.2) 5.6 7 (5.5) 29.2 STDs excluding HIV 1.5 1.1 13 (0.4) 0.3 1 (0.1) 0.1 12 (0.6) 0.6 0 (0.0) 0.0 HIV 3.3 6.0 48 (1.4) 1.6 14 (1.2) 1.5 29 (1.4) 1.8 5 (3.9) 1.2 Diarrhoeal diseases 6.7 4.2 34 (1.0) 1.9 17 (1.5) 2.2 16 (0.8) 1.8 1 (0.8) 0.3 Childhood cluster diseases 4.1 2.5 12 (0.4) 0.5 4 (0.4) 0.4 5 (0.2) 0.6 3 (2.3) 0.0 Respiratory infections 8.0 5.0 18 (0.5) 0.2 2 (0.2) 0.1 15 (0.7) 0.4 1 (0.8) 0.0 Lower respiratory infections 7.7 4.9 8 (0.2) 0.1 2 (0.2) 0.1 6 (0.3) 0.2 0 (0.0) 0.0 Maternal conditions 1.4 0.6 84 (2.5) 1.8 17 (1.5) 1.1 60 (2.9) 2.4 7 (5.5) 2.4 Perinatal conditions 6.1 3.9 25 (0.8) 0.4 1 (0.1) 0.1 23 (1.1) 0.8 1 (0.8) 1.0 Nutritional deficiencies 2.9 1.8 45 (1.4) 1.4 8 (0.7) 0.5 22 (1.1) 1.8 15 (11.7) 7.1 Protein energy malnutrition 1.2 0.7 6 (0.2) 0.2 1 (0.1) 0.0 1 (0.0) 0.0 4 (3.1) 3.4 Iron deficiency anaemia 1.5 1.0 10 (0.3) 0.2 1 (0.1) 0.3 6 (0.3) 0.2 3 (2.4) 0.0 Noncommunicable diseases 38.7 47.5 2344 (70.6) 62.0 732 (64.4) 56.5 1555 (75.6) 70.1 57 (44.5) 40.2 Malignant neoplasms 3.8 5.4 370 (11.1) 11.2 118 (10.4) 9.1 251 (12.2) 14.4 1 (0.8) 2.9 Diabetes mellitus 0.8 0.8 129 (3.9) 3.2 64 (5.6) 3.6 57 (2.8) 2.3 8 (6.3) 8.5 Neuro-psychiatric conditions 9.6 11.5 248 (7.5) 8.8 112 (9.9) 10.5 124 (6.0) 6.6 12 (9.4) 11.6 Unipolar major depression 4.0 5.0 0 (0.0) 0.0 0 (0.0) 0.0 0 (0.0) 0.0 0 (0.0) 0.0 Bipolar disorder 1.2 1.4 3 (0.1) 0.2 0 (0.0) 0.0 1 (0.0) 0.1 2 (1.6) 3.5 Sense organ diseases 1.5 2.1 185 (5.6) 4.8 25 (2.2) 2.1 148 (7.2) 7.5 12 (9.4) 8.8 Cataract 1.2 1.7 25 (0.8) 0.9 6 (0.5) 0.3 16 (0.8) 1.3 3 (2.3) 2.5 Cardiovascular diseases 11.4 14.6 203 (6.1) 3.6 38 (3.3) 2.3 159 (7.7) 5.2 6 (4.7) 0.7 Ischaemic heart disease 5.3 7.1 56 (1.7) 0.9 11 (1.0) 0.7 41 (2.0) 1.2 4 (3.1) 0.2 Cerebrovascular disease 2.1 2.7 20 (0.6) 0.3 3 (0.3) 0.2 17 (0.8) 0.4 0 (0.0) 0.0 Respiratory diseases 3.7 4.8 68 (2.0) 1.5 15 (1.3) 1.1 45 (2.2) 1.6 8 (6.3) 3.2 Chronic obstructive pulmonary disease 1.4 2.0 2 (0.1) 0.0 0 (0.0) 0.0 2 (0.1) 0.0 0 (0.0) 0.0 Digestive tract diseases 2.3 2.4 198 (6.0) 5.3 52 (4.6) 3.7 143 (6.9) 7.5 3 (2.3) 1.5 Cirrhosis of liver 1.1 1.2 12 (0.4) 0.6 1 (0.1) 0.6 11 (0.5) 0.6 0 (0.0) 0.0 Congenital anomalies 3.4 3.5 105 (3.2) 1.6 2 (0.2) 0.2 103 (5.0) 3.3 0 (0.0) 0.0 Injuries 17.2 18.4 28 (0.8) 0.7 7 (0.6) 0.6 19 (0.9) 0.8 2 (1.6) 0.4 Unintentional injuries 15.0 15.9 24 (0.7) 0.6 7 (0.6) 0.6 15 (0.7) 0.6 2 (1.6) 0.4 Road traffic injuries 3.5 5.1 2 (0.1) 0.0 0 (0.0) 0.0 2 (0.1) 0.0 0 (0.0) 0.0 Falls 3.5 3.0 0 (0.0) 0.0 0 (0.0) 0.0 0 (0.0) 0.0 0 (0.0) 0.0 Fires 2.1 2.0 4 (0.1) 0.1 2 (0.2) 0.1 2 (0.1) 0.2 0 (0.0) 0.0 Intentional injuries 2.1 2.5 1 (0.0) 0.1 0 (0.0) 0.0 1 (0.0) 0.1 0 (0.0) 0.0 Self-inflicted injuries 1.4 1.7 1 (0.0) 0.1 0 (0.0) 0.0 1 (0.0) 0.1 0 (0.0) 0.0 Total 100 100 3322 (100) 100 1136 (100) 100 2058 (100) 100 128 (100) 100 *According to the Global Burden of Disease Study [2]; only diseases/conditions with disease burden of >1% of the total are listed, plus diabetes mellitus; since all diseases/conditions are not listed, the sum of sub-categories shown may not add up to the total for their categories; DALY is disability-adjusted life year † Denominator for this percent calculation is 3322, which excludes 1172 papers that were not classifiable into specific disease/condition categories ‡ Based on the total impact factor of 3456.262 for the 3322 original health research papers included in this table §Denominator for this percent calculation is 1136, which excludes 1091 papers that were not classifiable into specific disease/condition categories ¶Based on the total impact factor of 1757.491 for the 1136 original basic health research papers included in this table #Denominator for this percent calculation is 2058, which excludes 61 papers that were not classifiable into specific disease/condition categories **Based on the total impact factor of 1557.811 for the 2058 original clinical health research papers included in this table †† Denominator for this percent calculation is 128, which excludes 20 papers that were not classifiable into specific disease/condition categories ‡‡Based on the total impact factor of 140.96 for the 128 original public health research papers included in this table Overall, the diseases/conditions that were substantially underrepresented in the relative proportion of quality-adjusted original research output as compared with their contribution to the disease burden were injuries, cardiovascular disease, respiratory infections, diarrhoeal diseases, perinatal conditions, childhood cluster diseases (including measles and tetanus), unipolar major depression, and HIV/AIDS (Table 2 ). As the research output was least in public health, a brief description follows to understand this deficiency better. Figure 1 shows the diseases/conditions that were estimated to contribute more than 4% of the total disease burden in 2000 or 2010, and for which the original research output in public health was less than one-third of their proportional contribution to the disease burden estimated for 2010, suggesting that these diseases/conditions needed particular attention. Table 3 shows the distribution of original research in the various areas of public health, which suggests that original research in human resources, health policy, and health economics is relatively more deficient within the already low public health research output. Only six of the original public health research papers were on assessing interventions across the various areas, suggesting that the existing public health research in India has not yet evolved to the stage of methodically assessing the impact of public health interventions, which is a necessary step in the evolution of effective public health action. Figure 1 Diseases/conditions poorly represented in original public health research relative to their contribution to the disease burden in India. Table 3 Distribution of the types of original public health research from India Type of public health research No. (%) of original research public health papers Total impact factor of original research public health papers in each type % quality-adjusted output for all original research public health papers† Epidemiology 72 (48.6) 59.6 38.9 Population epidemiology 69 (46.6) 56.5 36.9 Biostatistics / Methods 3 (2.0) 3.0 2.0 Environment / Social 31 (20.9) 29.3 19.1 Environmental sciences 14 (9.5) 16.7 10.9 Social aspects 17 (11.5) 12.7 8.3 Health Systems / Policy 45 (30.4) 64.4 42.0 Health services 42 (28.4) 63.3 41.3 Health economics* 8 (5.4) 5.7 3.7 Training / human resources* 5 (3.4) 0.6 0.4 Health policy 3 (2.0) 1.1 0.7 Total 148 (100) 153.3 100 *Health economics and training / human resources are sub-categories of health services † Based on the denominator of 153.3 Of the total 4876 health papers from India in PubMed for 2002, 1300 (26.7%) were published in Indian journals, but these papers accounted for only 1.5% of the total impact factor of all health papers from India due to the very low impact factors of Indian journals. Among the public health papers 44.4% were published in Indian journals, for clinical sciences papers this was 39.7%, whereas this proportion was much smaller for basic sciences (12.4%). The highest proportion of quality-adjusted basic research output was by university departments, institutions affiliated with the Council of Scientific and Industrial Research, and technical institutions; the predominant proportion of clinical research was by medical colleges / hospitals; and public health research by medical colleges / hospitals, government departments (due to one paper in a very high impact factor journal), and institutions affiliated with the Indian Council of Medical Research (Table 4 ). The National Capital Territory of Delhi accounted for the highest health research output among all states / union territories or cities (Table 5 ). The top ten research producing cities, with 6% of the population of India, produced 75.6% of the quality-adjusted research output, suggesting a concentration of quality research activity in parts of the country. Table 4 Distribution of health research output from various types of institutions in India Type of institution All health Basic science Clinical science Public health No. (%) of health papers % quality-adjusted output for health papers No. (%) of basic science papers % quality-adjusted output for basic science papers No. (%) of clinical science papers % quality-adjusted output for clinical science papers No. (%) of public health papers % quality-adjusted output for public health papers Medical college / Hospital 2571 (52.7) 33.4 407 (17.3) 11.9 2044 (89.0) 82.1 119 (55.1) 45.9 Indian Council of Medical Research* 159 (3.3) 4.0 52 (2.2) 2.3 78 (3.4) 6.7 28 (13.0) 13.1 Council of Scientific and Industrial Research † 387 (7.9) 13.3 361 (15.3) 18.9 21 (0.9) 1.7 5 (2.3) 0.6 Technical institutions ‡ 281 (5.8) 12.2 268 (11.4) 17.7 7 (0.3) 0.6 5 (2.3) 2.3 Paramedical college/institution 164 (3.4) 2.9 154 (6.5) 4.1 8 (0.3) 0.4 2 (0.9) 0.9 University department 813 (16.7) 16.4 743 (31.5) 23.0 53 (2.3) 2.5 16 (7.4) 4.7 NGO / Foundation / Society 69 (1.4) 1.2 12 (0.5) 0.6 33 (1.4) 1.6 22 (10.2) 8.0 Government department 4 (0.1) 0.6 0 (0.0) 0.0 0 (0.0) 0.0 4 (1.9) 17.9 § Industry 31 (0.6) 0.6 28 (1.2) 0.9 3 (0.1) 0.1 0 (0.0) 0.0 Other 397 (8.1) 15.4 333 (14.1) 20.6 49 (2.1) 4.3 15 (5.9) 6.7 Total 4876 (100) 100 2358 (100) 100 2296 (100) 100 216 (100) 100 *Institutions affiliated with the Indian Council of Medical Research [15] † Institutions affiliated with the Council of Scientific and Industrial Research [16] ‡ Indian Institutes of Technology, Indian Institute of Science, and other technical institutions § Percentage high due to one paper in a very high impact factor journal The total of basic, clinical and public health papers does not add up to the "all health" papers in all rows, as 6 "other" papers that could not be classified as basic, clinical or public health (Table 1) are not included in this table Table 5 Distribution of health research output from states and cities in India State / Union Territory* Population in millions† No. (%)‡ of health papers No. of health papers per million population Total impact factor of health papers % quality-adjusted health research output§ Total impact factor per million population National Capital Territory of Delhi 13.8 1014 (20.8) 73.5 1216.0 20.8 88.1 Karnataka 52.7 491 (10.1) 9.3 786.8 13.5 14.9 Maharashtra 96.7 573 (11.8) 5.9 710.1 12.2 7.3 Uttar Pradesh 166.0 484 (9.9) 2.9 591.0 10.1 3.6 West Bengal 80.0 362 (7.4) 4.5 510.6 8.7 6.4 Tamil Nadu 62.1 476 (9.8) 7.7 476.7 8.2 7.7 Andhra Pradesh 75.7 299 (6.1) 3.9 461.1 7.9 6.1 Union Territory of Chandigarh 0.9 364 (7.5) 404.4¶ 336.2 5.8 373.6¶ Kerala 31.8 183 (3.8) 5.8 177.5 3.0 5.6 Punjab 24.3 105 (2.2) 4.3 123.4 2.1 5.1 Gujarat 50.6 74 (1.5) 1.5 68.4 1.2 1.4 Madhya Pradesh 60.4 71 (1.5) 1.2 61.9 1.1 1.0 Union Territory of Pondicherry 1.0 66 (1.4) 66.0 53.7 0.9 53.7 Haryana 21.1 95 (1.9) 4.5 44.6 0.8 2.1 Orissa 36.7 41 (0.8) 1.1 43.2 0.7 1.2 Rajasthan 56.5 62 (1.3) 1.1 40.8 0.7 0.7 Jammu And Kashmir 10.1 20 (0.4) 2.0 28.6 0.5 2.8 Assam 26.6 18 (0.4) 0.7 26.0 0.4 1.0 Uttaranchal 8.5 21 (0.4) 2.5 25.7 0.4 3.0 Meghalaya 2.3 9 (0.2) 3.9 13.7 0.2 6.0 Himachal Pradesh 6.1 12 (0.2) 2.0 13.1 0.2 2.1 Andaman & Nicobar Islands 0.4 6 (0.1) 15.0 9.9 0.2 24.8 Goa 1.3 6 (0.1) 4.6 8.6 0.1 6.6 Bihar 82.9 5 (0.1) 0.1 5.5 0.1 0.1 Jharkhand 26.9 5 (0.1) 0.2 3.4 0.1 0.1 Sikkim 0.5 3 (0.1) 6.0 1.6 0.0 3.2 Manipur 2.4 4 (0.1) 1.7 1.5 0.0 0.6 Chhattisgarh 20.8 3 (0.1) 0.1 1.3 0.0 0.1 Arunachal Pradesh 1.1 2 (0.0) 1.8 0.9 0.0 0.8 Tripura 3.2 2 (0.0) 0.6 0.3 0.0 0.1 Top fifteen cities (State / Union Territory)* Delhi (National Capital Territory of Delhi) 13.8 1014 (20.8) 73.5 1216.0 20.8 88.1 Bangalore (Karnataka) 8.4 258 (5.3) 30.7 598.2 10.2 71.2 Mumbai (Maharashtra) 11.9 393 (8.1) 33.0 499.4 8.5 42.0 Kolkata (West Bengal) 4.6 299 (6.1) 65.0 463.6 7.9 100.8 Hyderabad (Andhra Pradesh) 3.7 233 (4.8) 63.0 404.2 6.9 109.2 Chandigarh (Union Territory of Chandigarh) 0.9 364 (7.5) 404.4¶ 336.2 5.8 373.6¶ Lucknow (Uttar Pradesh) 3.7 272 (5.6) 73.5 332.1 5.7 89.8 Chennai (Tamil Nadu) 4.2 246 (5.0) 58.6 267.6 4.6 63.7 Pune (Maharashtra) 7.2 108 (2.2) 15.0 163.0 2.8 22.6 Varanasi (Uttar Pradesh) 3.1 87 (1.8) 28.1 138.8 2.4 44.8 Thiruvananthapuram (Kerala) 3.2 121 (2.5) 37.8 135.7 2.3 42.4 Mysore (Karnataka) 2.6 74 (1.5) 28.5 96.2 1.6 37.0 Vellore (Tamil Nadu) 3.5 84 (1.7) 24.0 95.6 1.6 27.3 Pondicherry (Union Territory of Pondicherry) 0.7 66 (1.4) 94.3 53.7 0.9 76.7 Visakhapatnam (Andhra Pradesh) 2.2 34 (0.7) 15.5 38.5 0.7 17.5 * Listed in descending order of total impact factor of health papers; the states / union territories of Mizoram, Nagaland, Dadra & Nagar Haveli, Daman & Diu, and Lakshadweep had no publications in PubMed in 2002 † Population for 2001 from the Census of India [14] ‡ Percent of the total 4876 health research papers from India in 2002 § Percent of the total impact factor of 5842.055 for all 4876 health research papers from India ¶This high per capita output is likely related to the small population of Chandigarh and the high concentration of academic institutions Search of websites of major academic institutions in India, international agencies, and publishing houses revealed that substantial original public health research output that was accessible in the public domain was not readily available from these sources. Among the major academic institutions in India involved with public health research, only one was found to have a few reports on health research accessible on its website [ 9 ] and another had some health research abstracts on its website [ 10 ]. The international agencies had some reports on their websites on India-related health research that were mostly authored by non-Indian authors. In the April-June quarter of 2002, 1905 health papers published from Australia were located on PubMed, of which 722 (37.9%) were in basic sciences, 954 (50.1%) in clinical sciences, and 229 (12%) in public health. Taking into account the population and total gross domestic product (GDP) adjusted for purchasing power parity (PPP) of Australia and India [ 1 ], the quality-adjusted health research output and public health research output were 19.6 and 31 times higher from Australia than India, respectively, per unit GDP adjusted for PPP (Table 6 ). Table 6 Comparison of health research output from India and Australia in 2002 India Australia Australia-India ratio Total Per million population* Per billion GDP-PPP† Total‡ Per million population* Per billion GDP-PPP† Per million population Per billion GDP-PPP No. of health papers 4876 4.72 1.66 7620 392.78 15.49 83.2 9.3 Impact factor for health papers 5842 5.65 1.99 19231 991.27 39.10 175.3 19.6 No. of basic science papers 2358 2.28 0.80 2888 148.87 5.87 65.2 7.3 Impact factor for basic science papers 3944 3.82 1.35 10598 546.31 21.55 143.1 16.0 No. of clinical science papers 2296 2.22 0.78 3816 196.70 7.76 88.5 9.9 Impact factor for clinical papers 1698 1.64 0.58 7624 393.01 15.50 239.1 26.7 No. of public health papers 216 0.21 0.07 916 47.22 1.86 225.9 25.3 Impact factor for public health papers 193 0.19 0.07 1008 51.95 2.05 277.6 31.0 *Based on 1033.4 million population for India and 19.4 million for Australia in 2001 [1] † Based on the gross domestic product adjusted for purchasing power parity (GDP-PPP) of US$ 2930 billion for India and US$ 491.8 billion for Australia in 2001 [1] ‡ Based on multiplying the number of papers and their total impact factor for the April-June 2002 quarter by four to obtain the estimate for the year 2002 The total of the number and impact factor for basic, clinical and public health papers does not add up to that for the health papers, as 6 "other" papers (with total impact factor 6.012) that could not be classified as basic, clinical or public health (Table 1) are not included in this table Discussion The data presented in this paper suggest that the health research output from India is not commensurate with the magnitude and distribution of disease burden. The research output in public health is particularly meagre, which is a major concern as public health sciences are a necessary tool to facilitate improvement in population health. Within this low research output, several diseases/conditions contributing substantially to the disease burden and several major areas of public health importance have relatively less representation. Without dynamic, relevant, good quality and adequate original research in the various aspects of public health it is difficult to imagine how the sub-optimal health status of the Indian population would improve on rhetoric or theoretical concepts alone [ 14 , 15 ]. In this paper we used impact factors for journals as a measure of the quality of papers published in those journals. Although impact factors are not without their limitations, they still offer a tangible, and perhaps the best available, option to compare the quality of publications in journals [ 6 ]. We explored several sources where information about health research output from India could be available in the public domain, as the utilisation of research findings is facilitated most if they are readily accessible in the public domain. However, we did not find any source that would add substantially to the information available in the PubMed database. Indeed, there are more Indian health journals than are included in PubMed, but their quality in general is not as high as those included in PubMed with none of them having an impact factor above zero. Non-inclusion in our analysis of the papers published in these journals, therefore, did not bias our assessment of quality-adjusted research output based on impact factors. The relative low quality and impact factor of a large proportion of Indian journals has been discussed previously [ 16 , 17 ]. PubMed lists affiliation of the first author only, and therefore, the analysis presented in this paper includes only those publications in which the first author had Indian affiliation. There would be other publications with non-Indians as first author and Indians as co-author(s), which we estimate to be a very small fraction of those with Indians as first author. In the general context, the PubMed/MEDLINE database has been used previously to assess the health research output from several countries [ 18 - 25 ]. We used the disease burden in India as estimated by the Global Burden of Disease Study [ 2 ]. Although the limitations of this Study have been debated previously in the literature, we could not find a better alternative for use for our study, as these were the most comprehensive estimates available for India. In any case, these estimates can be taken only as indicative, and therefore, we highlight only gross deviations of health research output from these trends. There has been a previous attempt to assess the health research output from India using the Science Citation Index of 1981–85 and relating the number of papers published in journals of various medical/health specialities with the perceived areas of major disease burden [ 26 ]. However, review of all published abstracts to classify each paper in various categories, the approach used by us, has not been used previously to assess health research output from India to our knowledge. Systematic tracking of health research output, and its relation to the estimated trends in disease burden, are necessary for guiding further appropriate development of health research in India. In addition to the overview of research needs identified in this paper, more in-depth assessment of research needs for major diseases/conditions would also be necessary, as was reported recently for the evidence base needed to control HIV/AIDS in India [ 27 ]. Since public health sciences seem to be the weakest link in improving health in India currently, it is imperative that a strategic framework for developing original public health research in India be evolved. To do so, the demand , supply and environment issues would have to be addressed: • Demand. Among the multitude of factors that influence the demand for relevant public health research, the role of policy makers and senior health academics is of particular importance. This is seriously sub-optimal in India at present. Political compulsions push many policy makers into short-term gains instead of investments in comprehensive research for long-term benefits. Although there has recently been an increasing trend in India towards commissioned research by government and international agencies in some aspects of public health, this by itself is not enough to boost comprehensive public health research in India, and the reports of such studies are many times not available in the public domain which reduces the chance of their widespread utilisation. Many senior health academics in India continue to disregard public health research as a less-respectful cousin of basic and clinical research. Systematic efforts are needed to demonstrate to these groups the linkages between all aspects of health research (basic, clinical and public health), and the linkages between public health research and improvements in population health, in order to boost the demand for relevant and good-quality public health research in India. • Supply. Enhancing the output of public health research will require effort on various fronts. Establishing schools of public health and other institutions to train quality scientists in public health is a priority, as India has a surprisingly few number of institutions that can provide proper training in public health research. Another area that needs quick attention is to make public health exposure in medical and paramedical colleges more practical to encourage hands-on investigative thinking, as currently it is so theoretical that it rarely inspires enthusiasm in young professionals towards public health research. Setting higher standards for the research dissertations currently required for post-graduate degrees in preventive and social medicine would also encourage better quality and practically relevant public health research. It is also necessary to systematically develop performance-based opportunities to public health research scholars for career enhancement. Another element that would help develop public health research capacity in India is evolving mechanisms to encourage contribution to this effort by the many Indian public health researchers living abroad. • Environment. A conducive environment is necessary for the demand and supply of public health research to function optimally. Efforts are needed to develop this by attempting to develop broad-based coalitions, that include health care providers, civil society and non-governmental sector, for-profit private sector and industry, and national and international agencies providing financial support, which would understand and support the need for vibrant public health research as a vital element of societal development. This is a necessary element that has so far received scant attention, which must be addressed if sustainable development of public health research to improve population health is to become possible in India. An environment of good-quality and comprehensive public health research in India would also infuse the much-needed originality in teaching public health sciences and their practical application to the local context. Evolving such frameworks would require building up a critical momentum for this effort through perseverance and wisdom. One such opportunity is provided by the recent initiative of the Indian Ministry of Health and Family Welfare to develop more effective institutes of public health in India, with relevant public health research and its utilisation an important key to improving population health [ 28 ]. The recent attention towards revitalising the academic aspects of health care / medicine through evidence [ 29 ] and evidence-based global health [ 30 ] is particularly relevant for developing nations. Evolving a strong, dynamic and locally-relevant evidence base is even more important for developing nations as this is likely to yield relatively higher returns by contributing to improvements in the health, lives and economy of a larger proportion of the world's population. For this to happen, theoretical concepts alone would obviously be not enough. The practical solutions for this effort would have to be developed wisely. The data and its interpretation presented in this paper are, we hope, an example of how the deficiencies in the evidence-base needed for adequate health care in developing nations can be understood objectively in order to plan its strengthening. Conclusions • Publications from India in PubMed were 11 times less in public health than those in basic sciences and in clinical sciences in 2002. • Injuries, cardiovascular diseases, respiratory infections, diarrhoeal diseases, perinatal conditions, childhood cluster diseases, unipolar major depression, and HIV/AIDS had substantially less proportion of quality-adjusted original research output in India as compared with their contribution to the disease burden. • India produced 20 times less quality-adjusted health research output than Australia per unit gross domestic product adjusted for purchasing power parity, and this ratio for public health research output was even higher at 31 times. • Good-quality public health research output from India is grossly inadequate, and strategic planning to improve it is necessary if substantial enhancement of population health were to be made possible. Competing interests The author(s) declare that they have no competing interests. Authors' contributions LD conceived the idea of this study, guided the design, data collection and analysis, and wrote the initial draft of this paper. YSS contributed to the design, data collection and analysis. MNJ contributed to data collection and analysis. VSUB contributed to data management and analysis. RD contributed to the idea of this study, design and data analysis. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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212703

Large-Scale Association Study Confirms Genetic Complexity Underlying Type 2 Diabetes

A leading cause of death and disability, diabetes affects some 16 million Americans and up to 135 million people worldwide. While environmental factors such as diet and lifestyle are known to influence an individual's risk of getting adult-onset, or Type 2, diabetes, there is also a substantial inherited component, though many of the genetic pathways involved remain unidentified. The challenge of defining these genetic pathways lies in the fact that diabetes is what is known as a “complex trait”: not only is it likely that variations in many different genes or some combination of genes contribute to an increased risk, but there are probably different genes associated with diabetes in different populations. Tackling the monumental task of deciphering this genetic puzzle in the largest known study of its kind, Inês Barroso and colleagues confirm the genetic complexity of the disease and clearly demonstrate that untangling the genetics will require even larger studies. In diabetes, defects in both the secretion and function of insulin—which is produced by the pancreas—impair the body's ability to metabolize glucose. Based on what is known about the biology of pancreatic function and diabetes, the researchers chose 71 potential suspect genes that could reasonably be expected to contribute to the disease if they malfunctioned. Some of these genes are involved in the function of pancreatic beta-cells, which secrete insulin; a second group influences the function of insulin and glucose metabolism; and a third plays a broader role in energy metabolism. The results show that none of the genes on their own had a large effect, though a number of gene variations increased risk slightly. They also suggest the existence of variations in several genes that influence the risk of Type 2 diabetes. The dataset will be valuable for future studies of diabetes and supports the view that variation in genes affecting insulin production as well as insulin action can influence the risk of Type 2 diabetes. However, the genetic complexity of the disease—with a number of genes conveying a slightly increased risk— demands additional studies of larger populations to reliably identify the genes involved and the genetic variations that, alone or in combination, increase or lower an individual's risk of developing the disease.

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548130

Identification of novel prognostic markers in cervical intraepithelial neoplasia using LDMAS (LOH Data Management and Analysis Software)

Background Detection of Loss of Heterozygosity (LOH) is one of the most common molecular applications in the study of human diseases, in particular cancer. The technique is commonly used to examine whether a known tumour suppressor gene is inactivated or to map unknown tumour suppressor gene(s). However, with the increasing number of samples analysed using different software, no tool is currently available to integrate and facilitate the extensive and efficient data retrieval and analyses, such as correlation of LOH data with various clinical data sets. Results An algorithm to identify prognostic disease markers is devised and implemented as novel software called LDMAS. LDMAS is a software suite designed for data retrieval, management and integrated analysis of the clinico-pathological data and molecular results from independent databases. LDMAS is used in stratification of disease stages according to clinical stage or histological features and correlation of various clinico-pathological features with molecular findings to obtain relevant prognostic markers such as those used in predicting the outcome of cervical intraepithelial neoplasia (CIN). This approach lead to the identification of novel prognostic cervical cancer markers and extraction of useful clinical information such as correlation of Human Papilloma Virus (HPV) status with CIN lesions. Conclusions A novel software called LDMAS is implemented and used to extract and identify prognostic disease markers. The software is used to successfully identify 4 novel prognostic markers that can be used to predict the outcome of CIN. LDMAS provides an essential platform for the extraction of useful information from large amount of data generated by LOH studies. LDMAS provides three unique and novel features for LOH analysis : (1) automatic extraction of relevant data from patient records and reports (2) correlation of LOH data with clinico-pathological data and (3) storage of complex data in flexible format. The first feature automates the creation of database of clinically relevant information from huge amount of data, the second feature extracts useful biomedical information such as prognostic markers in CIN and the third feature simplifies the statistical analyses of the data and allows non-statisticians to carry out the analysis. Additionally, LDMAS can be used to extract clinically useful markers from other diseases and interface to high throughput genotyping analysis software such as GDAS used to generate LOH data from Affymetrix ® GeneChip Mapping arrays.

Background Detection of LOH is one of the most common molecular applications in the study of human diseases, in particular cancer. It is commonly used to examine whether a known tumour suppressor gene is inactivated or to map unknown tumour suppressor gene(s). Detection of LOH not only helps in understanding the molecular mechanisms underlying the development of cancer, but also provides important information useful for disease diagnosis and prognosis. LOH detection is commonly carried out by the analysis of microsatellite markers using an automated DNA sequencer. With the raw data from the sequencer being stored in one file per lane together with corresponding clinical information and patient follow up data, each LOH study [ 1 , 2 ] generates hundred of files that need to be organised and related in a structured format. However, with the increasing number of samples analysed using different software, no tool is currently available to facilitate the extensive and efficient data retrieval and analyses, such as correlation of LOH data with various clinical data sets. We have developed a novel software package: LOH Data Management and Analysis Software (LDMAS) in order to satisfy these needs. LDMAS can retrieve LOH data from automated DNA sequencer platform and clinical data from any patient record system and correlate different data sets according to the user's choice. Here we present how LDMAS interfaces to Genotyper software (ABI, Foster City, CA) which is used to determine the presence of LOH, and the patient record system SunQuest (San Francisco, CA), facilitating the identification of LOH markers associated with the development of CIN [ 3 ]. CIN show variable clinical behaviour despite morphological homogeneity within each subgroup. Clinically, it is vital to distinguish CIN lesions with different behaviour and identify those likely to persist and progress despite treatment. Implementation System architecture LDMAS package is composed of three modules: (1) MRES (Medical Report Extractor Software) which parses patient report files, extracts the information of interest and organises it into a structured format, applicable to LDAS (2) LDAS (LOH Data Analysis Software) which obtains LOH data from Genotyper (Applied Biosystems, California) or GDAS (Affymetrix, California) software and correlates it to clinical data obtained from MRES (3) LDMS (LOH Data Management Software) which is used to gather patients' clinico-pathological data and extract significant relationship between the various data sets LDAS and LDMS work synergistically to manage and analyse LOH data. The MRES source code for automatic parsing of patient reports is written in C++ using C++ Builder 5.0 (Borland Software Corporation, Scotts Valley, CA), LDAS is written in Visual Basic for Application as an Excel 2000 add-in and LDMS is written as Visual Basic for Application modules embedded within Access 2000 as fully functional software. These modules can be run independently and used for applications other than LOH. LDMAS runs on Microsoft ® Windows 2000 or Windows XP operating system. Figure 1 shows LDMAS architecture. Type of input data for LDMAS modules The MRES module takes its input from any patient report file containing clinical details such as diagnosis, stage of the disease, treatment and follow up results, parses and formats patient's data into a structured format that can be saved as Excel spreadsheet. In this case, data were taken from SunQuest patient record system and MRES converted and produced the data as: (1) Hospital Number (2) Hospital ID (3) Patient Name (4) Date of Birth (5) Pathological specimen Number (6) Date of Diagnosis (7) Histological diagnosis. The user can manually check the data and use it as template for analysis. Data analysis is carried out using LDAS which obtains LOH data from Genotyper and correlates it to the clinical data obtained from MRES. LDAS obtains data in plain text format and can thus be easily interfaced to any LOH platform generating software such as Genotyper and GDAS. Finally all data are entered into LDMS for storage and intelligent mining of data. Database query results can be exported back to LDAS allowing correlation between LOH and various clinico-pathological parameters such as age, histological grade, treatment modality and their responses, and HPV status as well as carry out multivariate analysis to determine the sensitivity and specificity of the markers involved in the LOH study. A more detailed description of all the modules is provided in LDMAS user's guide [Additional file 1] and the example below. Advantages of LDMAS LDMAS offers several advantages to users. It is user friendly and its architecture is modular allowing versatility of use. It enforces the standardisation of procedures for studies involving large cohorts of individuals. The data is well organized since LDMAS systematically assigns LOH results of each case to its corresponding clinical information. Additionally, LDAS standardizes LOH data analysis implicitly and allows the user to edit the data manually if needed. Microsoft Excel has been chosen to implement LDAS because of its wide use, versatility and convenient statistical analysis features facilitating the implementation of multivariate analysis and correlation testing between LOH and clinico-pathological parameters. Results and discussion LDMAS application in identification of LOH markers associated with persistence / progression of cervical intraepithelial neoplasia We divided the CIN groups into disease free indicating cases that become CIN free after treatment, and disease persistence/progression indicating cases that develop show progression or persistence of CIN despite treatment. We used LDMAS to retrospectively examine the prognostic value of LOH at 12 microsatellite markers including 10 from 3p14, 3p22-21, 6p21 and 11q23 which are frequently deleted in cervical cancer [ 3 , 4 ], in 164 cases of CIN lesions using archival cytological/histological specimens. LOH was further correlated with high risk HPV infection. Initially MRES was used to automatically parse 4300 patient records and extract clinico-pathological data including age, diagnosis, method of treatment and treatment response during follow up. Out of those, 164 cases with follow up of 3 or more years were chosen for the study and their clinico-pathological information was imported into LDAS. Initially, 71 out of the 164 selected cases were examined for LOH using 12 fluorescent microsatellite markers ran on ABI377 DNA Sequencer. LDAS was then used to identify the microsatellite markers for which LOH was significantly associated with disease persistence/progression of CIN using two tailed student t-test. Figure 2 generated using LDAS shows that microsatellite markers D3S1300 (3p14.2), D3S1260 (3p22.2), D11S35 (11q22.1) and D11S528 (11q23.3) have the highest LOH in CIN lesions displaying persistence/progression than those who were disease free during follow up [ 5 ]. Validation of prognostic markers associated with persistence / progression of CIN To validate this finding, LOH at these four markers was investigated in a further series of 93 cases. Compatible results were obtained from these additional cases. The two sets of data were combined and further compared using LDMS. Methodologies included : 1) comparison using χ 2 (chi-squared) test of LOH at each of the four microsatellite markers with age, various methods of treatment, different subtypes of HPV infection and between CINs showing disease free or disease persistence/progression. 2) correlation of LOH data with histological grade of CIN, treatment response and various HPV subtypes. Through such complex analysis, we showed that concurrent LOH at two of the four microsatellite markers could identify 47% of CINs that showed disease persistence/progression with 100% specificity [ 5 ]. Furthermore, LOH at D3S1300 was found to be significantly associated with HPV16 infection. Part of this data analysis is supplied in the LDMAS guide [see Additional file 1]. More detailed analysis of this study is described in [ 5 ]. Algorithm for identifying prognostic disease markers Based on the above example, an algorithm can be developed to extract prognostic markers for other diseases. The algorithm can be summarised in the following pseudocode : (1) Divide the disease in groups according to the pathology staging (2) Parse patient data from clinical records and use the groups defined in part (1) (3) FOR each microsatellite marker carry out a two tailed student t-test between the disease groups using LOH data IF t-test p ≤ 0.05 Marker is significant in prognosis of the disease ELSE Marker is not significant in prognosis of the disease (4) Validate the prognostic markers using χ 2 (chi-squared) test of LOH with clinico-pathological data and correlation of LOH data with histological grade of CIN, treatment response and various HPV subtypes. LDMAS has been implemented using the above pseudocode. Conclusions We have devised an effective algorithm to identify and extract useful markers that can be used to predict the outcome of disease and used the algorithm to successfully identify 4 novel prognostic markers that can be used to predict the outcome of CIN. The algorithm was implemented in a novel software called LDMAS which provides an essential platform for the extraction of useful information from large amount of data generated by LOH studies. Furthermore, LDMAS is used to efficiently store, manage and track the data. Its flexible nature allows the easy manipulation of data facilitating complex analysis as demonstrated in the current study. The various modules of LDMAS can be easily adapted and used with other applications such as high throughput LOH and genotyping using SNPs on Affymetrix ® GeneChip Mapping arrays and fingerprinting studies. Modules such as MRES can be used independently to parse medical records facilitating extraction of specific clinical information of interest. Additionally, LDMAS can be used to extract clinically useful markers for other diseases. Availability and requirements The source code and executable files for LDMAS modules as well as user manual including examples from real study data are freely available and can downloaded from our website at : Additionally examples of input files are provided from our website for users to test the software and assess its functionality. Authors' contributions RH designed and developed and implemented LDMAS software and the web site. AE and RH did the experimental work to generate the data necessary to test and validate LDMAS. MD supervised the study and designed the experimental work using CIN biopsies and smears. All authors read and approved the final manuscript.

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545602

Genome-wide mutagenesis of Zea mays L. using RescueMu transposons

The authors describe a large-scale transposon-tagging study in maize using the RescueMu system. The study provides a large resource of tagged and sequenced maize alleles, as well as some insights into the biology of RescueMu .

Background MuDR/Mu transposable elements are widely used for mutagenesis and as tags for gene cloning in maize [ 1 , 2 ]. The high efficiency of Mu insertional mutagenesis regulated by MuDR in highly active Mutator lines reflects four features of this transposon family. First, a plant typically has 10-50 copies of the mobile Mu elements [ 3 ], although some plants have over 100 copies. Second, they insert late in the maize life cycle, generating diverse mutant alleles transmitted in the gametes of an individual Mutator plant [ 1 ]. Third, they exhibit a high preference for insertion into genes [ 1 ]. And fourth, most maize genes are targets as judged by the facile recovery of Mu insertion alleles in targeted screens [ 1 , 4 - 6 ]. In directed tagging experiments, the frequency of Mu -induced mutations for a chosen target gene is 10 -3 -10 -5 [ 7 ]. Interestingly, a bronze1 exon [ 8 ] and the 5' untranslated region of glossy8 [ 9 ] contain hotspots for Mu insertion in specific regions, which may explain the higher frequency of mutable allele recovery for these genes. Somatic mutability, visualized as revertant sectors on a mutant background, is indicative of transposon mobility. By monitoring maintenance of a mutable phenotype, it was established that the Mutator transposon system is subject to abrupt epigenetic silencing, which affects some individuals in most families [ 10 , 11 ]. A molecular hallmark of silencing is that both the non-autonomous Mu elements and the regulatory MuDR element become hypermethylated [ 12 , 13 ]. Without selection for somatic instability of a visible reporter allele and/or hypo-methylation, Mutator lines inevitably lose Mu element mobility. The high efficiency of Mu mutagenesis has been exploited in several reverse genetics strategies. The first protocol described used PCR to screen plant DNA samples to find Mu insertions into specific genes using one primer reading out from the conserved Mu terminal inverted repeats (TIRs) and a gene-specific primer [ 14 - 17 ]. Alternatively, survey sequencing of maize genomic DNA flanking Mu insertions yields a list of tagged genes in each plant [ 18 , 19 ]. A third method uses RescueMu , a Mu1 element containing a pBluescript plasmid, to conduct plasmid rescue by transformation of Escherichia coli with total maize DNA samples. To identify insertions in genes of interest, RescueMu plasmids can be screened or the contiguous host genomic DNA can be sequenced using primers permitting selective sequencing from the right or left TIRs of Mu1 [ 20 ]. Here we describe the initial results of a large scale RescueMu tagging effort conducted by the Maize Gene Discovery Project. The tagging strategy employed grids of up to 2,304 plants organized into 48 rows and 48 columns. Plasmid rescue was undertaken from individual pools of up to 48 plants per row or column. Genomic sequences next to RescueMu insertion sites were obtained for all the rows and for a subset of columns of six grids. Maize genomic sequences were subsequently assembled into 14,887 unique genomic loci using computational approaches. These loci were analyzed for gene content, the presence of repetitive DNA and correspondence to mapped maize genes and ESTs. Gene models were built by co-assembling the genomic sequence with ESTs and cDNAs by spliced alignment and by ab initio gene prediction. Identified gene models were tentatively classified using gene ontology terms of potential homologs [ 21 ]. Many features of Mu element behavior have been examined previously using hundreds of tagged alleles or by analyzing the population of Mu elements in particular plants and a few descendants. With single founder individuals for the analyzed tagging grids, we could examine the distribution of new insertion sites of RescueMu in large progeny sets. The contiguous genomic sequences were analyzed to determine if there were insertion hotspots, preferential insertion site motifs, routine generation of the expected 9-base-pair (bp) direct target sequence duplication (TSD) and evidence of pre-meiotic insertion events. Like other Mu elements, RescueMu exhibits a strong bias for insertion into or near genes, as few insertions were recovered in retrotransposons or other repetitive DNA. In addition, for the set of RescueMu insertions into confirmed genes, a bias for insertions into exons (rather than introns) was observed, consistent with the well-established use of Mutator as a mutagen. The gene-enrichment exhibited by RescueMu was compared against two physical methods of gene enrichment, methyl filtration [ 22 ] and high C 0 t genome fractionation [ 23 ]. Results RescueMu transposition in active Mutator lines In standard Mutator lines, Mu1 elements maintain copy number through successive outcrosses, indicating that some type of duplicative transposition occurs [ 24 ] in the absence of genetic reversion [ 25 ]. Most new mutations are independent and occur late in the life cycle [ 26 , 27 ]. Consequently, a single pollen donor can be used to generate thousands of progeny with diverse Mu insertion events (Figure 1 ). Initially RescueMu germinal insertions were sought by direct mobilization of elements from transgene arrays containing multiple copies of the original 35S:RescueMu:Lc plasmid and the plasmid conferring resistance to the herbicide Basta used for selection of transformed callus [ 20 ]. Using eight different transgene arrays crossed with diverse active Mutator lines, the average germinal transposition frequency through pollen was only 0.07 (Table 1 , grid A); lines with a single MuDR element had no transposed RescueMu ( trRescueMu ). Materials were selected from the progeny of grid A plants for grids B through E using two criteria: there were visible seedling mutations in around 10% of progeny characteristic of a very active Mutator line [ 26 ] and the presence of trRescueMu . By DNA blot hybridization of individuals within grids B through E, the RescueMu transposition frequencies ranged from 0.1 to 0.26 (Table 1 ). By sequence analysis after plasmid rescue, trRescueMu were identified that had inserted into likely maize genes and generated the diagnostic 9-bp TSD characteristic of Mu transposition (data not shown). There were also events initially scored as transposition by blot hybridization that represented RescueMu rearrangements within the transgene array, and deleted forms of RescueMu were detected by blot hybridization and gel electrophoretic sizing of rescued plasmids (data not shown). Although RescueMu insertion frequency was low, overall Mu movement was very high in these grids; visible, independent seedling mutations were identified in 10.1-28.3% of the selfed progeny (Table 1 ), as high as the most active Mutator lines described to date [ 28 ]. In an effort to increase transposition frequency, lines with trRescueMu but no transgene array were selected. Plants with a verified trRescueMu were crossed to r-g and colorless kernels selected - these lack red spotting from RescueMu somatic excision from the 35S:RescueMu:Lc transgene. During subsequent plant growth Basta-sensitivity was scored as a second indicator that the transgene array was absent [ 20 ] and DNA blot hybridization then confirmed that a trRescueMu but not the Basta-resistance transgene was present in the plant. To guard against Mutator silencing, plants were also screened by DNA blot hybridization to verify that they contained unmethylated Mu1 and MuDR elements after digestion of genomic DNA with the methylation-sensitive enzymes Hin fI and Sst I, respectively (data not shown). Four plants each with a single trRescueMu were identified by these criteria and crossed to r-g . A DNA blot hybridization screen was conducted on 393 progeny of these four individuals. Seven progeny were identified with two new trRescueMu , seven plants were identified with three events, and 33 plants had a single trRescueMu ; the original, parental trRescueMu elements were shown to segregate as Mendelian factors in the populations screened (data not shown). The 14 plants with two or three new trRescueMu were each crossed by an anthocyanin tester and also crossed multiple times as pollen parents to tester lines to generate sufficient progeny to construct one grid from each founder plant. Inexplicably, in sampling seedling progeny from each outcross ear, some lineages had very few new trRescueMu . The lines with the highest transposition frequencies had two trRescueMu and were used in grids G through J; DNA blot hybridization analysis of 30-200 grid plants was used to estimate transposition frequencies within each grid, which ranged from 0.38 to 0.66 (Table 1 ), with an average of 0.58 per plant and 0.29 per parental RescueMu element. The two parental trRescueMu elements were shown to be segregating 1:1 and independently (Figure 2 for grid G, and data not shown for other families). Subsequently, surveys within each grid were used to identify plants with two or three newly trRescueMu and no evidence of Mutator silencing for construction of the next tagging populations. In this manner, the frequency of trRescueMu was increased in some grids to 1.0-1.4 per plant (Table 1 ) reflecting a frequency of 0.5-0.7 per parental element. Library plate preparation and gene representation As shown schematically in Figure 1 , the trRescueMu insertion sites have been immortalized by preparing libraries from each of the row and column leaf pools from 16 grids, with three additional grid libraries under construction (Table 1 ). Briefly, total maize DNA was digested with Bam HI and Bgl II, both of which recognize sites outside of RescueMu , and the fragment mixture was used to transform E. coli (see Materials and methods). The resulting library plates contain 56-96 individual row and column libraries representing the diversity of germinal trRescueMu and a sampling of somatic events present in the harvested leaf tissue (each well in a library plate is a pool of 20-48 plants from a row or column). The parental RescueMu insertion sites inherited from the grid founder(s) are present in every library. Library plates contain a high diversity of genomic sequences. In a row of 48 plants, assuming random insertion, two segregating founder elements and a transposition frequency of 1.0, there will be 50 different plasmid types in the heritable class. Including heritable and somatic insertions, we estimate that each row or column library contains about 100-200 distinct plasmid types. Given these parameters, a library plate from a 48 row × 48 column grid with an average of 150 somatic plasmids per row or column library would contain 14,400 somatic insertion sites plus 2,304 germinal events and the two parental insertion sites. Because RescueMu shows a strong bias for insertion into genes [ 20 ], each library plate contains a substantial fraction of the predicted 50,000 genes of maize [ 29 ], provided the insertion sites are random. Ultimately, library plates for 19 grids derived from 33,000 plants and containing an estimated 30,108 heritable trRescueMu insertion sites (grid size × transposition frequency from Table 1 ) will be available online from the Maize Gene Discovery project through MaizeGDB [ 30 ]. Plasmid recovery analysis and identification of probable germinal insertions (PGIs) Based on gel electrophoretic analysis of nearly 1,000 rescued plasmids, the genomic DNA flanking RescueMu averaged 3.5 kilobases (kb), with a range of 0.4-15 kb (data not shown). To accommodate the large size of some plasmids, a PCR template preparation protocol was devised to amplify genomic inserts of up to 16 kb for high-throughput sequencing [ 31 ]; primers were designed to amplify from within the right and left TIRs reading outward into the maize genomic DNA such that high quality sequence would be available to identify the TSDs flanking RescueMu insertion sites. Plasmids from all rows plus several columns of a grid were sequenced, with a routine yield of 80-92% success. A subset of plasmids could not be bidirectionally sequenced, because they lacked the TIRs at one or both ends. Deleted forms of trRescueMu were detected in several percent of the individuals surveyed by DNA blot hybridization (see Figure 2 for an example). If such derivatives retained the origin of replication and ampicillin-resistance marker, they could be cloned by plasmid rescue; if the TIRs were absent, they could not be sequenced. Previous analysis of trRescueMu demonstrated that somatic insertion events, typically found in a tiny leaf sector, were sequenced just once from a leaf DNA sample while multiple instances of the germinal events could be recovered [ 20 ]. Out of 28,988 non-parental plasmids sequenced, 41% (11,749) were recovered once (new trRescueMu somatic plus germinal insertion events) for each grid, and 59% (17,239) were recovered multiple times (probable new trRescueMu germinal insertion events). In addition, a total of 24,875 parental plasmids were transmitted from the founder plants. The percentage of parental plasmids within each grid varied from 17% for grid G to 61% for grid P. Some grids had more parentals than other grids and some parental plasmids were preferentially sequenced for unknown reasons. The parental insertion sites include the two or three known parental sites that each segregated into 50% of the progeny. Somatic sectors in the tassel or ear of the parental plant that generated plasmids found in multiple individuals within the grid are analyzed in a later section. Grid sequence data were used to cross-check the transposition frequency estimated from DNA blot hybridization (Table 1 ) using both a row and column matching method and a more general multiple recovery method. Analysis of 80 individuals from six contributing outcross ears in grid G identified 54 that were newly trRescueMu , equivalent to a frequency of 0.68 new insertions per plant. Using a Poisson model based on this transposition frequency for an individual grid (Table 1 ), the sequencing goal was established to reach a depth sufficient to insure that with 95% confidence, each probable germinal insertion would be recovered at least once. In the Poisson model, the 5% probability for the zero class (in other words, the 95% probability of finding all PGIs at least once) occurs when the observed mean is -ln(0.05) or approximately 3. After sequencing several rows and at least one column for a grid, multiple occurrences of PGIs were counted and used to project the sequences required to obtain the desired average of 3 occurrences of each PGI. As a cross-check of this coverage using the row and column matching method, the sequenced row plasmids were compared to the sequences available from four columns of grid G and 149 matches were found. This is equivalent to a transposition frequency of 0.81 based on 149/(4 × 46 plants per row), somewhat higher than the estimate of 0.68 based on blot hybridization analysis of individual plants. Recovery in both a row and a column is highly indicative of a probable germinal insertion because the row and column plasmids were obtained from different leaves and only germinal insertions would be found throughout a plant. The results for each analyzed grid are shown in Table 2 . The low column sampling in grid K (only 192 plasmids were attempted for each of three columns) and grid M (96 plasmids for two columns and 192 plasmids for a third column) resulted in a lower than expected number of germinal insertions. Grid P had a low germinal insertion count with this method because a portion of the column sequences was from rows generated from different parental plants and subsequently excluded from the analysis. Analysis of the row and column sequence data within grids demonstrates that the row sequencing was too shallow to recover some probable germinal insertions more than once and that a fraction of germinal insertions were not sequenced. For example, within grid G, 385 plasmids were identified twice in the available column data but were missing from the row sequences; this is over twice the number of plasmids identified by row and column matching. From the number of plasmids successfully sequenced per row within grid G, we estimated a 70-95% probability of sequencing the likely germinal insertion events at least once in the rows. For other grids, the sampling efficiency ranged from 30 to 95% per row. Grids in which some rows had sampling efficiency less than 60% are listed as partial in Table 1 ; sequencing was terminated in portions of these grids because of technical difficulties such as an excess representation of a parental insertion site, a large number of rearranged RescueMu elements that could not be sequenced with the standard protocol, or poor yield of RescueMu plasmids for unknown reasons. The second method of identifying probable germinal insertions includes plasmids that were recovered multiple times, regardless of whether a column sequence was present. Almost all somatic insertions should only be recovered once due to their occurrence in just a few cells. The results using this method for each grid are shown in Table 3 . What these data mean in practice is that the 3,138 probable germinal insertions identified after sequencing the same RescueMu plasmid at least twice is not a comprehensive list of the heritable insertion events. On the basis of the number of grid plants and estimated transposition frequencies (Table 1 ), 8,311 probable germinal insertions were expected from the six grids (see Table 3 ). From this we estimate that the majority of the heritable insertion events are represented by only a single sequenced RescueMu plasmid. It is likely that nearly half of the plasmids recovered just once represent a germinal insertion (0.44 = (8,311-3,138/11,749)). By PCR screening of library plates containing the immortalized row and column plasmids, plants containing a specific insertion event can be verified (Figures 1 and 3 ). Selection against specific plasmids in E. coli probably contributed to non-recovery of certain insertion sites as sequencing templates, and these plasmids may also be under-represented in library plates. Verification of germinal transmission Individual grid plants with probable germinal insertions were identified on the basis of recovery of the same plasmid in both a row and a column. In addition, library plates containing all of the row and column libraries can be screened using PCR, with one primer designed to the Mu1 TIRs present in RescueMu and a second primer in the gene of interest, as illustrated in Figure 3 . A probable germinal insertion plasmid should yield the same size product in at least one row and one column library of that grid plate; the row and column identifiers specify the address of the plant(s) containing this insertion. To test this method, 11 instances of duplicate plasmid recovery in grid G (N. Arnoult and G-L.N., unpublished data) and 14 such cases in grid H (K. Goellner and V.W., unpublished data) were verified to be represented in both a row and a column library by PCR screening of the corresponding library plate. Seedling progeny from the identified row and column plants were evaluated for the presence of the expected RescueMu insertion site. A germinal insertion was verified for 16/16 cases examined by DNA blot hybridization and/or PCR of individual progeny plants in the family (see Additional data file 2 for methods and for plants used to verify germinal transmission [ 31 ]). Mutational spectrum of RescueMu As shown in Figure 4 , RescueMu insertions occur in diverse gene types. Illustrating the utility of Mu tagging, insertions are found in housekeeping genes, such as actin, as well as in regulatory genes such as those for transcription factors and protein kinases. Using the database of mapped maize genes and expressed sequence tags (ESTs) [ 30 ], RescueMu insertions are identified in genes on all 10 maize chromosomes [ 32 ]. These data confirm earlier studies tracking Mu insertions using DNA blot hybridization that established that these elements insert throughout the genome and do not show a measurable bias for insertion locally [ 1 ]. In addition, about 85% of RescueMu insertion sites that match maize ESTs correspond to genes of unknown function, suggesting the discovery of novel genes. Of the 14,887 RescueMu insertion sites identified in six grids (multiple insertions into a gene from the same grid being counted only once because the majority are the same insertion event), 88% represent single instances of transposon insertion locations. There were 596 instances of a specific genomic sequence having two or more RescueMu insertion events. If the maize genome contains 50,000 distinct genes that are targets of Mu insertional mutagenesis, then far fewer cases of duplicate recovery would be expected by chance alone, given the number of events analyzed ( p < 0.001); therefore, RescueMu exhibits some preference for particular genes. To determine if there were 'hotspots' for RescueMu insertion within particular genes, data were compared between grids with independent founder individuals. As summarized in Table 4 , 90% of the RescueMu insertion sites were found in just one grid. This was true for both probable germinal insertion events (plasmids found two or more times within a grid) as well as for singlet sites (a mixture of germinal and somatic events). The 10% of insertion sites found in two or more grids represent independent recovery of a RescueMu insertion into the same locus. In addition to the computational comparison in which an overlap of 50 bases (95% identity) was scored as insertion into the same gene, over 730 insertion sites were examined manually for 250 cases of genes with insertions from more than one grid. Of these insertion sites, 80% were at different locations within the same locus; we found 85 cases of insertions within a 1-10 bp region and 67 cases of insertions at the same base. Previously, Dietrich et al . [ 9 ] reported that 62 of 75 Mu insertions at glossy8 were in the 5' untranslated region, with 15 insertions at the same base; similarly, the beginning of exon 2 within bronze1 is the most frequent site of Mu insertion in that gene [ 8 ]. One RescueMu contig from the Genomic Survey Sequencing (GSS) section of GenBank, ZM_RM_GSStuc03-10-31.4765 [ 33 ], is a hotspot for RescueMu insertion, with six plasmids sequenced from row 42 of grid G and one each from grids H, I, and M. Insertion sites were identical across the grids. Sequences generated to both the left and right of the RescueMu element were aligned as demonstrated in Figure 3a . Many maize ESTs matching a maize acetohydroxyacid synthase were found near this insertion site; the closest (GenBank GI: 4966438) is less than 50 bp away. Because this RescueMu insertion site was recovered multiple times in grid G, a heritable insertion may exist. After PCR screening of grid G plasmid libraries, summarized in Figure 3a , the plant at row 42, column 22 was identified. To assess heritability of this RescueMu insertion site, total leaf DNA was extracted from selfed seed of this plant, namely G 42-22, obtained from the Maize Genetics Cooperation Stock Center. PCR screening of the DNA (Figure 3c ) indicated that plant 5 is homozygous for the insertion and plant 7 is homozygous wild type. DNA blot hybridization with a 0.6-kb purified PCR probe amplified with primer pair 1 + 5 confirmed plant 5 to contain the homozygous insertion allele, plant 7 to be wild-type, and the rest to be heterozygous for the insertion (Figure 3e ). Various mutant phenotypes were observed in plant 5 (Figure 3f ), including retarded seedling growth, reduced plant height, discolored streaks on adult leaves and sterile tassel and ear. Because there are multiple Mu elements in this line, further characterization of selfed progeny of its heterozygous siblings will be performed to determine the true phenotype caused by this insertion. Analysis of 9-bp TSD and insertion site preferences Because a 9-bp TSD is characteristic of Mu insertion events, the 9 bp next to the left and right TIRs of an individual RescueMu plasmid were used to join the right and left flanking sequence provided they were complementary (Figures 1 , 3 ); note that the sequences are complementary because they were generated from different strands. For non-parental plasmids, left and right sequence data were available for 13,966 plasmids, and the 9 bp was readily identified computationally for 47.2% (6,596) of these. The remaining non-parental plasmids did not have both right and left sequence data and/or the 9-bp motif could not be verified; 5.7% (1,816) contain only post-ligation sequences. Possible explanations for incomplete sequencing results include deletions next to Mu1 elements that remove a portion of the TIR as well as flanking host sequence [ 34 , 35 ]; these events occur with about a 10 -2 frequency at existing insertion sites and if they occurred during or subsequent to RescueMu insertion they would preclude identification of the 9-bp repeat. Alternatively, the lack of a 9-bp TSD could reflect sequencing error. Manual inspection of 300 of the unmatched cases indicated that for nearly 90% there was an 8/9-base repeat match with the mismatch being an undetermined base (an 'N') or a single missing or additional base. Given that all sequences were single pass but of high average quality (phred 35, equivalent to one base-calling error in 3,160 bases), we consider that 9-bp TSDs exist in virtually all trRescueMu insertion sites. A few cases showed anomalies in the TSDs, which probably reflect rearrangements near RescueMu . Several groups have reported weak consensus insertion site preferences for Mu based on smaller data sets [ 9 , 18 , 20 ]. We have derived a site-specific frequency profile of the bases from 3,999 RescueMu insertion regions [ 32 ]. The profile is in agreement with what has been reported earlier by Dietrich et al . [ 9 ], showing a strong bias for high G/C content in the 9-bp TSD within a flanking dyad-symmetrical consensus: CCT-(TSD)-AGG. The non-random insertion pattern strongly suggests that RescueMu targeting is at least partially dependent on sequence features. In addition, we have compared the profiles derived independently from insertion sites within confirmed exons, introns and uncharacterized regions, respectively, and found the same base preferences in all three sets (data not shown). Of 14,887 genomic loci, 62% matched maize or other plant EST/cDNAs. As more genomic sequence becomes available that can be assembled with ESTs to annotate the non-coding portions of maize genes, it will be interesting to determine if the RescueMu insertion sites that do not match an EST or gene in another species represent introns or other non-coding genic regions. On the basis of the gene structure annotated by maize EST matching, we have located 968 TSD sites within genes. Of these, 849 are inside exons. To check if RescueMu has a preference for insertion into exons (that is, the above observed high frequencies of exon insertions is not the result of potential high exon proportion in the maize genes), a standard binomial test with normal approximation was performed. On the basis of the matching to ESTs, the lengths of all exons and introns observed from all RescueMu contigs were counted as 2,182,954 bp and 439,403 bp, respectively. Assuming that RescueMu does not have a preference to insert into exons (null hypothesis), the probability of observing an exon insertion event is proportional to the length of exons (single binomial trial probability 0.832). The probability of observing at least 849 exon insertion events was calculated (less than 0.001; reject the null hypothesis). This result suggests that RescueMu has some preference to target exon regions within genes. As outlined in Materials and methods, the RescueMu GSS sequences were scanned and masked for repetitive elements as collected in The Institute for Genomic Research (TIGR) Cereal Repeat Database [ 36 ]. The repeat content was compared with results for GSS sequences derived by methylation filtration (MF) and high C 0 t selection (HC) using the same repeat-masking criteria [ 36 ]. The percentage of masked nucleotides was 16.5, 24.5 and 16.2% for RescueMu , MF and HC, respectively. Therefore, on the nucleotide level, RescueMu shows similar repeat content as the physical enrichment methods. However, after we assembled the RescueMu GSS sequences to remove redundancy, only about 3% of the RescueMu loci are composed of repetitive DNA (equal or greater than 75% masked, Table 5 ). If the maize genome is two-thirds retroelements [ 37 ], then there is an approximately eightfold insertion bias by RescueMu against this component of the genome. We also downloaded the latest MF and HC contigs (version 3.0) from TIGR [ 38 ] and applied the same repeat masking on those contigs. Our results show that 28% of the MF and 6% of HC contigs are repetitive DNA. Thus, RescueMu and HC have similar bias against repetitive DNA, superior to the MF bias. It should be noted, however, that the MF and HC GSS sequencing has generated, on average, much longer contigs than RescueMu (see Additional data file 2). In addition, only 0.4% of the RescueMu insertions were found in either the approximately 10,000 copies of the 9.1 kb 28S + 18S rRNA genes [ 39 ] comprising 3.6% of the 2.5 gigabase (Gb) maize genome, or in the large number of tRNA and 5S rRNA genes in the maize genome (Table 5 ). These results demonstrate a strong bias against insertion into genes transcribed by RNA polymerases I and III. Also shown in Table 5 , about 62% of the RescueMu loci match strongly to maize or other plant ESTs or appear to encode proteins with high similarity to known proteins. In addition, about another 5% of the loci were predicted to be genic regions with high stringency by ab initio gene prediction programs. As a control, we matched ESTs to contigs assembled from unfiltered (random) maize GSS sequences [ 38 ]. From about 33,000 of those unfiltered contigs, less than 20% of them show significant matching to ESTs. This shows that RescueMu contigs contain more than threefold enrichment of genic regions than random sequencing. This is consistent with our expectation that RescueMu preferentially inserts into genes. It is worth pointing out that plant EST collections contain ESTs from repetitive elements. Although we masked contigs using the annotated TIGR repeat database [ 38 ], it is possible that some contigs still contain unidentified repetitive elements, which might overestimate the number of genic regions by matching the same ESTs to different copies of repetitive elements. In particular, 18% of the EST matched regions show high similarity to transposon coding regions based on BLAST searches against the GenBank nucleotide and protein databases, suggesting that at most 14% of unfiltered contigs include protein-coding genes. The numbers of genic sequences from MF and HC was reported to be 27% and 22%, respectively [ 36 ]. However, these numbers are not directly comparable to our RescueMu results, because these authors used much higher stringency for the EST spliced alignments with the BLAT program [ 40 ], requiring 95 and 80% identity, respectively, when matching to the TIGR maize gene index or other plant indices. We used the GeneSeqer program for spliced alignment of the RescueMu data, which tolerates less sequence matching without compromising gene structure prediction accuracy [ 41 ]. The results using GeneSeqer for RescueMu , MF, and HC are very similar (data not shown). Palmer et al . [ 42 ] evaluated the gene discovery rates of MF, EST sequencing and RescueMu by comparing the respective sequence sets to rice gene models. They concluded that unique gene discovery is most efficient with MF at a sequencing depth when EST sampling saturates. However, their reported low gene discovery rate for RescueMu does not reflect the RescueMu insertion bias, because their dataset included all sequences deposited in GenBank. That is, they did not remove the redundancy resulting from multiple sequencing of parental insertions. Multiply recovered RescueMu insertion sites in the progeny of a single founder plant Probable germinal insertions involve plasmids recovered several times within a sequenced row and/or column, but in addition, some RescueMu insertion sites were found in two or more row libraries (Table 6 ). Although these could represent hotspots for Mu insertion at exactly the same base, we consider it more likely that they reflect the known ability of Mu elements to insert pre-meiotically, resulting in several progeny with the same newly generated mutation present as a sector on an ear indicative of a single insertion event [ 43 , 44 ]. Robertson estimated that 20% of Mu transpositions occur pre-meiotically, 60% occur during meiosis or immediately afterwards, and 20% occur after the mitosis that separates the two sperm in haploid pollen [ 1 ]. We infer that multiple row recovery of the same insertion site within a grid was indicative of a likely pre-meiotic insertion; in contrast, authentic hotspots have the same insertion site among grids. A second line of evidence is that DNA blot hybridization surveys to calculate transposition frequency within a grid identified many instances of a particular fragment size shared in two or more progeny (data not shown). Finally, phenotypic screening of grid progeny families identified numerous instances of identical phenotypes segregating in related families [ 45 ]; each such phenotypic class was counted just once in calculating the percentage of families with a new visible phenotypic mutation (Table 1 ). To calculate the extent and timing of pre-meiotic sectors, the sequenced plasmids from grids G, H, I, K, M and P were classified as occurring in a single row or in multiple rows. The development of the tassel and ear must be considered when evaluating these data. An insertion event that occurs during meiosis can be represented in two haploid cells. During microgametophyte (haploid plant) ontogeny, both of these cells survive, resulting in two pollen grains with the same event. In contrast, only one megagametophyte develops after megaspore meiosis; therefore, female meiotic and subsequent events in the haploid megagametophyte are always represented in just one progeny plant. Most grid plants resulted from male transmission of RescueMu and a minority (about 10%) from female transmission. Given that the founder plants produced copious pollen, there is a low probability that two grains carrying the same meiotic insertion will both result in seed; therefore, the same RescueMu insertion site found in two rows should usually be from a pre-meiotic transposition event. For all events found in three or more rows, the insertion event must be pre-meiotic. The 103 insertions sites found in three or more rows of grid G must be pre-meiotic events (see Table 6 ). They represent 9% of the probable germinal insertion events (103/1,091) identified by the criterion of recovery of the same plasmid twice or more (see Table 3 ). The percentage was similar for all six grids: there were 321 events identified in three or more rows out of 3,138 probable germinal insertions. Surprisingly, 138 contigs were found in four or more rows in these six grids, including 34 events in 10 or more rows (Table 6 ). Therefore, occasionally there is a RescueMu insertion event very early in the somatic development of the inflorescence or in the apical meristems. The majority of trRescueMu insertion sites are found in only one row (92% of germinal plus somatic insertion sites, Table 6 ). As a cross-check on the analysis of pre-meiotic events presented in Table 6 , we evaluated the actual number of individual plants containing the same insertion site for a subset of each grid, using the sequence data from columns. Using this method we confirmed that among 184 plants in grid G with both row and column sequence data, there were 65 cases of insertion sites found in two or more rows or in two or more columns (Table 7 ). Similar results were obtained for the other five grids. From these calculations and the data in Table 6 it appears that RescueMu insertions must occur routinely before meiosis and that, although rare, there are a significant number of early somatic insertion events that are transmitted to multiple progeny. Discussion RescueMu was introduced into maize by particle bombardment resulting in complex transgene loci containing multiple copies of the transposon and the Basta-resistance plasmid used for selection of transgenic lines [ 20 ]. After crossing with an active Mutator line, RescueMu exhibited somatic excision from a 35S:Lc reporter allele resulting in a red-spotted aleurone but the heritable insertion frequency was very low. Progeny screening identified individuals containing two or three trRescueMu elements lacking the original transgene array by genetic segregation and unmethylated Mu1 and MuDR elements. Some of these individuals and subsequent derivatives with the same characteristics were used as founder plants to construct grids of plants organized into rows and columns for efficient generation and analysis of germinal mutations. Tagging maize sequences with RescueMu followed by plasmid rescue and sequencing of the flanking host DNA has identified 3,138 insertion locales from 17,239 plasmids (see Table 3 ). These plasmids represent 59.5% (17,239/28,988) of the total non-parental plasmids of the genomic loci found in each grid. Because sequencing depth was too shallow to identify all likely germinal insertions, the 40.5% of non-parental plasmids recovered just once (11,749 from Table 3 ) represent a mixture of somatic and germinal events. On the basis of the estimation of germinal insertion frequency from DNA blot hybridization, the six grids should contain more than 8,000 heritable trRescueMu insertion sites, but the sequencing depth was too shallow to identify all of these by multiple recovery of the same plasmid two or more times. RescueMu is suited for both reverse and forward genetic strategies. Given the genomic sequence contiguous to any trRescueMu , a PCR screen can be designed to identify which plant contains the insertion of interest using 96-well plates containing the immortalized collection of row and column rescued plasmids. The row and column plant address can be used to order seed for further genetic and phenotypic analysis as illustrated by the RescueMu insertion into the acetolactate synthase gene (Figure 3 ). Alternatively, the phenotype database, which is organized by individual plant, can be searched to identify individuals segregating for mutations of interest. Active Mutator lines with multiple mobile Mu elements were used so most mutations will be caused by these Mu elements because they increase mutation frequency 50-100-fold above spontaneous levels [ 1 ]. The high forward mutation frequency reflects the copy number of the elements and their preference for insertion into or near transcription units [ 1 ]. From the DNA hybridization blots (data not shown) used to verify that grid founder plants had unmethylated Mu elements, the copy number of unmethylated Mu elements was estimated at 20-40 per founder; therefore, two mobile RescueMu elements would be expected to account for 5-10% of the newly generated mutations. Seed was ordered through the Maize Genetics Cooperation Stock Center [ 46 ] for further characterization. RescueMu insertions were found in genes and ESTs mapped to all 10 maize chromosomes [ 31 ], and were found in all of the gene classifications for maize (Figure 4 ). These data confirm the empirical observations of maize geneticists that MuDR/Mu transposons are general and efficient mutagens for maize genes [ 1 ]. Analysis of 14,887 loci defined by RescueMu insertions demonstrates that transposition is highly preferential for RNA polymerase II transcription units: about 62% of the sites match maize or plant ESTs. Because the EST collections are incomplete and lack intron and promoter sequences, it is likely that an even higher proportion of RescueMu insertion sites are in or near genes but cannot be currently assigned to a specific gene. Given the current efficiency, large tagging populations in excess of 200,000 plants would be required in order to recover RescueMu mutations in all maize genes (estimation is based on the calculation method in [ 47 ]). The numerous grids evaluated for phenotypic characteristics should approach saturation of visible mutations, although most of the mutations are caused by standard Mu elements. Given that the maize genome comprises approximately 70% retrotransposons and other highly repetitive sequences, including around 10,000 copies of the rRNA genes [ 37 ], these components of the maize genome are significantly under-represented in RescueMu insertion sites. Only about 8% of the RescueMu insertion sites match repetitive elements and few insertions (0.4%) were recovered in genes transcribed by RNA polymerase I or III. These results suggest that a chromatin component associated with polymerase II transcription units or the absence of a structure in other classes of genes is important in targeting RescueMu and other Mu elements to maize genes. Similarly, recombination during meiosis and transcription per se is targeted to genes. It is likely that the parasitic Mu elements exploit an element of host gene packaging that evolved for other reasons to facilitate transposition into genes. The biological specificity for maize genes exhibited by RescueMu is close to methyl filtration and high C 0 t fractionation. The probable germinal insertion class defines a collection of mutations of enormous potential for the phenotypic characterization of maize with specifically disrupted functions. However, the low cost of template production is a distinct advantage of both physical enrichment methods compared to the high cost of designing, sampling and self-pollinating tagging grids. Current levels of sample sequencing from the physical enrichment templates highlight the desired redundancy of the RescueMu method, which is important for distinguishing somatic from germinal insertions at individual loci. The physical enrichment methods are considerably below one times coverage of the transcriptome of around 250 Mb; hence the current efficiency of generating novel sequence (the likelihood that the next clone sequenced is new) is much higher with these methods than with RescueMu . Using the RescueMu insertion site data, several parameters of Mu transposition behavior were investigated. We confirm that a 9-bp TSD is characteristic of virtually all Mu insertion sites. We confirm that a small percentage of trRescueMu suffer deletions, including loss of a TIR, as noted in previous studies of Mu1 [ 35 ]. Through evaluation of several hundred Mu insertion sites [ 9 , 18 ], consensus motifs have been proposed for insertion sites. The sequence profile derived from the much larger population of RescueMu insertion sites is consistent with the previously proposed motifs. A bias exists for G+C-rich sequence, reflecting the composition of maize exons. We confirm that there are hotspots for Mu insertion, identified by finding identical trRescueMu insertion sites in independent grids. A few loci were recovered in four or more of the six grids analyzed, and many more in two (1,295 genes) or three (233 genes) grids. There is no strong DNA consensus motif at these hotspots, and we consider it more likely that a specific DNA structure or a protein associated with genes establishes conditions for efficient Mu insertion at particular sites. It is important to note that active transcription is not a requirement for Mu element insertion; otherwise Mu would preferentially insert into genes active late in floral development and in gametophytes. The trRescueMu insertion sites represent a mixture of non-heritable somatic insertions present in leaves, germinal insertions in single grid individuals, insertion events in pre-germinal sectors within flowers, and parental elements. Parental elements identified in a grid founder plant segregated 1:1 in the progeny as expected. In addition, some insertion events were found in three or more grid rows, and hence in three or more individuals, and must be pre-meiotic transposition events in the founder. This class represented 10.2% (321/3,138) of all the likely germinal insertions identified (calculated from Table 6 ). Given the clonal analysis model of the pattern of cell divisions establishing the ear and tassel of maize [ 48 - 50 ], the earliest events within the apical meristem could affect up to half of the ear or tassel, with subsequent events affecting progressively narrower portions of the inflorescence. The majority of the pre-meiotic events are consistent with RescueMu transposition in the floral cells a few cell divisions before the onset of meiosis, that is, in precursor cells that are still proliferating and could generate at least two and up to approximately 50 meiocytes. A smaller fraction of new insertions events occurred early enough to be represented in many progeny of a particular plant. These rare, early transposition events generate very large sectors within the developing inflorescence. Mu transposon mutagenesis is highly efficient, primarily because the transposon targets genes and it is usually found in 10-50 copies per genome. How does the plant tolerate the large number of mutations generated by this agent? Within the diploid somatic tissues, most new mutations lack a phenotype; however, the haploid gametophytes are subject to stringent selection. Unlike animals, in which the phenotypes of the sperm and egg are set by previous gene activity in the parent, many characteristics of the haploid phase of the plant life cycle reflect haploid genetic activity, which requires overlapping but distinctive suites of genes in the mega- and microgametophytes [ 51 ]. Consequently, the late timing of new Mu insertions generates gamete diversity, but the unfit genotypes are culled from the population before fertilization. Coe et al . [ 52 ] describe the general problem that lethals occur much more frequently in pollen than in the megagametophyte. Any method that relies on pollen transmission will therefore fail to recover certain types of mutations that would be recovered through female transmission. For this reason, a subset of maize genes required in both types of gametophyte is refractory to knockout mutagenesis. Conclusions A public resource of transposon-tagged maize alleles was constructed and evaluated. RescueMu is an efficient tag for mutagenizing and cloning maize genes, because 66% of insertion sites appear to be in genes. Sequencing from immortalized plasmid libraries organized into row and column plates reflecting the organization of fields of plants permit identification of probable germinal insertions; the library plates can be searched by PCR to verify germinal insertions and subsequently acquire seed of the corresponding plant. Alternatively, a searchable database of segregating plant phenotypes in seed, seedling, or adult tissues can be used to find plants carrying mutations of interest. Although RescueMu can target most, if not all, RNA polymerase II transcription units in the nuclear genome, the transposon does exhibit hotspots in particular genes. Neither the hotspots nor other insertion sites contain a motif(s) defining predictable insertion locations. RescueMu properties confirm attributes established with smaller populations of standard Mu elements. Materials and methods Biological materials RescueMu contains all of Mu1 plus a 400-bp segment of Sinorhizobium meliloti and pBluescript (Stratagene), as described previously by Raizada et al . [ 20 ]. The complete sequence of RescueMu was obtained in this study using PCR primers to amplify overlapping sections of the element [ 31 ] for bidirectional sequencing (GenBank accession AY301066). In the construct used to make transgenic plants, the RescueMu transposon was placed in the 5' untranslated region of a 35S:Lc expression plasmid where it blocked expression [ 20 ]. Lc is a member of the R family of transcriptional regulators of the anthocyanin pathway [ 53 ]. Transgenic maize lines in the A188 × B73 ( r-r/r-g , A1 , Bz1 , Bz2 ) hybrid background were crossed to r-g testers and subsequently with r-g Mutator lines containing multiple copies of MuDR to visualize RescueMu somatic excision as red anthocyanin sectors in an otherwise white aleurone. The tagging populations used here were developed by screening for transposition of RescueMu from the original, complex transgene arrays to diverse genomic locations. Using DNA blot hybridization, these once-transposed RescueMu ( trRescueMu ) were closely monitored for subsequent transposition, and lines were monitored for Mu1 and/or MuDR methylation in the TIRs, a sign of incipient Mutator silencing. Details of line development and evaluation, including DNA blot hybridization methods, will be presented elsewhere. The anthocyanin tester lines (recessive for r-g , a1 , bz1 or bz2 ) were in inbreds W23, K55, A188, or hybrid combinations of these lines. Some RescueMu lines used in tagging grids were crossed to inbreds A619 or B73, which are both r-g , A1 , Bz1 , Bz2 . Grid backgrounds are presented in detail at [ 31 ]. Plasmid rescue and DNA sequencing Detailed protocols are presented at [ 54 ], and a schematic is provided in Figure 1 . Briefly, leaf tissue was collected from all plants in each row and from a different leaf in each column of a grid. A separate plasmid rescue library was constructed after Bam HI plus Bgl II digestion of the genomic DNA preparations. These libraries were immortalized in library plates available from the project [ 31 ]. Plated colonies were picked, grown overnight in liquid media, and sequencing templates prepared by a direct PCR method suitable for amplifying genomic inserts of up to 16 kb. Cycle sequencing was performed using Big Dye Terminator chemistry to read out from a position around 110 within the left or right terminal inverted repeat (TIR) of RescueMu ; although the primers were selective for one TIR, there was some cross-priming. All grid rows plus several columns were sequenced. Three 96-well plates were normally sequenced for each row or column to obtain sequence information for a desired minimum of 200 plasmids; additional sequencing reactions were conducted if necessary. Matches of row and column sequences are designated as probable germinal insertions, because they represent an insertion site present in two leaves of that plant (designated by its row and column address); when only row sequences were available from a particular plasmid, probable germinal insertions were designated after recovery of the same sequence two or more times. Plasmid types recovered just once are a mixture of heritable and strictly somatic insertions. Parental RescueMu insertion sites present in a grid founder plant segregated in the grid progeny, and these insertion sites were expected to be found in all rows and columns. In some cases, particular parental plasmids were over-represented in the sequenced plasmid population. To reduce their contribution and increase recovery of new insertion sites, a rare-cutting restriction enzyme site was identified in the parental plasmid and the corresponding enzyme was included in the genomic DNA preparation to bias against recovery of that parental plasmid. PCR screening of a library plate to quantify a RescueMu insertion hotspot Six gene primers plus one RescueMu left readout primer were used in this study: 1. 5'-TTGGGAGGTTGAAGGTAAAGACAT-3' 2. 5'-GTGCTG GATTGGTTACTCCG-3' 3. 5'-CGATGATTCTAGTTGAGCGTCTG-3' 4. 5'-ACTCGCACCAACATGAATACC-3' 5. 5'-GTTTCCGAGGACGCAGAGG-3' 6. 5'-AGCGCCAGGGCCAGGGGATTC-3' L. 5'-CAT TTC GTC GAA TCC CCT TCC-3' ( RescueMu ) Locations and directions with respect to the insertion site of RescueMu are shown in Figure 3a . PCR conditions were as follows: 5-20 ng of each plasmid library, 2.0-2.5 mM Mg 2+ , 0.4 mM dNTPs, 0.8-1.0 μM gene primer and 4-5 μM RescueMu L primer in a 50 μl reaction was first denatured for 2 min at 95°C followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C and 2 min at 72°C, and a final 2 min extension at 72°C. The same PCR conditions were used for screening using 5-100 ng samples of maize total genomic DNA. DNA blot hybridization Total genomic DNA was extracted from leaf tissues using a modified urea method [ 55 ]. After overnight digestion, the restricted DNA was separated on a 0.8% agarose gel and transferred onto Hybond-N+ membrane (Amersham Biosciences) in 0.4 M NaOH. Blots were hybridized with non-radioactive probes labeled with AlkPhos DIRECT system (Amersham Biosciences) for chemiluminescence detection on X-ray film. Initial clustering and assembly of genomic sequences The sequences were screened to remove the TIR sequences using the program crossmatch [ 56 ] and then trimmed to achieve a minimum phred score >15 in sliding windows over 40 bases. Overall the quality scores averaged phred >35, or less than one error in 3,160 bases. The average length of the trimmed, high quality genomic sequence entering the assembly was 378 bases. The right-TIR primer yielded 22% more successful sequence than the left-TIR primer resulting in an excess of right side sequences. Trimmed sequences were then assembled into contigs using phrap [ 56 ] with the following parameters: -minmatch 35 -minscore 30 -node_seq 14 -node_space 9. The member sequences for each contig were extracted from the phrap output files and assigned to a row or column of a grid. Within each contig, only a single sequence from a plasmid was used to determine the row and column representation. For example, if both the left- and right-flanking sequence from a plasmid assembled into one contig, this was considered one recovery of the plasmid. If the left-flanking sequence from one plasmid and the right-flanking sequence from a separate plasmid assembled into the same contig, this was considered two independent recoveries of the same genomic locus. In the latter case, if the right- flanking sequence was from a different row, then the sequence was recovered in multiple rows as well. All sequences were deposited into the Genomic Survey Sequencing (GSS) section of GenBank [ 57 ]. Assembly of RescueMu -derived genomic sequence data As shown in Figure 1 , using the 9-bp TSD characteristically generated during Mu element insertion [ 1 ], the sequences to the right and left of a particular RescueMu element can be assembled into a continuous sequence. To do this, trimmed RescueMu GSS sequences were downloaded from GenBank [ 58 ], for comparison to raw GSS sequences containing the Mu1 TIR sequences. The TIRs were masked by the cross_match program [ 56 ] to determine the flanking 9-bp TSD sequences. The TSDs are the end-overlaps between GSS sequences generated from the left and right side of RescueMu insertion. Merging through TSDs using the reverse-complementary strand of the left and right sequences recovers the original genomic sequences flanking the RescueMu insertion. A special consideration in the assembly of the genomic sequences flanking the right- and left-TIRs of RescueMu is the presence of a GGATCC ( Bam HI), AGATCT ( Bgl II), or a GGATCT ( Bgl II/ Bam HI) or AGATCC ( Bam HI/ Bgl II) motif. The two restriction digestion sites represent a true ligation site of sequence that was non-contiguous in the maize genome, but the post-restriction site sequences can unambiguously be assigned to the right or the left of RescueMu . On the other hand, the GGATCT or AGATCC motif could be contiguous genomic sequence or could have been generated during the ligation step of the plasmid rescue. Consequently, assignment of the position of the sequence beyond the GGATCT or AGATCC motif is ambiguous. If the RescueMu insertion site matched EST sequence across and beyond the GGATCT or AGATCC motif, the post-ligation sequence could be properly assigned (Figure 1 ). In the RescueMu plasmid sequences considered here, the average number of sequences reported to GenBank was 2.3 (131,364/57,022) per plasmid. The 131,364 RescueMu GSS sequences deposited at GenBank were screened for vector sequences against the UniVec database at the National Center for Biotechnology Information (NCBI) [ 59 ] using the crossmatch program: -mismatch 12 -penalty -2 -minscore 20. The resulting 130,861 vector-trimmed sequences were then screened against the maize repeat database annotated by TIGR [ 60 ] using the Vmatch program [ 61 ] with the parameters -l 50 -h 3 -identity 95. The 127,708 repeat-free sequences were then used to identify parental insertions. Any given RescueMu -transformed plant contains the parental RescueMu elements that were recovered at a high frequency during sequencing (from every sequenced row or column). Because our goal is to analyze the gene discovery by newly inserted RescueMu (that is, we are interested in where those non-parentals inserted into the maize genome), we decided to filter out the parental sequences as much as possible. We used Vmatch to cluster near-identical left and right sequences for each grid. A parental cluster contains sequences from nearly all the row or column sequences. A total of 59,069 parental sequences were identified and were excluded from the subsequent assembly. All the non-parental sequences were first preassembled for each plasmid using the left and right 9-bp TSD overlap. The merged GSSs were first clustered by PaCE [ 62 ] (minimum exact match 36 bp, minimum score threshold 30%) and then consensus sequences (contigs) for each cluster were generated by CAP3 [ 63 ] (overlap 40 bp; 90% identity cutoff). Because PaCE and CAP3 only pair sequence with the minimal overlap required to establish statistically significant identity, the number of contigs is probably an overestimate of the number of independent RescueMu insertion sites. For the particular case where TSDs were not recovered during sequencing, the left and right sequences could not be assembled together, even though they were from the same plasmid. Therefore, a Perl script was developed to conduct single-linkage clustering based on clone-pair constraints to assemble the GSS to the same 'genomic loci' if they were derived from the same plasmid clone. Classification of insertion site context To be successful as a gene-discovery tool, the transposon insertions must be predominantly into the genic regions of the maize genome. To quantify the potential enrichment of the RescueMu flanking sequences for genic regions, we matched all assembled contig sequences against various classes of known repetitive sequences, including retrotransposons, DNA transposons, centromeric and telomeric repeats, rRNA genes and plastid DNA. For this analysis, the non-parental sequences were used in their original form, with only vector sequences but not repeat sequences trimmed. The sequences previously discarded for analysis because they consist almost entirely of repetitive elements were assembled using the same procedure as described above for the repeat-trimmed sequences. Note, however, that this number of loci is unreliable and probably an underestimate of the true number of loci recovered because of the intrinsic difficulty with assembling repetitive DNA. To identify the repetitive elements in the contigs, Vmatch (-seedlength 14 -hxdrop3 -l 30 -identity 70) was used in combination with the TIGR cereal repeat database (version 2 consisting of maize, rice, barley, sorghum and wheat repeats). The contigs were also scanned from tRNA genes by tRANscan-SE program [ 64 ] with its default parameters. Gene discovery in GSS contigs Both similarity-based and ab initio approaches have been used to detect gene structures of the GSS contigs. For the similarity-based approach, GeneSeqer [ 65 ] programs were used to match plant EST contigs and cDNAs to GSS contigs. The plant EST contigs were regularly assembled by PlantGDB [ 66 ]. For the ab initio prediction, GENSCAN [ 67 ] (with default parameter settings for maize) was used and only high exon score predications (≥0.90) were selected. The GSS contigs were compared against SPTR [ 68 ], a nonredundant protein data set collected by the European Bioinformatics Institute (EBI), using BLASTX [ 69 ] with an E-value ≤ e-20. The BLASTX top protein hits were used to assign putative functions to the unique regions and for classification into functional categories based on annotation in the Gene Ontology [ 21 ] database. The genetically mapped maize ESTs were retrieved from MaizeGDB [ 70 ]. These ESTs were spliced-aligned to GSS contigs using GeneSeqer as described above. The matched GSS contigs were then plotted on the maize IBM Neighbor genetic map [ 30 ]. Analysis of 9-bp TSD and insertion site preferences For the analysis of RescueMu target sites, we retrieved the 9-bp TSD sequences from the confirmed insertion sites where both the left and right sequences match on the 9-bp TSD. We also retrieved the 20 bp up- and downstream sequences around the TSD. Then a 15-base long profile (9-base TSD and its three up- and downstream neighbors) was derived from the sequences and their reverse-complement orientation determined using the Expectation Maximization Algorithm [ 71 ]. Analysis of tentative unique contigs containing GSS sequences from multiple grids The GSS seqeunces present in each tentative unique contig (TUCs) were extracted from [ 31 ] and assigned to a row or column within a grid. A sample of TUCs with GSS sequences from multiple grids was then selected for detailed analysis. For each GSS in the TUC (excluding post-ligation sequences), the exact location of the TSD was determined by visual examination of the sequence alignment file for the TUC and the untrimmed GSS sequence data. The number of GSS sequences for each grid at each transposition site was recorded. Phenotypic analysis Grid plants were self-pollinated unless male or female-sterile. The resulting F1 families were evaluated by inspection of ears and kernels, at weekly intervals for five weeks after germination in a sand bench in a greenhouse, and at weekly intervals throughout the life cycle in the field. Phenotypes observed were recorded and are assembled into a searchable database at [ 31 ]. Unique phenotypes were documented with a digital image, and there are links to corresponding RescueMu flanking sequences where established. Instructions on how to obtain seed of grid plants is also provided. Additional data files The following additional data are available with the online version of this article: a table listing the internal primers used in sequencing RescueMu (Additional data file 1 ), supplementary material for this paper, including details of methods (Additional data file 2 ). Supplementary Material Additional data file 1 A table listing the internal primers used in sequencing RescueMu Click here for additional data file Additional data file 2 Supplementary material for this paper, including details of methods Click here for additional data file

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Tissue engineering, stem cells, cloning, and parthenogenesis: new paradigms for therapy

Patients suffering from diseased and injured organs may be treated with transplanted organs. However, there is a severe shortage of donor organs which is worsening yearly due to the aging population. Scientists in the field of tissue engineering apply the principles of cell transplantation, materials science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Both therapeutic cloning (nucleus from a donor cell is transferred into an enucleated oocyte), and parthenogenesis (oocyte is activated and stimulated to divide), permit extraction of pluripotent embryonic stem cells, and offer a potentially limitless source of cells for tissue engineering applications. The stem cell field is also advancing rapidly, opening new options for therapy. The present article reviews recent progress in tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure.

Introduction The goal of tissue engineering is to repair organ pathologies such as those acquired congenitally or by cancer, trauma, infection, or inflammation. It is based upon the foundations of cell transplantation and materials science. Tissue can be engineered 1) in vivo - by stimulating the body's own regeneration response with the appropriate biomaterial, or 2) ex vivo - cells can be expanded in culture, attached to a scaffold and then reimplanted into the host. Cells may be heterologous (different species), allogeneic (same species, different individual), or autologous (same individual). Autologous cells are preferred because they will not evoke an immunologic response and thus the deleterious side effects of immunosuppressive agents can be avoided. The ideal autologous cells can often be found within the organ itself. These cells (committed precursors) may be isolated, expanded and transplanted back into the same patient, thus representing an autologous transplantation resource. Previously, urothelial cells could be grown in the laboratory setting with only limited expansion. Several protocols were developed over the last 20 years which identified the undifferentiated cells and kept them undifferentiated during their growth phase [ 1 - 4 ]. Using such cell culture methods it is now possible to expand a urothelial strain from a single specimen which initially covered a surface area of 1 cm 2 to one that covers a surface area of >4000 m 2 (an area equivalent to one football field) within 8–14 weeks. These studies indicate the possibility of collecting autologous bladder cells from human patients, expanding them in culture, and returning them to the human donor in sufficient quantities for reconstructive purposes [ 1 , 3 - 11 ]. Major advances have been achieved within the past decade regarding possible expansion of several primary human cell types with specific techniques that employ autologous cells for clinical application. While autologous cells are recognized as the ideal transplantation resource, many patients with end-stage organ disease are unable to yield sufficient cells for expansion and transplantation. Furthermore, some primary autologous human cells cannot be expanded from particular organs ( i.e . pancreas, liver). Stem cells are envisioned as being an alternate source of cells from which the desired tissue can be derived. Human embryonic stem cells (HESC) can be derived from discarded non transferred embryos and have the advantage of being pluripotential (the ability to differentiate into all tissues of the embryo) and able to self-renew indefinitely. However, their clinical application is limited because they represent an allogeneic resource and thus their use would require high dose immunosuppressant therapy. New stem cell technologies such as somatic cell nuclear transfer (therapeutic cloning) and parthenogenesis offer an exciting alternative to create an inexhaustible supply of ESC that can differentiate into all cell types of the embryo, while not being rejected by the patient's immune system. Although many tissues have been created with ESC, they are not used clinically because of an inability to control differentiation. Hence, their ability to form multiple tissue types also becomes their limitation. New genomics and bioinformatics technologies have and will continue to offer new insights into the understanding of ESC growth and differentiation and their application to engineering tissues. In the near future, these new technologies will allow for the generation of an unlimited supply of any cell type in the body. Stem cells The political and ethical controversy surrounding stem cells began in 1998 with the creation of HESC derived from discarded, non-transferred human embryos[ 12 ]. The HESC were isolated from the inner cell mass of a blastocyst (5 days post-fertilization embryo) using an immunosurgical technique whereby the blastocyst was incubated with antibodies specific to trophectoderm. Complement proteins then resulted in lysis of the trophectoderm so that the only surviving cells were the inner cell mass [ 13 ]. Given that some cells can not be expanded ex vivo , ESC can potentially be the ideal resource for tissue engineering because of two fundamental properties, 1) the ability to self-renew indefinitely, and 2) the ability to differentiate into all three germ layers. With the current restrictions surrounding HESC work, many proponents of stem cell research have sought to modify the ban to incorporate the thousands of non-transferred frozen embryos resulting from IVF to be used for the creation of more HESC lines. A SART-RAND study identified approximately 400,000 frozen embryos in storage since the late 1970s [ 14 ]. However, only 2.8% of these have been designated for research. Of the 11,000 embryos designated for research, only 65% of these ( n = 7,334) are expected to survive the freeze/thaw process. From this, 25% are expected to develop to blastocyst stage ( n = 1, 834). If one assumes a 15% efficiency rate for establishment of a HESC line from blastocysts (as suggested by previous studies [ 12 , 15 ]), it may be estimated that approximately 275 HESC could be created from excess frozen embryos. However, the real number of HESC line generated would actually be much lower since not all frozen embryos allocated for research would be used to create HESC lines. Furthermore, even if the maximum possible number of HESC lines could be derived from human frozen embryos, the clinical application of such cells would be limited by the potential rejection from another individual's immune system. New stem cell technologies (such as somatic cell nuclear transfer and parthenogenesis) promise to overcome this limitation. Somatic cell nuclear transfer (therapeutic cloning) Somatic cell nuclear transfer (SCNT) entails the removal of an oocyte nucleus followed by its replacement with a nucleus derived from a somatic cell obtained from that patient. Activation with chemicals or electric shock stimulates cell division up to the blastocyst stage at which time the inner cell mass is isolated and cultured, resulting in ESC. This approach is distinct from reproductive cloning because the blasotcyst is not transplanted back to the uterus. Hence, development does not proceed beyond the 100 cell stage. This process also differs from fertilization since no sperm is used in this process. The resulting ESC are perfectly matched to the patients immune system and no immunosuppressants would therefore be required to prevent rejection. While interest in the field of nuclear cloning remains high since the birth of Dolly (1997), the first successful nuclear transfer was actually reported over fifty years ago by Briggs and King [ 16 ]. Cloned frogs, which were the first vertebrates derived from nuclear transfer, were subsequently reported by Gurdon in 1962 [ 17 ] although the nuclei were derived from non-adult sources. Indeed, in just the past six years alone important advances in nuclear cloning technology have been reported – a pace of discovery that betokens the relative immaturity of this research arena. In fact Dolly was not the first cloned mammal to be produced from adult cells. Live lambs were produced in 1996 using nuclear transfer and differentiated epithelial cells, although these were derived from embryonic discs [ 18 ]. To be sure, the significance of the Dolly report was that this described the first mammal to be derived from an adult somatic cell using nuclear transfer [ 19 ]. Subsequently, animals from several species have been grown using nuclear transfer technology, including cattle [ 20 ], goats [ 21 , 22 ], mice [ 23 ], and pigs [ 24 - 27 ]. A better understanding of the differences between reproductive cloning and therapeutic cloning may help alleviate some of the controversy surrounding these technologies [ 28 , 29 ]. Banned in most countries for human applications, reproductive cloning is used to generate an embryo that has the identical genetic material as its cell source. Such an embryo could then be implanted into the uterus of a female to give rise to a liveborn infant that is a clone of the donor. In contrast, therapeutic cloning is used to generate only ESC lines whose genetic material is identical to that of its source. These autologous stem cells have the potential to become almost any type of cell in the adult body, and thus would be useful in tissue and organ replacement applications [ 30 ]. Therefore, therapeutic cloning (SCNT) may provide an alternative source of transplantable cells. Figure 1 shows the strategy of combining therapeutic cloning with tissue engineering to develop tissues and organs. It has been estimated that approximately 3,000 people die every day in USA of diseases that could have been treated with stem cells-derived tissues [ 31 ]. With current allogeneic tissue transplantation protocols, rejection is a frequent complication because of immunologic incompatibility and thus immunosuppressive drugs are generally required to manage host-versus-graft disease [ 30 ]. The use of transplantable tissue and organs derived from therapeutic cloning could obviate unwanted immune responses typically associated with transplantation of non-autologous tissues [ 32 ]. Figure 1 Strategy for therapeutic cloning and tissue engineering While promising, somatic cell nuclear transfer technology has certain limitations requiring further improvement before it can be applied widely in clinical practice. Currently, the efficiency of the overall cloning process is quite low as the majority of embryos derived from animal cloning do not survive after implantation [ 33 - 35 ]. In practical terms, multiple nuclear transfers must be performed in order to produce one live offspring for animal cloning applications. The potential for cloned embryos to grow into live offspring ranges between <1 and 18% for sheep, pigs, and mice [ 36 ]. However, greater success (~ 80%) has been reported in cattle [ 37 ], a result which may in part be due to availability of advanced laboratory technologies specifically developed for this species for agricultural/breeding purposes. To improve cloning efficiencies, further improvements are required in the multiple complex steps of nuclear transfer such as enucleation and reconstruction, oocyte activation, and synchronization of cell cycle between donor cells and recipient oocytes [ 38 ]. It must be noted that abnormalities have been found in liveborn clones including macrosomia with an enlarged placenta ("large-offspring syndrome") [ 39 ], respiratory distress, defects of the kidney, liver, heart, and brain [ 40 ], obesity [ 41 ], and premature death [ 42 ]. These may be related to epigenetics of cloned cells which involve reversible modifications of DNA, while the original DNA (genetic) sequences remain intact. Faulty epigenetic modulation in clones may result from altered DNA methylation and/or histone modifications causing the overall chromatin structure of somatic nuclei not to be reprogrammed to an embryonic pattern of expression [ 30 ]. Reactivation of key embryonic genes at the blastocyst stage usually does not occur in embryos cloned from somatic cells, while embryos cloned from embryos consistently express early embryonic genes[ 43 , 44 ]. Proper epigenetic reprogramming to an embryonic state may help to improve the cloning efficiency and reduce the incidence of abnormal cloned cells. Novel applications of somatic cell nuclear transfer (therapeutic cloning) We applied principles of both tissue engineering and therapeutic cloning in an effort to produce genetically identical renal tissue in an animal model ( Bos taurus ) [ 45 ]. Bovine skin fibroblasts from adult Holstein steers were obtained by ear notch and single donor cells were isolated and microinjected into the perivitelline space of donor enucleated oocytes (nuclear transfer). The resulting blastocysts were transferred to the uterus of progestin-synchronized recipients permit further in vivo growth. After 12 weeks cloned renal cells were harvested, expanded in vitro , then seeded onto biodegradable scaffolds. The constructs (consisting of cells + scaffolds) were then implanted into the subcutaneous space of the same steer from which the cells were cloned to allow for tissue growth. The kidney is a complex organ with multiple cell types and a complex functional anatomy rendering it one of the most difficult organs to reconstruct [ 46 , 47 ]. Previous efforts in tissue engineering of the kidney have been directed toward development of extracorporeal renal support systems made of biological and synthetic components [ 48 - 54 ]. Although ex vivo renal replacement devices are known to be life-sustaining, there are obvious benefits for patients with end-stage kidney disease if such devices could be implanted long-term without the need for an extracorporeal perfusion circuit or immunosuppressive drugs. Cloned renal cells were seeded on scaffolds consisting of three collagen-coated cylindrical polycarbonate membranes (figure 2 ). The ends of the three membranes of each scaffold were connected to catheters terminating in a collecting reservoir. This created a renal neo-organ with a mechanism for collecting the excreted urinary fluid (figure 3 ). Scaffolds with the collecting devices were transplanted subcutaneously into the same steer from which the genetic material originated and retrieved 12 weeks after implantation. Figure 2 Combining therapeutic cloning and tissue engineering to produce kidney tissue, an illustration of the tissue-engineered renal unit. Figure 3 Renal unit seeded with cloned cells, three months after implantation, showing the accumulation of urinelike fluid. Chemical analysis of the urine-like fluid (for urea nitrogen/creatinine levels, electrolyte levels, specific gravity, and glucose concentration) revealed that the implanted renal cells possessed filtration, reabsorption, and secretory capabilities. Histological examination of the retrieved implants revealed extensive vascularization and self-organization of the cells into glomeruli- and tubule-like structures. A clear continuity between glomeruli, tubules, and the polycarbonate membrane was noted that allowed the passage of urine into the collecting reservoir (figure 4 ). Immunohistochemical analysis with kidney-specific antibodies revealed the presence of renal proteins, and RT-PCR analysis confirmed the transcription of renal specific RNA in the cloned specimens. Western blot analysis confirmed the presence of elevated renal-specific protein levels. Figure 4 Clear unidirectional continuity between the mature glomeruli, their tubules, and the polycarbonate membrane. As previous studies have confirmed bovine clones harbor mitochondrial DNA (mtDNA) of strictly oocyte origin [ 55 - 57 ], the donor egg's mtDNA was thought to be a potential source of immunologic incompatibility. Differences in mtDNA-encoded proteins expressed by cloned cells could stimulate a T-cell response specific for mt-DNA-encoded minor histocompatibility antigens when cloned cells are implanted back into the original nuclear donor [ 58 ]. We used nucleotide sequencing of the mtDNA genomes of the clone and fibroblast nuclear donor to identify potential antigens in the muscle constructs. Only two amino acid substitutions were noted to distinguish cells from the clone and the nuclear donor. Since peptide-binding motifs for bovine MHC class I molecules remain poorly understood, there is no reliable method to predict the impact of these amino acid substitutions on bovine histocompatibility. Oocyte-derived mtDNA was also considered to be a potential source of immunologic incompatibility in cloned renal cells. Maternally transmitted minor histocompatibility antigens in mice have been shown to stimulate both skin allograft rejection in vivo and cytotoxic T lymphocytes expansion in vitro [ 58 ] that could prevent the use of these cloned constructs in patients with chronic rejection of major histocompatibility-matched human renal transplants [ 59 , 60 ]. We tested for a possible T-cell response to the cloned renal devices using delayed-type hypersensitivity testing in vivo and Elispot analysis of interferon-gamma secreting T-cells in vitro . Both analyses revealed that the cloned renal cells showed no evidence of T-cell response, suggesting that rejection will not necessarily occur in the presence of oocyte-derived mtDNA (figure 5 ). This finding may represent a step forward in overcoming the histocompatibility problem of stem cell therapy [ 47 ]. Figure 5 Elispot analyses of the frequencies of T-cells that secrete IFN-gamma after primary and secondary stimulation with allogeneic renal cells, cloned renal cells, or nuclear donor fibroblasts. These studies demonstrated that cells derived from nuclear transfer can be successfully harvested, expanded in culture, and transplanted in vivo with the use of biodegradable scaffolds on which the single suspended cells can organize into tissue structures that are genetically identical to that of the host. These studies were the first demonstration of the use of therapeutic cloning for regeneration of tissues in vivo . Others in the field have created mouse SCNT derived c-kit-positive stem cells to restore infarcted myocardium [ 61 ], dopaminergic neurons to correct the phenotype of a mouse model of Parkinson disease [ 62 ]. The first HESC line derived from SCNT was created in February, 2004 [ 63 ]. Parthenogenesis Parthenogenesis (< Gr . "virgin birth") is production of offspring by a female with no genetic contribution from a male and without meiotic chromosome reduction. The process is common reproductive strategy among insects such as aphids, flies, ants, and honeybees, but is also known to occur in vertebrates including lizards, snakes, fish, birds, and amphibians. The first demonstration of artificially-stimulated parthenogenesis in vitro was made by Jacques Loeb (1899), who was able to activate oocytes from sea urchins and frogs by pricking them with a needle or by changing the ambient salt concentration. Pincus (1939) demonstrated parthenogenetic activation of mammalian eggs using temperature and chemical stimuli. Thus far, parthenogenetic activation of eggs has been studied in a variety of mammals including mice, goats, cows, monkeys, and humans. Plachot et al . described parthenogenesis in humans by examining 800 human oocytes and showed that 12 activated parthenogenetically and four underwent normal cleavage[ 64 ]. Although there have been no reports of naturally-occurring human parthenotes, a human parthenogenetic chimera has been described [ 65 ]. The juvenile patient presented with developmental delay, apparent sex reversal, and entirely parthenogenetic blood leukocytes. This finding confirmed the viability of chimeras in higher mammals as presaged by successful murine experiments over the previous two decades (see below). There is no confirmed example of de novo mammalian parthenogenetic reproduction, but mammalian oocytes can be artificially induced to undergo parthenogenesis in vitro by a two-step protocol involving electroporation and/or treatment with a chemical agent (ionomycin, ethanol, or inositol 1,4,5-triphosphate) to elevate Ca 2+ levels transiently, followed by application of an inhibitor of protein synthesis (cycloheximide) or protein phosphorylation (6-dimethylaminopurine). Success rates and viability appear to be organism dependent. Mouse parthenotes are capable of developing beyond the post-implantation stage in vivo [ 66 , 67 ]; porcine parthenotes have developed up to post-activation day 29 (limb bud stage, past the early heart beating stage); rabbit parthenotes until day 10–11 [ 68 ]; primates ( Callithrix jacchus ) have only been shown to implant [ 69 ]. The reason for this arrested development is believed to be due to genetic imprinting. In normal zygotes maternal and paternal haploid genomes are epigenetically distinct, and both sets are required for successful development [ 70 , 71 ]. Indeed, unstable chromosome modifications in the form of DNA methylation or histone modification are distinctly different in human sperm, compared to eggs. Therefore each gamete carries unique patterns of gene expression into the embryo. Since all genetic material in parthenotes is of maternal origin, there is no paternal imprinting component and this prevents proper development of extraembryonic tissues whose expression is regulated by the male genome [ 72 ]. In most mammals – including primates – oocytes are arrested at metaphase II just before ovulation. Cytogenetic microscopy shows the presence of a 2n polar body under the zona pellucida and a 2n protonucleus in the cytoplasm. After chemical activation to mimic the effects of sperm penetration on changes in cellular Ca 2+ gradient, the cell fails to complete meiosis II. Instead, the second polar body is never extruded, resulting in a diploid protonucleus derived from two sets of sister chromatids. These chromatids then begin to undergo mitosis resulting in a parthenote manifesting uniparental disomy. Although the derivation of embryonic-like stem cells from oocytes (parthenogenetic stem cells, PSC) is relatively inefficient (perhaps due to complexities of genomic imprinting), when they are differentiated into adult tissues, they appear fully functional. In spite of non-viability of monkey parthenotes, the extracted stem cells seem to assume the morphology and functional behavior of HESC and express appropriate ESC markers. They have embryonic-like replicative ability and have been propagated in vitro in an undifferentiated state for up to 14 months. In vitro , they have been differentiated into cardiomyocyte-like cells, smooth muscle, beating ciliated epithelia, adipocytes, several types of epithelial cells, as well as dopaminergic and serotoninergic neurons. Almost all of these neurons express TUJ1 (beta-tubulin III), and up to 25% of the TUJ1+ cells co-express tyrosine-hydroxylase. This latter enzyme marker is considered diagnostic for catecholaminergic neurons (dopamine, norepinephrine, and epinephrine [ 73 ]). Furthermore, HPLC analysis of culture media following a depolarizing KCl-buffer identifies the release of the neurotransmitters dopamine and serotonin from the cells. Ater two weeks of differentiation, about half of the cells demonstrate neuronal morphology and begin to express voltage-dependent sodium channels that can be blocked by tetrodotoxin. These observations are recapitulated in vivo , since injection of monkey PSC into immunocompromised mice induces formation of benign teratomas containing tissue derivatives from all three germ layers (ectoderm, endoderm and mesoderm) including cartilage, muscle, bone, neurons, skin, hair follicles, and intestinal epithelia [ 74 , 75 ]. Of particular note is the apparent tendency of these cells to differentiate into neuronal tissues, as has been noted by chimera studies [ 67 ]. The reasons for this underlying preference are not well understood although one possible explanation is that it is a consequence of purely maternal genomic imprinting, reflecting a lack of epigenetic balance that would be conferred by paternally-imprinted genes. To be sure, parthenotes are not free from ethical controversy and are viewed by some in society as artificial entities that in some sense represent 'tampering with nature.' Since a parthenote is analogous to a mature ovarian teratoma (a spontaneous in vivo tumorigenic event) the de facto acceptance of experiments using teratoma tumor tissue lends some legitimacy to experimentation on parthenotes. These contradictions await reconciliation in a comprehensive ethical framework. Stem cell genomics The pluripotentiality of stem cells is also their limitation, and explains why they are not used clinically today. Although ESC can be differentiated into skin, neurons, blood, cardiac cells, cartilage, endothelial cells, muscle, hepatocytes, and pancreatic cells, the efficiency can be quite limited for certain cell types. Another difficulty is studying the quality of differentiation: are the neurons derived from stem cells bona fide neurons, or merely neuronal-like cells? To address this question we developed high throughput methodologies using microarrays to evaluate new stem cell derivatives [ 76 ]. We differentiated HESC into retinal pigmented epithelial cells (RPE) (the site of lesions in macular degeneration and retinitis pigmentosa) and used microarrays to identify their genetic signature. We then compared their gene signature to those derived from two established RPE cell lines (one of which has been successfully used clinically). A bronchial epithelial cell line served as a negative control and a freshly isolated human RPE served as a positive control. We demonstrated similarity between our HESC derived RPE and the freshly isolated RPE. The bronchial epithelial and two other established RPE lines were less similar. Interestingly, the data set that represented the genes common to freshly isolated RPE and HESC derived RPE (but not in the two established lines), contained many retinal specific genes. This finding provided further support of the benefits of HESC: the ability to generate a limitless number of HESC with the potential to differentiate along specific lineages to allow creation of RPE cells in quantities necessary for clinical transplantation. The next step would be to couple this technology to ESC derived from SCNT (or parthenogenesis) to create the ideal treatment for macular degeneration and retinitis pigmentosa. Another technology currently under development at our institution is "genomics guided tissue engineering." Here we perform microarrays periodically during stem cell differentiation. For example, microarrays are performed on undifferentiated monkey PSC, PSC derived neural precursors (PSC-NP), and NP that were further differentiated for 8 days (PSC-neurons). We have identified numerous targets such as receptors and ligands present at each of these distinct time points, and are modifying our culture system in order to improve the quality and quantity of differentiation. Furthermore, we are comparing the gene expression profiles of PSC derived neurons to gene expression profiles of reference neurons. Not only will this provide new insight into the type of neurons that may be generated, but it offers clues into what our stem cell derived neurons might be lacking. We can then go back to the culture system and try to target these specific genes/signaling pathways. Further study of stem cell genomics will give additional insight into pluripotentiality. An understanding of pluripotentiality might allow for a somatic cell to be de-differentiated into an intermediate stage, which could then be expanded, differentiated and transplanted back into the patient. We are presently characterizing the genetic signature of pluripoteniality by analyzing gene expression among primate stem cells derived from a variety of methods (IVF, parthenogenesis, and adult stem cells). By identifying "stemness" genes by comparing undifferentiated stem cells to their differentiated counterpart, and comparing this to stem cells of different origins, a core set of pluripotential target genes may be mapped. Of particular interest are the 1,075 genes that are similarly down-regulated in IVF derived human ESC and monkey PSC. Furthermore, we have detected paternally imprinted genes in our HESC but not in our PSC data sets. From this we conclude that paternal imprinting might not be necessary for pluriopotentiality. Conclusion Our systems biology approach incorporates the fields of genomics, cell biology, nuclear transfer, and materials science, and utilizes personnel who have mastered the techniques of bioinformatics, cell harvest, culture, expansion, transplantation, as well as polymer design essential for the successful application of these technologies. Experimental efforts are currently underway involving virtually every type of tissue and organ of the human body. Various tissues are at different stages of development with some already being used clinically, a few in pre-clinical trials, and some in the discovery stage. Recent progress suggests that engineered tissues may have an expanded clinical applicability in the future and may represent a viable therapeutic option for those who require tissue replacement or repair.

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Specialized microbial databases for inductive exploration of microbial genome sequences

Background The enormous amount of genome sequence data asks for user-oriented databases to manage sequences and annotations. Queries must include search tools permitting function identification through exploration of related objects. Methods The GenoList package for collecting and mining microbial genome databases has been rewritten using MySQL as the database management system. Functions that were not available in MySQL, such as nested subquery, have been implemented. Results Inductive reasoning in the study of genomes starts from "islands of knowledge", centered around genes with some known background. With this concept of "neighborhood" in mind, a modified version of the GenoList structure has been used for organizing sequence data from prokaryotic genomes of particular interest in China. GenoChore , a set of 17 specialized end-user-oriented microbial databases (including one instance of Microsporidia, Encephalitozoon cuniculi , a member of Eukarya) has been made publicly available. These databases allow the user to browse genome sequence and annotation data using standard queries. In addition they provide a weekly update of searches against the world-wide protein sequences data libraries, allowing one to monitor annotation updates on genes of interest. Finally, they allow users to search for patterns in DNA or protein sequences, taking into account a clustering of genes into formal operons, as well as providing extra facilities to query sequences using predefined sequence patterns. Conclusion This growing set of specialized microbial databases organize data created by the first Chinese bacterial genome programs (ThermaList, Thermoanaerobacter tencongensis , LeptoList, with two different genomes of Leptospira interrogans and SepiList, Staphylococcus epidermidis ) associated to related organisms for comparison.

Background We are facing a deluge of genome sequences. As of January 14th, 2005, the GOLD site identified 1248 completed or ongoing genome programs , and this certainly reflects only a partial view of the existing programs. While this shows that we implicitely possess an enormous wealth of information about the functions carried out by genes and genomes, the very fact that this amount is enormous makes it extremely difficult to mine that information easily. The role of specialized databases is to make this task easier for end-users. Many types of microbial genome databases exist. Most of them have been developed in a context of bioinformatics centres or laboratories purely favoring in silico research rather than the coupling between experiments using computers and experiments at the bench, and this is reflected in the structure and aims of the databases [ 1 - 8 ]. In contrast, at the onset of genome programs, we decided to set up a data structure for bacterial genomes that would help experimentalists to access knowledge on genes and genomes in an end user-oriented fashion. This was first the aim of the Colibri project, with the goal to organize Escherichia coli genome data, well before the whole genome sequence was known [ 9 ]. Later on, the SubtiList database was at the core of the Bacillus subtilis genome program data access [ 10 ]. Many databases constructed on the GenoList data schema were subsequently constructed ( , [ 11 ]). However, with the exponentially growing set of genome sequences, it became important to divide up the work while maintaining the main goal of the project, that of being end-user-driven and of course, user-friendly. While an ongoing effort aims at integrating all bacterial genomes within the GenoList frame into a single database, it is important to create individual databases that could be regularly updated by a selected team of scientists (preferably those that initiated the corresponding genome program). This is particularly important for countries that are beginning to develop at a high speed into the genomics era. We took the opportunity of the creation of the HKU-Pasteur Research Centre in Hong Kong (China) to set up genome databases for the microbial sequencing projects developed in China (with databases for related organisms for comparison). Within this economic context, it was also important to take into account the cost of development. The paradigm GenoList databases are based on commercial DataBase Management Systems (DBMS) [ 11 ] and we decided to shift from a commercial DBMS to a non-commercial one, providing more freedom for the future of the project. In the present set of databases (GenoChore), emphasis is placed on retrieval of information centered on the gene as the central object, with exploration methods that query simple properties of the gene products (such as molecular mass or isoelectric point) in addition to more complex features such as the class of codon usage bias used in the gene [ 12 ]. Furthermore, queries can be made on the sequence itself using large scale analyses such as BLAST, and search for word patterns present in DNA and protein sequences. Construction and content Data schema Because we wished to shift from a commercial DBMS to an open-source one, there were some applications that could not be implemented readily due to the lack of certain advantages possessed by the commercial DBMSs. Hence, we had to alter the data structure in order to cope with this situation. The core data schema used in this work was that of GenoList version 3.1 [ 11 ], with slight modifications (Figure 1 ). Figure 1 Data Schema of the Databases. The core object of the schema is the Genomic_object, as in GenoList. It uses pointers in the sequence that delimits several categories of objects, including protein Coding DNA Sequences (CDSs), RNAs and other objects such as transcription terminators or riboswitches. Database management system In the present GenoList databases, the DBMS used is Sybase™. While this is convenient because of excellent stability and maintenance, this may pose problems in terms of commercial policies, especially if the structure has to be exported. We therefore decided to rewrite the management of the GenoList structure using MySQL . Most function transfers were straightforward. However some functions such as nested subquery that were not available in MySQL had to be dealt with indirectly. The nested subquery has been entirely circumvented in the PERL code and is dealt with in the Extended Search algorithm by concatenating different SQL queries simply using the "AND" or "OR"connector. Data input Sequence and annotation data were parsed from the files extracted from the International Nucleotide Sequences Database (INSD: DDBJ/EMBL-EBI/GenBank [ 13 , 14 ]) with the following procedure. To get access to the INSD, the authors of a genome sequence must follow the specification of the Feature Table Definition (FTD) jointly issued by the INSD partners . The current version is Version 6.2 Oct 15, 2004. While this specification is rigid, there is still a significant degree of freedom in annotation, so that a large number of individual situations have to be taken care of semi-automatically. The basic idea of the parser is firstly to read through the input file at the INSD and check file formats. Subsequently, the information is collected and distributed into several temporary files using a set of predefined keywords and their qualifiers (i.e. those characterizing the data schema). Subsequently, a check process is initiated to identify all situations that do not fit the specifications, so that they can be corrected manually. Usually, most of the process of creating tables is automatic and only a few exceptions have to be corrected individually. A second type of input is also provided as an interactive interface to tell the database curator what information has to be collected: once collected this information can be loaded into the databases directly (Figure 2 ). Teraprot data are obtained from Infobiogen . Figure 2 Implementation of a Database Curator Page. In order to help users who would participate in the improvement of the database annotation a Curator Page is provided permitting input of updated information. It is available to users after acceptation of their collaboration, through a password protected access. Once data consistency has been verified the new annotations are implemented in the current database. Query methods and interface We kept the interface of GenoList as published, except that a box providing access to protected curation of annotations is now provided, aiming initially at helping the first party (sequencing teams) annotators. The front page is made of three frames. Briefly, the vertical frame on the left contains the controls necessary to get access to the content of the database. The upper part of this frame contains text fields for querying the database according to five types of queries: gene name(s), chromosome region around a gene, chromosome region defined by positions, free text, functional classification (more detailed information about each type of query can be obtained by clicking on the question mark near the query title). The "Extended Search" button gives access to a search form allowing the user to perform multicriteria searches on all the database fields. The lower part of this frame allows one to launch sequence analysis tools: BLAST and FASTA database searches (on the sequence data), and DNA or protein pattern searches. In the former case, the user can choose to explore sequences located upstream of putative operons. In the latter case, the user can search for patterns anywhere in proteins, but also restrict the search to the beginning or end of the protein. The upper frame on the right can contain various types of information, depending on the genome and on the query. It can contain a graphical representation of a chromosome region, that can be obtained in several ways: usually from a gene in the bottom frame. This frame may contain launch forms and result lists from the sequence analysis tools available (pattern search, BLAST or FASTA scanning). The bottom frame on the right always contains detailed information about one given gene, including regularly updated BLAST searches and Teraprot reports as well as related bibliographic references. The original package managing the interface of GenoList databases was written in C/C++, following the first database schema [ 10 ], that had been adapted for use with the Sybase™ DBMS (the initial platform was using the DBMS 4 th Dimension™). The modification of the database schema needed for using MySQL required additional adaptations of the application interfaces. Using the original package would have required iterative work that was systematically adding complexity into the system. Current best Web interfaces and application interfaces (i.e. friendly for sharing parties) are often based on Perl scripts. For this reason a new core management script was recreated, written in Perl, while keeping the package architecture and the Web interfaces. Among other languages that have comparable functionalities, the choice of Perl to create the system was motivated by its powerful capability to glue different programs or scripts together. In addition it is widely used by the INSD, and at the European Bioinformatics Institute in particular within the BioSapiens program . Furthermore, this choice allowed us to keep the optimized fast C code that has been constructed for searching pattern (strings of symbols) inside the DNA or amino acids sequences. The GenoList C/C++ package chose to use the GD library for generating graphic representations of genome regions. The GD graphics library is an open source library which allows programmers to easily generate PNG, JPEG, and WBMP images from many different programming languages. We used here a newer version of the same library (perl module perl-GD version 2.11) to make use of its improvements in creating dynamic pictures. In rewriting the core of the program we used the Perl module DBI . A DBI is a middle layer between the outside applications and the communicator (DBD). Different DBMSs have their own communication mechanism to talk with outside applications, and in the present version the choice of the DBI module has been implemented in such a way that we could change the DBMS if necessary with minimal work. In this way, when changing the DBMS, it will only be necessary to tell the DBI about the specifications of the new DBMS without having to modify any other code. Finally, we used the Perl module CGI to facilitate the production of the WebPage interfaces. As a consequence further developments of the GenoChore package should be performed with minimal effort. Utility and discussion Data schema In the original GenoList structure, the central table corresponding to genomic objects carried all relevant features that are associated to genes and gene products. For the sake of future developments and to accommodate new feature annotation present in genome flatfiles, we separated this table into several gene product tables, specific for RNAs and proteins. The current data structure remains open to include tables for other types of data, such as regulation properties annotations when they will become available. Figure 1 displays a diagram of the current generic database schema (we did not show tables that remain empty for want of annotation data). As expected for a database meant to provide knowledge from genome programs, the central tables are focussing on genomic objects, the main one corresponding to protein Coding DNA Sequences (CDSs). To match this structure, the information present in the flat files created by the sequencing consortia, and present in the INSD, is split into three parts, namely, a) genomic objects, i.e. what we see in a chromosome, at precisely identified positions in the genome sequence (depending on the annotation tools available to the consortia), such as a CDS, a promoter, a terminator, a tRNA, an sRNA etc.; b) genome annotations, i.e. protein, RNA and other bio-molecules' products, functions, comments and so on; c) relations between genomic objects: e.g. the typical concept of gene requires its association to a promoter, a terminator and usually a CDS. In this representation, a set of genome objects' identifiers (ids) is used to represent a gene. This facilitates the association of genomic objects together with much more sophisticated relationships into more complex structures, when required. It is important here to notice that, in contrast to a rather ubiquitous practice, we explicitely separate between Open Reading Frames (ORFs) that are simply sequences multiple of 3 between two termination codons (TAA, TAG and TGA) and CDSs, that begin with a specific codon, usually ATG (in the DNA text), preceded by a ribosome binding site (RBS), typically AAGGAGGT in many bacterial genomes. One must remember that in most genomes the beginning of CDSs has not been experimentally identified. Identification of CDS starts is however much easier in low G+C Firmicutes that do not possess a counterpart of ribosomal protein S1 found in gamma proteobacteria [ 15 ]. In the same way, G+C-rich organisms have usually long ORFs, but the CDSs they harbour are usually highly enriched in A+T at the third codon position. Some caution, therefore, should be exerted by users when using the information collected in the databases about the beginning of proteins in these organisms (for example in the Streptomyces coelicolor database, CoeliList). Nomenclature: naming genes Users know that the system used for naming genes in genome databases is extremely unwieldy and completely lacks standardization. This is usually because genes are simply labelled in databases by access numbers corresponding to the annotation phase of the relevant genome program (e.g. PA3004 for a gene found in the genome of Pseudomonas aeruginosa ). In the absence of knowledge of a gene name it takes some time to identify it (often using BlastP), for example when aiming at the study of its neighborhood ((i.e. proximity of an object or a relationship with others sharing the same conceptual space, including presence in a common article [ 12 ]). Naturally, because most genes have never been experimentally identified in the majority of the newly sequenced genomes, this approach is certainly safer than giving a name without proper identification criteria. However it is extremely useful for scientists studying a genome to start from "islands of knowledge", with genes with a known background, reflected by a known gene (and a gene name has usually been coined by experimentalists for that gene). For this reason, we decided to use a conservative approach, using bidirectional best Blast hits of the genome of interest with model genome ( Escherichia coli K12 and Bacillus subtilis 168). Orthologues were identified as reciprocal best hits [ 16 ] (using a global alignment where the gaps on the edges of the largest sequence are ignored) with at least 50% identity in amino acid sequence and less than 20% difference in protein length. When possible, in order to increase the likelihood of the putative identification we used a second well known representative of the genome under study and looked for orthologues between every pair of each of the two triplets (i.e. between each pair of the three organisms: the organism of which the database is constructed, B. subtilis for Firmicutes and another organism of the same family, such as Listeria monocytogenes , and E. coli for gamma-proteobacteria, with another one of the same family, such as Photorhabdus luminescens ). Finding putative orthologues in the three organisms was considered as substantiating evidence for the use of a gene name. Then, in each triplet, we did not transfer the model organism gene name to all orthologues that were not simultaneously present in the three genomes or that gave different correspondences in different comparisons. In another comparison where the orthologues were found with at least 50% similarity, the model organism gene names to be transferred were preceded by the letter ' y '. In order to help users recognize gene names (and all the knowledge they associate with those names) we used as reference names those in the model bacteria, trying to comply as much as possible with the names used at SwissProt in the HAMAP project [ 17 ]. This allows the users to have "anchor" points to start to use the databases in a more efficient way. Naturally, the names previously used in the corresponding genome programs are kept as synonyms, so that access to the sequences with these names is still allowed. For example, in AeruList, gene rpsA can be accessed directly or using its synonym PA3162 : it is then found downstream of cmk (a context similar to that found in many Gram negative bacteria) and upstream of himD . We are aware that some erroneous identification (or propagation of erroneous identifications) must have occurred in some cases, but we think that this is a trade-off (which will be continuously corrected) for a much more user-friendly usage of the databases. A ' y ' letter starting a gene name indicates that it has not been experimentally identified, nor convincingly identified after in silico analysis yet. We provide curation pages (see below) to help users to correct annotation errors and improve annotation in a continuous way. Functional categories and bioprocesses An important feature for allowing users to explore biological functions is to investigate the genes neighborhoods [ 12 ]. Related functions are often coded by genes in close vicinity in the chromosome. We therefore used the GenoList table for functional categories, that allows the user to make links with the roles of proteins in the cell. The functional classification used in some of the present databases has been created by superimposing the functional classification (ontology) created for SubtiList, and that of Escherichia coli created by Monica Riley and her collaborators [ 18 ] (Additional file). In addition we created a field for the ontology describing underlying bioprocesses: explore, sense, shape, circulate, excrete, replicate, grow, respire, manage energy, store, scavenge, maintain, protect, control. They will be used in the future to color the arrows indicating genes in the picture of the region surrounding a gene of interest, allowing the user, at a glance, to have a rough idea of the processes encoded in the corresponding region. Queries using mining algorithms In addition to using keyword queries or sequence tags (such as molecular mass or isoelectric point of a protein) the database provides a versatile way to identify sequences from the biological knowledge viewpoint. In particular, as in many other databases, it allows the user to use Fasta, BlastP and BlastN to compare a sequence of interest to that of those present in the database. Furthermore, in contrast to most cases, it allows the user to extract information using motifs, that can be either continuous or discontinuous (e.g. finding all proteins with motif CXXCHX 12–25 C). This facility has already, in a quite unobtrusive but efficient way, permitted discovery of many unexpected functions. We have also provided means to explore the beginning and the end of protein sequences, as well as DNA regions upstream of putative operons, computed as strings of genes transcribed in the same orientation and separated by a maximum number of nucleotides (60 nt by default). Automatic updates Genome annotation is continuously updated by scientists all over the world, at a time when new genome sequences appear every three days or so. In order to cope with this enormous flux of information, a facility for browsing automatically new entries in major data libraries has been implemented. In the gene information panel, where each gene of interest is described after being identified as the result of a query (including resulting from a Blast or Pattern search), an "Automatic Blast" link provides a list of updated blast searches against the UniProt library (SWISSPROT+TREMBL). In addition, when the genome belongs to the 'Teraprot' Smith and Waterman Z-score family , the corresponding links (that are statistically much more significant than the results of Blast searches) are provided, allowing the user to look for remote kinships. To discuss the use of the databases we shall restrict our exploration to two databases from the package. LeptoList, that comprises two genomes (each one having two chromosomes) for Bacteria, and CunicuList, that describes the genome sequence and annotation of a small eukaryote. An example: LeptoList LeptoList is the reference database dedicated to the genome of Leptospira interrogans serovar Lai, the paradigm of leptospirosis causative agents [ 19 ]. It is presented together with a second sequence, that of L. interrogans serovar Copenhageni in order to allow easy comparison [ 20 ]. The WWW interface takes into account the fact that L. interrogans has two chromosomes (this feature was not yet displayed in GenoList databases). Using the regular comparison to the CDS to the non-redundant INSD protein database allowed us to suspect that a significant proportion of the short putative CDSs in the genome are likely ORFs and not authentic CDSs. This fits with the recent sequencing of the second Leptospira genome [ 20 , 21 ]. A couple of examples of its use are given here. We looked for counterparts of RRF, the ribosome release factor. In order to find the gene we used a known sequence, from B. subtilis ( frr gene product) and compared it using BLAST with the functionality implemented in LeptoList. This search led to a single gene, LA3295, located downstream of gene pyrH (as in most other bacterial genomes). This synteny is obviously highly significant. In the same way, the gene immediately upstream from pyrH (LA3297), as in other bacteria, is likely to be coding for elongation factor EFTs ( tsf ). When curating the database, we suggest to the curator that it would be of excellent policy to replace the gene numbers by the corresponding gene name. In another type of investigation, looking for patterns of the type TTGACA (1 ambiguity) – 17 nt – TATAAT# (1 ambiguity) (consensus sequence of the σ 70 -type promoter) in the 300 nt region upstream of genes revealed 70 sequences in chromosome I, many of which are likely to be promoters (at least they would be good guesses to start investigating promoters in L. interrogans ). In the same way, the putative DNA binding site located in the 300 nt nucleotide region upstream of genes, TGTGA (1 ambiguity) – 2 nt – KK – 2 nt – TCACA (1 ambiguity) (consensus sequence of the CAP/FNR family of transcriptional regulators), yielded 130 matches in chromosome I of serovar Lai and 72 matches in serovar Copenhageni and 2 in chromosome II of serovar Lai and 0 in serovar Copenhageni, allowing one to start investigating possible regulatory elements. This result is interesting as it suggests that chromosome I genes are submitted to a regulation recognizing that particular DNA-protein binding site. Furthermore, most genes found with the site in serovar Copenhageni are also found in serovar Lai, with sometimes several repeats in the latter, occuring upstream of some genes (such as fadH or prfC ), accounting for the higher total number of putative binding sites in that organism. It seems most interesting that genes involved in the control of respiration (cytochrome c biosynthesis), control of the TCA cycle (pyruvate dehydrogenase synthesis), control of the coupling between translation and transcription (stringent control) or translation itself (release factor 3 synthesis) are present in the list. While there are several putative adenylyl cyclase genes present in the organism, as well as several homologs of crp , it is plausible to propose that cAMP plays an important role in the life cycle of L. interrogans , perhaps suggesting ways to allow multiplication on plates of this elusive organism. LeptoList is accessible at the URL CunicuList: a database for a small eukaryote genome The GenoList structure has been initially constructed for organizing sequence data from prokaryotic genomes. However it may be extended to other organisms as well (the "genomic object" type must be extended accordingly). We have therefore tested the implementation of the structure for the genome of Encephalitozoon cuniculi , belonging to the Microsporidia taxon. Eleven chromosomes are present in this organism. Extraction of information is similar to that from other databases. For example we looked for counterparts of genes involved in tRNA modification (often essential genes). Using MesJ (TilS) [ 22 ] as well as TrmU [ 23 ] we found that gene Ecu03_1240 is most probably involved in driving the codon and amino acid specificity of a tRNA (possibly isoleucine or lysine tRNA). In the same way we could predict that gene Ecu07_1610 codes for synthesis of dihydrouridine in tRNA, a general feature of tRNA structure, because of its similarity with the yacF B. subtilis gene. Looking for counterparts of genes in the methionine salvage pathway [ 24 ], we failed to identify any gene that would code for the enzymes of the pathway, indicating that the parasite obtains all the metabolites derived from S-adenosylmethionine from its host. This is substantiated by the fact that the genes needed to synthesize queuosine [ 25 ] are apparently absent from the genome. Some organisms do not use this major tRNA modification, but this could be an interesting information for identification of drug targets against the parasite, since this suggests that those metabolites have to be transported into the cell by specific permeases. Database curation Several other similar bacterial databases are accessible at URL . Table 1 presents the list of microbial databases that are available at the Bioinfo server of the University of Hong Kong. Table 1 List of databases present at the Bioinfo server The GenoChore suite presented here manage bacterial genome data, except for CunicuList, which presents the sequence and annotation data of the small eukaryote Encephalitozoon cuniculi . AeruList Pseudomonas aeruginosa PA01 EMBL:AE004091 AnthraList Bacillus anthracis str. Ames EMBL:AE016879 CampyloList Campylobacter jejuni NCTC 11168 EMBL:AL111168 CereList Bacillus cereus ATCC 14579 EMBL:AE016877 CholeList Vibrio cholerae EMBL:AE003852 , EMBL:AE003853 CoeliList Streptomyces coelicolor A3(2) EMBL:AL645882 DiphteList Corynebacterium diphtheriae NCTC 13129 EMBL:BX248353 CunicuList Encephalitozoon cuniculi EMBL:AL391737 , EMBL:AL590442 , EMBL:AL590443 , EMBL:AL590444 , EMBL:AL590445 , EMBL:AL590446 , EMBL:AL590447 , EMBL:AL590448 , EMBL:AL590449 , EMBL:AL590450 , EMBL:AL590451 InfluList Haemophilus influenzae Rd KW20 EMBL:L42023 LeptoList Leptospira interrogans Lai str. 56601 EMBL:AE010300 , EMBL:AE010301 Leptospira interrogans Fiocruz L1-130 EMBL:AE016823 , EMBL:AE016824 MeningoList Neisseria meningitidis MC58 EMBL:AE002098 PutidaList Pseudomonas putida KT2440 EMBL:AE015451 SepiList Staphylococcus epidermidis ATCC 12228 EMBL:AE015929 SubtiList Bacillus subtilis str. 168 EMBL:AL009126 ThermaList Thermoanaerobacter tencongensis MB4 EMBL:AE008691 VulnifiList Vibrio vulnificus YJ016 EMBL:BA000037 , EMBL:BA000038 XylelList Xylella fastidiosa 9a5c EMBL:AE003849 Despite of – or because of – the large number of genome programs, once a sequence has been deposited at the INSD, its annotation is seldom updated. This is because the cost of curating annotations is extremely high, and usually not considered, despite its enormous importance. One of our aims was therefore to allow curation by selected teams by creating a curator page where such teams would input their annotations, that would then be propagated to the databases. The basic schema of the curator interface is shown in Figure 2 . In order to preserve the quality of the input data, potential users are asked to write to the database's webmaster to ask for account and passwords. We kept the interface of GenoList as published, except that a box providing access to protected curation of annotations is now provided, aiming initially at helping the first party (sequencing teams) annotators. If this works to our satisfaction this will be extended to selected third party annotators. Subsequently, on a yearly basis (or more frequently if needed) the collected re-annotation of the curators would be submitted as a new version of the same genome to the INSD. We hope that this service will be useful for the scientific community as a whole. Conclusions A set of 17 specialized end-user-oriented microbial databases (including one instance of Microsporidia) has been implemented in Hong Kong. They allow one to browse genome sequence and annotation data using the most frequent queries that end-users would like to ask. In addition they provide a weekly update of searches against the world-wide protein sequences data libraries, allowing one to monitor annotation on genes of interest. Finally, they allow users to search for patterns in DNA or protein sequences present in the databases. All comments, bug reports and suggestions for improvement are more than welcome: this work is meant to be useful for the community of microbiologists interested in genomics. Competing interests The author(s) declare that they have no competing interests. Authors' contributions GF wrote the parsers used to create the preliminary C/C++ MySQL package, and created important sections of the Perl package; CH created the procedure for renaming orthologs with reference to accepted names for model bacteria ( Bacillus subtilis and Escherichia coli ), created the link to Teraprot for identification of gene functions, and implemented parts of the PERL package; YQ implemented parts of the Perl package; CC and VC implemented most of the databases into the core structure; ZY wrote part of the new parsers, implemented the two chromosomes of LeptoList by changing the data structure in the database and set up with CC the first LeptoList database; FC set up and administered the Apache web server and MySQL database; IM over the years designed most of the GenoList data schema and user interface; AD was at the origin of the project, participated in the design and evolution of the data schema, was the systematic tester and end-user and wrote the core of the article. Supplementary Material Additional File 1 Functional categories. The genes' roles are listed into six major categories. The three first ones are directly linked to biological roles, while the remaining categories are created ad hoc : adaptation to atypical conditions correspond to miscellaneous roles, while the two last categories correspond to roles that have not yet been ascribed to genes because of lack of in vivo or in silico data Click here for file

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549560.xml

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Phylogenetic inference in Rafflesiales: the influence of rate heterogeneity and horizontal gene transfer

Background The phylogenetic relationships among the holoparasites of Rafflesiales have remained enigmatic for over a century. Recent molecular phylogenetic studies using the mitochondrial matR gene placed Rafflesia , Rhizanthes and Sapria (Rafflesiaceae s. str.) in the angiosperm order Malpighiales and Mitrastema (Mitrastemonaceae) in Ericales. These phylogenetic studies did not, however, sample two additional groups traditionally classified within Rafflesiales (Apodantheaceae and Cytinaceae). Here we provide molecular phylogenetic evidence using DNA sequence data from mitochondrial and nuclear genes for representatives of all genera in Rafflesiales. Results Our analyses indicate that the phylogenetic affinities of the large-flowered clade and Mitrastema , ascertained using mitochondrial matR , are congruent with results from nuclear SSU rDNA when these data are analyzed using maximum likelihood and Bayesian methods. The relationship of Cytinaceae to Malvales was recovered in all analyses. Relationships between Apodanthaceae and photosynthetic angiosperms varied depending upon the data partition: Malvales (3-gene), Cucurbitales ( matR ) or Fabales ( atp1 ). The latter incongruencies suggest that horizontal gene transfer (HGT) may be affecting the mitochondrial gene topologies. The lack of association between Mitrastema and Ericales using atp1 is suggestive of HGT, but greater sampling within eudicots is needed to test this hypothesis further. Conclusions Rafflesiales are not monophyletic but composed of three or four independent lineages (families): Rafflesiaceae, Mitrastemonaceae, Apodanthaceae and Cytinaceae. Long-branch attraction appears to be misleading parsimony analyses of nuclear small-subunit rDNA data, but model-based methods (maximum likelihood and Bayesian analyses) recover a topology that is congruent with the mitochondrial matR gene tree, thus providing compelling evidence for organismal relationships. Horizontal gene transfer appears to be influencing only some taxa and some mitochondrial genes, thus indicating that the process is acting at the single gene (not whole genome) level.

Background Combining gene sequences from multiple subcellular compartments continues to provide increasingly well-resolved flowering plant phylogenies [ 1 ] and these have precipitated a new classification for angiosperms [ 2 ]. Whereas most groups have been placed at the ordinal level, seven of the 18 "taxa of uncertain position" are holoparasitic, nonphotosynthetic flowering plants. These parasites have been difficult to ally with green plants owing to extreme reduction and/or loss of morphological features [ 3 ]. Chloroplast genes commonly used to infer land plant phylogenetic relationships either show elevated substitution rates or are absent in these holoparasites [ 3 - 5 ]. Moreover, nuclear ribosomal genes also show greatly increased rates [ 6 ], thus analytical methods that accommodate such among-lineage rate heterogeneity must be used. Rafflesiales are a fascinating and enigmatic group of holoparasitic plants that includes Rafflesia , whose meter-wide flowers are the largest among all angiosperms, and Pilostyles , whose flowers are less than a centimeter in diameter. Such wide morphological variation has resulted in classifications that comprise four families: 1) the "small-flowered clade" (Apodanthaceae) with Apodanthes , Berlinianche , and Pilostyles , 2) the "large-flowered clade" (Rafflesiaceae s. str.) with Rafflesia , Rhizanthes , and Sapria , 3) the "inflorescence clade" (Cytinaceae) with Bdallophyton and Cytinus , and 4) the "hypogynous clade" (Mitrastemonaceae) with Mitrastema [ 7 , 8 ]. Recently, Barkman et al. [ 9 ] used DNA sequences of the mitochondrial gene matR to identify the closest photosynthetic relatives of two clades within Rafflesiales. Three genera, representing two of the four families in the order, were used in that study: Rafflesia and Rhizanthes (Rafflesiaceae s. str.) and Mitrastema (Mitrastemonaceae). Analyses of the matR data placed Rafflesiaceae s. str. within Malpighiales, an order that includes passionflowers ( Passiflora ), willow ( Salix ), and violet ( Viola ). Mitrastemonaceae was placed within Ericales, an order containing blueberries ( Vaccinium ), primroses ( Primula ), and tea ( Camellia ). The authors argued that these results were robust because they were congruent using different analytical methods (parsimony, neighbor-joining, Bayesian) and were not affected by long-branch attraction artifacts [ 10 ]. Moreover, because sequences from host plant lineages were included, and the parasites did not emerge as sister to these lineages, contamination and horizontal gene transfer (HGT) were discounted. In this study we expand upon the previous analysis [ 9 ] by including representatives of all Rafflesiales genera and families, thus allowing us to address the question of monophyly of the order. Moreover, parsimony, likelihood and Bayesian analyses were conducted on genes derived from all three subcellular compartments. These results were compared to assess the impact of artifacts such as long-branch attraction and HGT on various relationships. The data sets used were 1) mitochondrial matR , 2) mitochondrial atp1 and 3) a "3-gene" data set consisting of nuclear SSU rDNA plus two chloroplast genes: rbcL and atpB (the latter two only from nonparasites). Results Maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) analyses of mitochondrial matR resulted in trees congruent with each other and with those previously generated [ 9 ] (Figure 1 and additional data file 1 ). As shown on the ML tree (Figure 1 ), Rafflesia , Rhizanthes , and Sapria were placed with strong support in Malpighiales. Mitrastema was placed in Ericales sister to Vaccinium . The Cytinus and Bdallophyton clade (Cytinaceae) was strongly supported and this clade was sister to one composed of four genera of Malvales, an order that contains cotton ( Gossypium ), rockrose ( Cistus ) and chocolate ( Theobroma ). For Apodanthaceae, Apodanthes and Pilostyles were sister taxa and derived from within Cucurbitales, an order that contains squash/pumpkin ( Cucurbita ) and Begonia . For Berlinianche , sequences homologous to matR could not be obtained using several primer combinations. Figure 1 ML strict consensus tree from mitochondrial matR . Strict consensus of two trees obtained from ML analysis of the 77-taxon mitochondrial matR matrix. Clades with Bayesian posterior probabilities between 0.9 and 1.0 are indicated by thick lines. Bootstrap percentages from MP analysis shown above lines. Rafflesiales taxa are shown in bold italics. Arrow represents a putative cases of horizontal gene transfer. The small phylogram is included to demonstrate branch length heterogeneity. All three analytical methods of the atp1 data produced trees that were generally congruent, thus the ML tree is illustrative (Figure 2 , additional data file 2 ). Clades among the monosulcates generally follow previously reported relationships, whereas the topology of the eudicot portion of the tree does not clearly reflect accepted clades, possibly owing to poor sampling within rosids and asterids (sequences for these taxa were not available from GenBank). Despite these shortcomings, this gene provides additional evidence useful in assessing the phylogeny and molecular evolution of Rafflesiales. With all three analytical methods, Mitrastema forms a clade with Beta (Caryophyllales), although this relationship does not receive strong support. This is remarkable given that 15 taxa from Ericales were included, yet a relationship with this order (as seen with matR ) was not obtained with atp1 . The large-flowered clade was strongly supported as monophyletic in all analyses, however, its position within the eudicots did not receive strong support. Parsimony analysis placed Pilostyles as sister to Pisum (Fabales) and this clade was sister to Berlinianche , but both with low bootstrap support. Apodanthes was strongly suported (90% bootstrap) as sister to Polemonium (Ericales) with MP but with ML this long-branch clade received lower support (Figure 2 ). The two genera of Cytinaceae, Cytinus and Bdallophyton , were sister to Malvales, with moderate (MP) to strong (BI) support. Figure 2 ML tree from mitochondrial atp1 . Phylogram obtained from ML analysis of the 71-taxon mitochondrial atp1 matrix. Clades with Bayesian posterior probabilities between 0.9 and 1.0 are indicated by thick lines. Rafflesiales taxa are shown in bold italics. Note that the clade with Apodanthes and Polemonium (asterisk) is poorly supported with a posterior probability of 0.54. Maximum parsimony analyses of the full-length (103 taxon) and reduced (77 taxon) 3-gene matrices were generally congruent and both resulted in all taxa of Rafflesiales being associated with Malvales (Figure 3 ), although with low bootstrap support for the monophyly of this clade. The two accessions of Pilostyles were sister to a clade composed of Pavonia and Gossypium , also with low bootstrap support. In constrast, BI analysis of the 3-gene matrix placed Mitrastema with Ericales and the large-flowered clade was a component of Malpighiales, the latter with strong support. The inflorescence clade ( Cytinus and Bdallophyton ) and the small-flowered clade ( Pilostyles ) were allied with Malvales (see additional data file 3 ), although posterior probablilities of this association were lower. Figure 3 Unconstrained MP tree from the 3-gene data matrix. Strict consensus of 12 trees obtained from an unconstrained maximum parsimony analysis of the 77-taxon "3-gene" matrix (nuclear SSU rDNA, rbcL , atpB ). Bootstrap support is shown above the lines. Rafflesiales taxa are shown in bold italics. Parsimony analysis of the nuclear SSU rDNA matrix, constrained to an accepted topology for nonparasites, showed the same pattern of relationships as the unconstrained 3-gene MP analysis, i.e., all Rafflesiales taxa were associated with Malvales (see additional data file 4 ). In contrast, the tree (Figure 4 ) resulting from ML analysis using the same constraint tree showed the same relationships as the BI tree for the 3-gene data set. Figure 4 Constrained ML tree from nuclear SSU rDNA. Tree resulting from the constrained ML analysis of the 77-taxon nuclear SSU rDNA matrix. Rafflesiales taxa are shown in bold italics. None of the consensus trees generated from MP analysis of the 100 nuclear SSU rDNA data sets simulated on 20-taxon trees matched the topology of the model tree. 58 of the 100 MP consensus trees showed a Mitrastema + Rafflesia/Rhizanthes/Sapria clade and 17 showed a Bdallophyton/Cytinus + Rafflesia/Rhizanthes/Sapria clade (Figure 5 ). Two other combinations, Bdallophyton/Cytinus + Pilostyles and Bdallophyton/Cytinus + Mitrastema + Rafflesia/Rhizanthes/Sapria accounted for 6% and 2% of the MP consensus trees, respectively. Thus, 83% of the MP trees contained incorrect clades, and most of these can be attributed to the long-branch Rafflesia clade. However, only two of the 100 MP trees showed all six long-branch taxa as monophyletic, a result seen on the original MP tree for the full 77-taxon data set. Results of parsimony analyses of data sets simulated on the full 77-taxon tree showed a similar pattern – 58 of the MP consensus trees showed a Mitrastema + Rafflesia/Rhizanthes/Sapria clade, 7 showed a Bdallophyton/Cytinus + Rafflesia/Rhizanthes/Sapria clade, and 14 showed a Bdallophyton/Cytinus + Pilostyles clade (Figure 5 ). In other words, MP returned an incorrect "long-branch" clade for 79% of the data sets simulated on the full 77-taxon model tree. In contrast, far fewer incorrect long-branch clades were recovered by ML for the 20-taxon simulations, and most (56%) ML trees matched the model tree in that the Rafflesia clade was sister to Passiflora , Mitrastema was sister to Helianthus/Nicotiana , and Pilostyles , Bdallophyton and Cytinus were associated with Gossypium . Figure 5 Rafflesiales long branches mislead MP. Proportion of simulated data sets (replicates) for which incorrect "long-branch" clades are recovered in maximum parsimony (black bars, 77 taxa), maximum parsimony (grey bars, 20 taxa), and maximum likelihood (open bars, 20 taxa) analyses. Inset is the model tree used to generate the simulated data sets. M = Mitrastema , B = Bdallophyton + Cytinus , R = Rafflesia + Rhizanthes + Sapria , P = Pilostyles . MP analyses of SSU data sets from which all but one parasite group had been removed resulted in phylogenetic placements that matched those found in the ML tree. MP analysis of a data set from which all Rafflesiales except Mitrastema had been removed resulted in trees that placed Mitrastema in Ericales. Removal of all parasites except Pilostyles or Bdallophyton + Cytinus individually placed both of these groups in Malvales. Finally, removal of all parasites except the large-flowered clade ( Rafflesia , Rhizanthes and Sapria ) placed this clade in Malpighiales. Thus, the positions of the parasite clades inferred in four separate MP analyses matched the positions found for these clades in the single ML tree. Discussion Rate heterogeneity and long-branch attraction artifacts Determining the photosynthetic relatives of Rafflesiales has long presented a challenge owing to the extreme reduction and/or modification of morphological structures that have accompanied the evolution of this lineage [ 3 , 11 ]. Molecular phylogenetic approaches, although providing great promise in resolving such questions, also come with their own set of challenges that includes losses of some genes, substitution rate increases in other genes, and horizontal gene transfer. Examples of the first process can be seen in chloroplast genes such as rbcL that are typically used to infer phylogenetic relationships among angiosperms but have not yet been amplified from any Rafflesiales and are presumed lost [ 5 ]. Increased substitution rates in the normally conservative plastid rDNA has been demonstrated in these holoparasites [ 4 , 12 ]. Similarly, accelerated rates in mitochondrial SSU rDNA, typically very conservative in many photosynthetic angiosperms, occur in Rafflesia and Cytinus [ 13 ]. Despite these complications, molecular phylogenetic analyses of some holoparasite lineages with comparatively lower rates have been tractable. For example, the mitochondrial genes atp1 and matR were used, in combination with nuclear rDNA and chloroplast genes, to reliably place Hydnoraceae with Aristolochiaceae [ 11 ]. Long-branch attraction, a bias in certain phylogenetic inference methods in which similarity due to convergent or parallel changes produces an erroneous phylogenetic grouping of taxa [ 10 ], is often implicated as the reason for anomalous phylogenetic groupings [ 14 ]. It has been suggested that some data sets with marked among-lineage rate heterogeneity cannot be applied to particular phylogenetic problems owing to hypothesized long-branch attraction artifacts [ 15 ]. In their unconstrained parsimony analysis of several angiosperm SSU rDNA sequences, Barkman et al. [ 9 ] found that the branch leading to Rafflesia was several times longer than any other branch, and that this branch was attracted to the second-longest branch in the tree – the one between gymnosperms and angiosperms. For these reasons, they argued that nuclear SSU rDNA sequences are of limited utility for assessing the phylogenetic position of Rafflesia . Barkman et al. [ 9 ] analyzed their SSU rDNA data using only parsimony, not model-based methods (e.g., ML or BI methods) that are less likely to be misled by long-branch attraction [ 16 ]. Our ML analysis of the SSU rDNA data recovers a topology that closely matches the matR topology presented by Barkman et al. [ 9 ] in which Rafflesia is closely related to Malpighiales and Mitrastema is a member of Ericales (Figure 4 ). These results highlight the requirement to analyze SSU rDNA data with methods less biased by long-branch attraction than parsimony, as well as the advantage gained by independent confirmation of results obtained from a single gene. Several authors have suggested that adding taxa can "break up" long branches and allow parsimony to recover the correct topology [ 17 - 19 ]. Our parsimony analysis of the 103- and 77-taxon SSU rDNA data sets, in which we included representatives of all genera of Rafflesiales (i.e., sequences that could potentially break the Rafflesia long branch), recovers a nearly monophyletic Rafflesiales containing all of the longest terminal branches in the tree (see additional data file 3 ). Based on our simulation study and MP analyses of data sets from which all but one parasite group was removed, we believe that this topology represents a case of long-branch attraction. These simulation results support the contention that the branches leading to the parasitic taxa are long enough to attract one another (Figure 5 ), a result in agreement with previous work [ 3 , 6 ]. Taxon sampling is not a cure-all for long-branch attraction problems [ 20 ]. Even for the data sets simulated on the full 77-taxon tree, MP returned incorrect long-branch clades nearly 80% of the time. MP did nearly as poorly with data sets simulated on a 77-taxon tree as it did on data sets simulated on a 20-taxon tree. Evaluation of the ML tree for the SSU data (Figure 4 ) shows that increasing the number of taxa from 20 to 77 did not improve the result because the long parasite branches were not broken. Instead, shorter (nonparasite) branches were broken which did not help MP recover the true topology for the simulated data sets. MP analyses of the full 77-taxon SSU data set that included all parasite clades resulted in a worse estimate of the phylogeny than MP analyses of smaller data sets in which only single parasite clades were included. Thus, the frequently stated view that increased taxon sampling can help MP avoid long-branch attraction problems may only be true if the added taxa are not distantly related long-branch clades themselves. Phylogenetic relationships of the four Rafflesiales clades Rafflesiaceae (the large-flowered clade) The results from analyses of Rafflesiales using independent data sets are summarized in Table 1 . For Rafflesiaceae s. str., placement in Malpighiales is supported by ML and BI analyses of the 3-gene and nuclear SSU rDNA data sets as well as mitochondrial matR . This placement in Malpighiales is also supported by a molecular phylogenetic study that used a single copy nuclear gene phytochrome C [ 21 ]. These authors proposed that Rafflesiaceae are most closely related to Ochnaceae or Clusiaceae which contrasts with presumed synapomorphies with Passiflora given by Barkman et al. [ 9 ]. Within Malpighiales, tremendous morphological diversity exists among the 27 families and 16,000 species. Moreover, relationships among the major clades are still poorly resolved [ 22 ]. Although the evidence for a malpighialean affinity of Rafflesiaceae appears strong, it is possible that the molecular data have only identified the stem group that represents the sister to the parasitic lineage. Table 1 Summary of phylogenetic analyses of Rafflesiales using different data partitions and methods of analysis. 3-Gene* 3-Gene nuSSU rDNA nuSSU rDNA matR matR atp1 atp1 Parsimony Bayesian Parsimony constrained Likelihood constrained Parsimony Likelihood & Bayesian Parsimony Likelihood & Bayesian Mitrastema Malvales Ericales Malvales Ericales Ericales Ericales Caryophyllales Caryophyllales Cytinus Malvales Malvales Malvales Malvales Malvales Malvales Malvales Malvales Bdallophyton Malvales Malvales Malvales Malvales Malvales Malvales Malvales Malvales Apodanthes N/A N/A N/A N/A Cucurbitales Cucurbitales Polemonium Polemonium Pilostyles Malvales Malvales Malvales Malvales Cucurbitales Cucurbitales Fabales Fabales Berlinianche N/A N/A N/A N/A N/A N/A Ericales/Fabales Ericales/Fabales Rafflesia Malvales Malpighiales Malvales Malpighiales Malpighiales Malpighiales Eudicots Eudicots Rhizanthes Malvales Malpighiales Malvales Malpighiales Malpighiales Malpighiales Eudicots Eudicots Sapria Malvales Malpighiales Malvales Malpighiales Malpighiales Malpighiales Eudicots Eudicots *Nuclear SSU rDNA plus chloroplast rbcL & atpB . Possible HGT events Long-branch artifact Barkman et al. [ 9 ] suggested that the floral similarities between Rafflesia and Passiflora , first noted by Robert Brown [ 23 ] represent morphological synapomorphies that support the results obtained from the matR gene tree. Arguments in favor of a number of other, equally credible relationships within eudicots could be made based on hypothetical evolutionary transformation series of morphological characters. Indeed Brown concluded that Rafflesia may have affinity with Passifloraceae (Malpighiales) but he also considered other groups such as Aristolochiaceae ("Asarinae", Piperales), Sterculiaceae (Malvales) and Cucurbitaceae (Cucurbitales). In general, different characters supported relationships with one or another group and therefore he left the subject as unresolved. Three proposed synapomorphies between Passifloraceae and Rafflesia were cited by Barkman et al. [ 9 ]: a hypanthium (perigone tube in Rafflesia ), an androgynophore (gynostemium or column in Rafflesia ), and an annular corona (diaphragm in Rafflesia ). Whether these structures are homologous is not clear and will likely require further morphological studies, possibly examining the floral development genes themselves. These hypotheses require scrutiny because the apparent similarities in structure are not clear when examined in detail. For example, the androgynophore of Passiflora is composed of a stalk that bears the androecium and gynoecium. In Rafflesia , the ovary is inferior (with no stalk), hence the central column must involve other gynoecial parts. The corona of Passiflora is very different in structure and function from the diaphragm of Rafflesia [ 24 ]. The observation of a physical union between Passiflora caerulea and Euonymus [ 25 ] was discussed by Barkman et al. [ 9 ] as a possible clue to the origin of parasitism in Rafflesia . Whether this association represents parasitism or not is a matter of semantics [ 26 ], for other similar associations exist such as Cissus and Opuntia growing on Yucca and Opuntia on Cercidium and Idria . In all of these cases, a true haustorium does not form and more likely these represent forms of grafting. It is difficult to state whether such rare occurrences have any bearing on the origin of parasitism in Rafflesiales or other parasitic flowering plants. Mitrastemonaceae (the hypogynous clade) Maximum likelihood and Bayesian analyses of the 3-gene and nuclear SSU rDNA data partitions placed Mitrastema in Ericales, a result congruent with that obtained using mitochondrial matR . As noted by Barkman et al. [ 9 ], this relationship within the asterids had not previously been proposed. Mitrastema has bisexual, protandrous flowers with a collar-shaped, four-merous perianth tube. The stamens are connate into a tube (androphore) crowned by a fertile zone of pollen-bearing locules. The staminal tube, open at the top by a small hole, circumscissally separates from the flower as it is pushed up by the growing gynoecium. The apical portion of the staminal tube is sterile, but below this is a series of vertical rings of ca. ten minute, pollen sacs each. The gynoecium is hypogynous, one-locular, with a thick, conical stigma. Placentation is parietal with 8–15 (-20) unequal placental lobes filling the locule. The numerous ovules are small (190 by 120 μm), anatropous, unitegmic (but with two cell layers), and tenuinucellar. Although some floral morphological features of Mitrastema are not in conflict with those seen in Ericales, such as extrorse anthers and cellular endosperm, features such as decussate leaves, circumscissile fruit dehiscence, and parietal placentation are too general to draw specific associations. Given that Mitrastema is an achlorophyllous holoparasite and that one clade of Ericaceae (Monotropoideae) contains achlorophyllous mycotrophs, it is intriguing to ask whether these groups share a common ancestor or evolved independently. The most specialized morphological feature found in Mitrastemonaceae, the athecal androecium, is not found in Ericales but in Malvaceae, the only angiosperm family that shows the entire gamut from taxa with normal stamens, to taxa with stamens deviating only slightly from the common pattern [ 27 , 28 ], to athecal androecia [ 29 ]. Cytinaceae (the inflorescence clade) The most consistent phylogenetic signal that is seen across all data sets and types of analyses is a relationship between Cytinaceae and Malvales (Table 1 ). Because the relationship between Cytinaceae and Malvales is the strongest among all four Rafflesiales clades, it is possible that this clade acts as an "attracter" for the other three Rafflesiales clades in some analyses. This is seen when using nuclear SSU rDNA sequences, either alone or with the topology of the tree stabilized through the addition of two chloroplast genes. In both cases, parsimony produces a monophyletic Rafflesiales within Malvales which contrasts with the result seen with the constrained ML SSU rDNA and the matR results. These results and those obtained from the simulation study indicate that the large-flowered clade and Mitrastema are artifactually attracted to Cytinaceae when parsimony is utilized. Unlike other Rafflesiales, members of Cytinaceae have multiple flowers arranged in an inflorescence. The floral structure called the diaphragm, seen in Rafflesia and Sapria (but not Rhizanthes ), is lacking in Cytinaceae. Bdallophyton is dioecious and Cytinus is either dioecious ( C. capensis , C. sanguineus ) or monoecious ( C. hypocistis ). The perianth is tubular, composed of four to nine imbricate organs. The androecium is connate, forming a compact synandrium with extrorse anthers and the pollen is 2-, 3-, or 4-porate. The female flower is epigynous with a columnar style terminated by a globose or capitate, viscous stigma with commissural lobes [ 30 ]. The ovary is unilocular with 8–14 deeply intrusive, discrete parietal placentae that bear numerous, orthotropous, tenuinucellate ovules. Apodanthaceae (the small-flowered clade) Maximum parsimony and likelihood analyses of the 3-gene data set and nuclear SSU rDNA sequences alone also place Pilostyles (the only Apodanthaceae for which SSU rDNA sequences are available) within Malvales, however, a sister relationship with Cytinaceae is not consistently obtained. A 3-gene alignment that included additional representatives of Malvales (16 taxa) gave similar results as shown in Figure 3 (i.e., Pilostyles on a clade separate from other Rafflesiales). These data, in conjunction with the results from the mitochondrial genes, support an evolution of Apodanthaceae independent from Rafflesiaceae s. str. The well-supported relationship between Pilostyles and Apodanthes using matR is expected given their very similar floral morphology [ 31 ], yet this clade is sister to two representatives of Cucurbitales ( Begonia and Cucurbita ). Contamination with host tissue is excluded because neither parasite is known to currently occur on a member of Cucurbitales. Apodanthaceae are grouped with Pisum (Fabales) and Polemonium (Ericales) on the atp1 tree, but no atp1 sequences from representatives of Cucurbitales were available from GenBank to test the matR result. The sister relationship between Apodanthes and Polemonium is strongly supported on the MP tree (bootstrap support value = 90%; additional data file 3 ), but this pairing must be viewed with caution given the low Bayesian posterior probability of the clade (0.54) and that both taxa are very long branches (Figure 2 ). Although ML is less susceptible to long-branch attraction artifacts than MP, it is not immune to it; thus, it remains unclear whether or not this relationship is artifactual. Moreover, the Polemonium sequence is separate from the clade containing 12 other members of this order, thus raising the possibility that the sequence results from contamination or HGT (see below). Additional sampling within the eudicots will be required to better understand the atp1 gene tree topology. Morphological features shared between Apodanthaceae and Cytinaceae are: unisexual flowers, a connate androecium, an inferior ovary, and a unilocular ovary with four parietal placentae bearing numerous, anatropous, tenuinucellate ovules [ 30 , 31 ]. Floral morphological features that might link Apodanthaceae and Cytinaceae with Malvales [ 31 ] include an androecial tube (e.g., Malvaceae), a trend toward synandria without anthers and thecae (e.g., Malvaceae) [ 29 ], tri- to hexamerous flowers (e.g., Thymelaeaceae), and parietal placentae (e.g., Cistaceae). The floral conditions of unisexuality and epigyny do occur in Malvales, albeit rarely. Unisexual flowers pose some difficulties for interpreting the morphological homologies of various floral organs. For Pilostyles and Apodanthes male flowers, a tubular synandrium surrounds and fuses with a central structure that could be interpreted as a sterile gynoecium. Support for the concept that such a central structure is a pistillode comes from Berlinianche where the upper portion of the synandrium is free from the central part. In female flowers of Apodanthaceae, there is no rudiment of an androecium, hence the central tissue is apparently entirely gynoecial. In contrast to the above discussion, the matR data indicate Apodanthaceae are related to Cucurbitales, an order with seven families, 129 genera and 2300 species. Hosts for Apodanthaceae are generally legumes, although Apodanthes occurs most frequently on Casearia (Salicaceae, Malpighiales). Thus, neither recent HGT nor contamination explains this result. Apodanthaceae shares some morphological features with members of Cucurbitaceae, subfamily Cucurbitoideae: unisexual, five-merous flowers ( Berlinianche ); carpellate flower with a unilocular, inferior ovary with parietal placentation; anatropous, bitegmic ovules; staminate flower with connate filaments (monadelphous) and a rudimentary gynoecium (pistillode) [ 32 ]. Conflicting characters also occur, such as a three-carpellate gynoecium in Cucurbitoideae (vs. four-carpellate in Apodanthaceae) and a valvate perianth (vs. imbricate). All of these characters, however, are less specialized than those shared between Apodanthaceae and Malvales. Background on horizontal gene transfer A requirement of the molecular phylogenetic approach to inferring evolutionary histories of organisms is vertical transmission of genetic material from parent to offspring. In contrast, horizontal gene transfer (HGT) describes the movement of genetic material between organisms of no direct ancestor-descendant relationship. Although the frequency of HGT is currently not well understood among prokaryotic and eukaryotic organisms, it is clear that HGT can compromise accurate inference of genealogical history. In plants, lateral movement of genetic material has been documented for mobile genetic elements such as introns [ 33 - 37 ] but only recently has convincing evidence emerged documenting HGT of mitochondrial genes [ 38 , 39 ]. Genes of the mitochondrion are extensively used to infer evolutionary relationships in plants [ 40 - 42 ], thus highlighting the importance of characterizing the frequency of HGT across genes and taxa. Incongruence among gene trees derived from different data sets can derive from a number of factors such as technical causes (insufficient data, gene choice, sequencing error, taxon sampling and identification), gene/genome-level processes, and organism-level processes (e.g., hybridization/introgression, lineage sorting, and HGT) [ 43 ]. HGT has only recently been recognized as a potentially important force in the evolution of plant mitochondrial genomes and detecting HGT is highly dependent upon the presence of multiple gene data sets with robust taxon sampling [ 38 , 39 ]. Evidence for horizontal gene transfer in parasitic plants We believe that incongruence between the the mitochondrial and the nuclear gene trees (Table 1 ) stem not just from long-branch attraction artifacts but also from cases of HGT. The placement of Apodanthes and Pilostyles on the atp1 tree as sister to Pisum (a legume, the family of hosts for Pilostyles ) represents a likely case of HGT. The atp1 data conflict with those from matR that associates Apodanthaceae with Cucurbitales. Moreover, we infer that the SSU rDNA tree better represents the organismal phylogeny because it seems less likely that nuclear genes would be influenced by HGT [ 44 , 45 ]. The main rationale for this is that nuclear rDNA cistrons are repeated hundreds to thousands of times in tandem arrays at nucleolar organizing regions of the chromosomes. Although it can be envisioned that concerted evolution could homogenize all rDNAs in the parasite with a form obtained via HGT, the probability of this happening is small given the vastly different number of starting copies. In their study of Rafflesiaceae s. str. and Mitrastemonaceae, Barkman et al. [ 9 ] discounted HGT as a possible explanation for their results because they state the phenomenon is rare and the overall topology of the matR tree closely matched results from other molecular phylogenetic investigations of angiosperms. The present study confirms that HGT is not implicated for the two lineages studied by Barkman as well as Cytinaceae, but this process could be invoked for Apodanthaceae. More recent work by these authors [ 46 ] identified several cases of HGT from host to parasite for atp1 . These included Dalea to Pilostyles , Tetrastigma to Rafflesia , and Lithocarpus to Mitrastema . In addition, HGT of another mitochondrial gene, nad1 , has been reported for Rafflesia and Sapria , both of which occur on the same clade as their hosts ( Tetrastigma ) on a gene tree [ 20 ]. These examples demonstrating the presence of host genes in parasitic plants provide the most compelling evidence for HGT. This form of transfer is intuitively logical given the intimate contact between cells of the two organisms via the endophytic haustorium. However, parasitic plants exist in complex ecosystems where they are in physical contact with many other organisms (bacteria, fungi, phytophagous and pollinating animals, etc.) that could potentially affect HGT. That such nonhost HGT may also be occurring is evidenced by the presence of an apparent cucurbitalean matR gene in Pilostyles and Apodanthes . Moreover, present-day hosts of parasitic angiosperms do not represent the only conduit for HGT if host choice has shifted through time as the parasite lineage evolves. For example, Barkman et al. [ 9 ] state that Mitrastema only parasitizes Fagales (e.g., Lithocarpus and Castanopsis , both Fagaceae) but this parasite has also been recorded from Aquifoliaceae, Asteraceae, Elaeocarpaceae, Juglandaceae, and Myrtaceae [ 47 ]. Host latitude for this species would be broader if rare hosts and hosts of parasite ancestors were fully known, thus expanding the taxonomic spectrum of potential HGT sources. Formidable contamination issues Contamination of parasite DNA with DNA from the host plant is an issue that must be given serious attention. Indeed, two sequences shown on the matR tree (Figure 1 ), Tetrastigma2 and Julbernardia are hosts for Rafflesia tuan-mudae and Berlinianche , respectively. These sequences were obtained by PCR amplification and sequencing from what was originally thought to be pure parasite genomic DNA. Sequences of the host (obtained from separate samples) were found to be identical to these "parasite" sequences, strongly suggesting contamination. In the case of Rafflesia , the DNA was obtained from a bud still attached to the host vine, both of which had been sectioned longitudinally. Disruption of these tissues likely resulted in transfer of host sap to the bud region where the tissue was sampled. Other samples of R. tuan-mudae from the same population, obtained as floral bracts with no host tissue, resulted in matR sequences that were similar to the other two Rafflesia species. For Berlinianche , whose flowers are much smaller than those of Rafflesia (5 mm in diameter), extreme care (using a stereo microscope) was exercised to remove floral parts devoid of any host tissue. Despite this, the matR sequence obtained from the first sample was that of the host, Julbernardia . Later, silica gel dried samples of other populations of the parasite were extracted, again using extreme care in avoiding host contamination. PCR products were obtained using several mitochondrial matR primers, but none were found to be homologous to this gene following BLAST searches. This result shows that host DNA was not present in this sample in sufficient amounts to amplify and that the parasite matR gene, if present, is highly divergent at the priming sites used. For all three Apodanthaceae genera, the conical style in female flowers is papillate and heavily secretory [ 31 ]. This sticky surface tends to capture a variety of environmental debris, likely including extraneous pollen, fungal spores, and host tissues that have been disrupted upon collecting. Obtaining a proper nuclear SSU rDNA sequence for Pilostyles was extremely difficult. Despite PCR products of the correct sizes using a variety of primer combinations, the sequences obtained from genomic DNA derived from flowers were deemed contaminants following BLAST searches that showed them to be most similar to monocots, fungi, etc. Only when sequences from two accessions of Pilostyles (Texas and California) both were most similar to Malvales was this considered good evidence for their true phylogenetic affiliation. Retrospectively, it is likely that the sticky flowers had accumulated wind-dispersed pollen (e.g., grasses) and that this DNA, despite being in low concentration, had less divergent priming sites than the parasite target DNA, allowing PCR to preferentially amplify the contaminant DNA. The mechanism of horizontal gene transfer: some considerations Given the accumulating molecular evidence for HGT from host to parasitic plant, it is worthwhile to consider potential mechanisms, along with their constraints, that may suggest further research. Relatively little information exists on the structure of the endophyte of Rafflesiales. Ultrastructural studies have been conducted on two species of Pilostyles : P. hamiltonii [ 48 ] and P. thurberi [ 49 ]. These authors conflict, however, as to whether there exists symplastic continuity between host and parasite via plasmodesmata; the former indicated that such connections are the major path of nutrient uptake by the parasite whereas the latter rejected this idea. Despite this controversy, heteroplastic plasmodesmatal connections have been documented in another parasitic plant, Cuscuta [ 50 ] and indeed such connections can even form in heterografts between distantly related plant taxa [ 51 ]. Given this, we assume that host-parasite plasmodesmatal connections exist in the endophytes of Rafflesiales. Transmission electron micrographs of Pilostyles suggest that intact, mature mitochondria are too large to pass through heteroplastic plasmodesmata, however, mitochondrial genomes or portions of the genome are certainly small enough for transmission. Once inside the parasite cell, there are various fates for the host gene. It could become incorporated into the parasite mitochondrial genome, and then either replace the parasite copy or exist as a duplicate, or the host gene could reside in the parasite nuclear genome. For the latter case, the gene would likely become a pseudogene given the requirement of mitochondrial-specific patterns of RNA editing. Two forms of atp1 are present in the primitive angiosperm Amborella trichopoda [ 38 ], one of which is derived from a HGT event from a eudicot. It is not known whether both forms of the gene exist in a single mitochondrial genome, in different mitochondrial genomes within the cell (i.e., heteroplasmy), or if one is nuclear and the other mitochondrial. Future work to address these questions would involve sequencing flanking regions of purported horizontally transferred genes to determine their subcellular location. Additionally, cDNA sequences obtained from matR mRNA would be useful to determine whether the gene is expressed and whether mitochondrial-specific RNA editing patterns are present. Conclusions In this study we have used data derived from nuclear, mitochondrial and chloroplast DNA and a variety of analytical approaches to address long-standing questions about the holoparasitic flowering plant order Rafflesiales. We show that Rafflesiales are not monophyletic but composed of at least three and possibly four independent lineages. Rafflesiaceae ( Rafflesia , Rhizanthes , and Sapria ) representing the large-flowered clade are monophyletic and are related to Malpighiales. The monogeneric family Mitrastemonaceae, the only member of the order with a superior ovary, is related to Ericales. The first of the remaining two families that have previously not been sampled is Cytinaceae ( Bdallophyton and Cytinus ) which is strongly supported as a member of Malvales. The last remaining unsampled family, Apodanthaceae ( Apodanthes , Berlinianche , and Pilostyles ) is either related to Malvales or Cucurbitales. Our simulation studies indicate that Mitrastema , Bdallophyton/Cytinus , and Rafflesia/Rhizanthes/Sapria have branches that are long enough to mislead parsimony. All of these relatively long branches appear to be attracted toward the Cytinaceae clade within Malvales. When nuclear SSU rDNA sequences are analyzed with ML, results fully congruent with those previously reported for two Rafflesiales clades using mitochondrial matR are obtained. If the phylogenetic affinityof Apodanthaceae are with Malvales, the results from the mitochondrial matR gene must represent a case of horizontal gene transfer (HGT) from Cucurbitales. If this proves to be the case, this provides an example of HGT from a nonhost plant to a parasitic angiosperm. To properly discern phylogenetic relationships in enigmatic parasitic taxa, our results demonstrate the need for robust taxon sampling, gene sequences from multiple subcellular compartments, and the use of analytical methods that accommodate rate heterogeneity and avoid the pitfalls of long-branch attraction. When the phylogenetic relationships among such holoparasitic taxa are poorly known, the strongest phylogenetic signal that can be obtained is congruence among results derived from independent sources (i.e., genes from different subcellular compartments). Comparisons among gene trees allows for the identification of HGT, a phenomenon that requires further investigation to determine its modes of action and frequency among taxa and through evolutionary time. Methods DNA extraction, PCR, sequencing DNA was extracted, amplified, cloned, and sequenced by using methods formerly reported [ 52 - 54 ]. The nuclear and mitochondrial sequences were PCR-amplified using primers reported elsewhere [ 6 , 55 , 56 ] and are also given on the first author's web site [ 57 ]. Sequencing was conducted using manual and automated methods (ABI Prism ® 377 automated DNA sequencer, Applied Biosystems) according to manufacturer's protocols. DNA alignments The initial matR alignment incorporated all of the Rafflesiales parasites and the nonparasite sequences previously published [ 9 ] as well as our newly generated sequences. The 106-taxon matrix represented over 40 orders and contained three gymnosperm outgroup taxa ( Ginkgo , Pinus , and Zamia ), 28 monosulcates, 63 nonparasitic eudicots, and 15 Rafflesiales. For two taxa ( Mitrastema and Rhizanthes ), our sequences, as well as those previously published, were from the same species but different accessions to test for consistency. Taking into account codon information, an alignment of 2177 sites was constructed manually using SeAl version 2.0 [ 58 ]. The full matrix was used for parsimony analyses whereas another, truncated to 77 taxa by removing all but three monosulcate taxa (Laurales used as outgroup), was constructed to facilitate likelihood analyses. This operation was justified because monosulcates were never implicated as relatives of Rafflesiales in any analyses. A 71-taxon, 1265-site atp1 alignment was similarly constructed and included the same gymnosperm outgroup genera as above, 24 monosulcates, 32 nonparasitic eudicots and 12 Rafflesiales. All of the monosulcate genera in the atp1 alignment were also represented in the matR data set, whereas eudicot sampling for atp1 was constrained by sequences available on GenBank (12 of the same genera as with matR or placeholders from same family). To test the position obtained for Rafflesiales taxa using mitochondrial genes with an independent data set derived from different compartments, a 4646-site "3-gene" matrix combining sequences from nuclear SSU rDNA and chloroplast rbcL and atpB was constructed that included 103 taxa (3 gymnosperms, 28 monosulcates, 58 nonparasitic eudicots, and 14 Rafflesiales). Sampling across angiosperm orders was very similar to the matR matrix, differing only by the presence of 11 placeholders and a second accession of Pilostyles . For the holoparasites, only nuclear SSU rDNA sequences were included; the chloroplast gene data for these taxa were coded as missing. The two chloroplast genes were included to add stability to the tree topology given that nuclear SSU has been shown to contain lower phylogenetic signal when used alone [ 15 ]. As with matR , the 103-taxon matrix was truncated to 77 taxa by removing all but five monosulcate taxa to facilitate likelihood analyses. All alignments reported in this paper have been deposited with TreeBASE [ 59 ]: study accession number S1177, matrix accession numbers = M2034–M2037 . Data analysis All three data sets were analyzed using maximum parsimony (MP) and maximum likelihood (ML) methods in PAUP* 4.0b10 [ 60 ] and Bayesian inference (BI) methods in MrBayes 3.0b4 [ 61 ]. Maximum parsimony All MP searches were performed using 100 random addition sequence replicates with tree-bisection-reconnection (TBR) branch-swapping, holding ten trees at each addition step, with all sites equally weighted. For the 77-taxon SSU data set, a series of four MP analyses were performed in which all but one parasite group ( Bdallophyton + Cytinus , Mitrastema , Pilostyles or the large-flowered clade comprising Rafflesia , Rhizanthes and Sapria ) was removed to determine the position of each parasite group in the absence of other long-branch parasite taxa in the analysis. This is a form of the test proposed by Siddall and Whiting [ 62 ]. Maximum likelihood For ML analyses, a MP tree was used in PAUP* to evaluate 56 nucleotide substitution models. ModelTest 3.06 [ 63 ] was used to select an appropriate model from the PAUP* output using hierarchical likelihood-ratio tests (hLRT's) and the Akaike Information Criterion (AIC). The general time-reversible (GTR) substitution model with among-site rate heterogeneity modeled with a "gamma + invariant sites distribution" (Γ + I) was chosen via the AIC as the best-fitting model for the atp1 data set. Investigation of the likelihood score output from PAUP* suggested that a simpler model not evaluated by ModelTest was not significantly worse than the GTR+Γ + I model (LRT; p = 0.520824). This submodel employed four (rather than six) relative rate parameters: one for A-C transversions and A-G transitions, one for A-T and C-G transversions, one for C-T transitions, and one for G-T transversions; the PAUP* LSET option used for analysis was "RCLASS = (a a b b c d)". Likewise, the models chosen by ModelTest for the matR data set were TVM+Γ (hLRT) and TIM+Γ (AIC), but a simpler statistically equal model (LRT; p = 0.583393) was used for analysis. This model employed three relative rate parameters: one for A-C, A-G, and G-T substitutions; one for A-T and C-G substitutions; and one for C-T substitutions; "RCLASS = (a a b b c a)", with among-site rate heterogeneity modeled with a gamma distribution. These simplified models were chosen to reduce computational time and to avoid estimation of unnecessary parameters, which can lead to greater variance in parameter estimates and higher topological uncertainty. A successive approximations approach was used for all ML analyses [ 19 , 64 ]. Substitution model parameters were estimated from the data on a MP tree. With parameter estimates fixed, starting trees for ML analyses were produced via random stepwise addition using five starting seeds, with each tree subjected to a round of tree bisection-reconnection (TBR) branch swapping. Substitution model parameters were then re-estimated on all resulting trees, followed by another round of random stepwise addition and TBR swapping. The tree with the highest likelihood was accepted as the ML tree. Nodal support Nodal support for all data sets was estimated using one or more of the following methods: equal-weights MP bootstrap analysis (100 pseudoreplicates, each consisting of a heuristic search using 100 random sequence addition replicates), ML bootstrap analysis (100 pseudoreplicates generated with SEQBOOT in PHYLIP and analyzed using successive approximations in PAUP*) [ 65 , 66 ], and Bayesian analysis (10 million generations, with the first one or two million discarded as burn-in and trees sampled every 500 generations for the matR and atp 1 data sets; 10 million generations, with the first 5 million discarded as burn-in and trees sampled every 500 generations for the 3-gene data set) [ 61 ]. The GTR+Γ + I submodels used in PAUP* are not available in MrBayes; a standard GTR+Γ + I model was used for the matR and atp1 data sets instead. A partitioned model was used for the 3-gene data set (see below). Two Bayesian runs were performed for all analyses in an attempt to determine if stationarity was reached, and plots of log likelihood and parameter convergence were also evaluated; log-likelihood plots alone are insufficient for monitoring chain mixing and convergence [ 67 , 68 ]. Partitioned analyses The 3-gene data set was also analyzed in MrBayes 3.0. A "fully partitioned" analysis was used in which the 3-gene data set was divided into seven partitions: nuclear SSU; atpB first, second and third codon positions; rbcL first, second and third codon positions. Appropriate substitution models for each data partition were chosen by computing likelihood scores for each partition on a MP tree for the 3-gene data set under 56 substitution models in PAUP* and comparing the scores in ModelTest. The GTR+Γ + I model was the best-fitting model for all partitions. The Bayesian analysis was performed with all model parameters (except branch lengths) unlinked across partitions. Constraints For the nuclear SSU rDNA data, constrained analyses were also performed. A constraint tree for 63 nonparasitic taxa was constructed using the MP topology of the "B series" tree from Soltis et al. [ 1 ] with relationships for poorly supported clades left unresolved. This tree was used as a backbone constraint for MP and ML analyses of 77 taxa including Rafflesiales. MP analyses were performed as described above. ML analyses followed a successive approximations approach similar to that described above. Simulations To investigate possible long-branch attraction in parsimony analyses of the SSU rDNA data set, two sets of simulations were performed. For the first set of simulations, a reduced data set of SSU rDNA sequences for 20 taxa (13 nonparasites and 7 Rafflesiales) was constructed and analyzed under ML (GTR+Γ + I model) in PAUP*. The tree resulting from this analysis, with its associated ML branch lengths and model parameters, was used as the model tree on which 100 data sets of length 1766 (the length of the original SSU rDNA data set) were simulated in Seq-Gen 1.2.7 [ 69 ]. For the second set of simulations, the ML tree for the full 77-taxon data set, with associated branch lengths and model parameters, was used as a model tree to simulate 100 data sets of length 1766 in Seq-Gen 1.2.7. Either MP and ML trees (20-taxon simulation) or just MP trees (77-taxon simulation) were estimated for all 100 simulated data sets. The trees (or strict consensus trees, if more than one MP or ML tree was recovered for a given simulated data set) were then inspected to determine the presence of "incorrect" clades (containing two or more "long-branch" Rafflesiales taxa) that were not present on the model tree. We do not expect to recover such clades at high frequencies unless long-branch attraction is biasing the analyses. List of abbreviations Γ + I – gamma + invariant sites distribution atp1 – ATP synthase alpha subunit atpB – ATP synthase beta subunit BI – Bayesian inference GTR – general time reversible model HGT – horizontal gene transfer matK – maturase K matR – maturase R ML – maximum likelihood MP – maximum parsimony rbcL – ribulose bisphosphate carboxylase/oxygenase, large subunit SSU – small subunit TBR – tree bisection-reconnection branch swapping Authors' contributions DLN coordinated all aspects of the study, obtained many of the genomic DNAs, generated all the nuclear SSU rDNA, conducted the sequence alignments, and drafted the manuscript. AB conducted the majority of the mitochondrial atp1 and matR sequencing and revised the text regarding morphological character comparisons. YQ provided primers, introduced AB to the field of molecular systematics, and supervised his Ph.D thesis. RVR conducted the PCR experiments showing host contamination of Rafflesia DNA and generated the matR sequences for several taxa. FEA performed the phylogenetic analyses. All authors read and approved the final manuscript. Supplementary Material Additional File 1 MP strict consensus tree from mitochondrial matR Strict consensus of 200,000+ trees obtained from maximum parsimony (unconstrained MP) analysis of the 77-taxon mitochondrial matR matrix. Bootstrap percentages are shown above the lines. Rafflesiales taxa are shown in bold italics. Click here for file Additional File 2 Strict consensus MP tree from mitochondrial atp1 Strict consensus of 328 trees resulting from a MP analysis of the 71-taxon mitochondrial atp1 matrix. Rafflesiales taxa are shown in bold italics. Bootstrap percentages are given above the branches. Click here for file Additional File 3 Majority rule consensus BI tree from 3-gene data set Majority rule consensus of 20,000 trees (10 million generations, 5 million burn-in) resulting from Bayesian analysis of the 77-taxon nuclear 3-gene matrix. Clades with Bayesian posterior probabilities are indicated above the clades. Rafflesiales taxa are shown in bold italics. Click here for file Additional File 4 Strict consensus constrained MP tree from nuclear SSU rDNA Strict consensus of 6 trees resulting from the constrained MP analysis of the 77-taxon nuclear SSU rDNA matrix. Rafflesiales taxa are shown in bold italics. Bootstrap percentages are given above selected nodes (Rafflesiales). Click here for file Additional File 5 Taxa used in this study MS Excel file giving taxon names and GenBank numbers for all genes used. Click here for file

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521179

Shut Down, Don't Stress Out

Among the many stresses faced by a cell, one of the most serious is exposure to oxidizing agents. An invading organism, for example, must defend itself against the oxidative assault mounted by a host's immune system. Since oxidation can rapidly destroy many types of molecules, cells have developed multiple means of protecting against it. Rapid mobilization of these defenses requires diversion of resources and temporary suspension of many normal cellular functions, including protein synthesis. In a new study, Elise Hondorp and Rowena Matthews show that when the Escherichia coli bacterium confronts oxidative stress, an enzyme that stands at a central point in the amino acid supply line for protein synthesis is rapidly and reversibly inactivated. Of the twenty amino acids that make up proteins, methionine plays a special role. It is the first amino acid added to every polypeptide chain, and without it, protein synthesis grinds quickly to a halt. Methionine is formed in E. coli through the action of the enzyme cobalamin-independent methionine synthase (MetE), which makes up between one and five percent of all protein in the cell. Thus, by turning off MetE in the face of oxidative stress, protein synthesis can be slowed or stopped, freeing cellular resources to be used elsewhere. Hondorp and Matthews show that in E. coli , MetE is acutely vulnerable to oxidation under a variety of conditions. These results are in accord with a companion study by Leichert and Jakob, also in PLoS Biology, showing that MetE is one of the proteins most sensitive to oxidative damage. When the active site of MetE is stressed by an oxidant, Hondorp and Matthews show, it is temporarily blocked by the attachment of a glutathione subunit. Glutathione is a small molecule that includes a reactive sulfur atom. During “glutathionylation” of MetE, a sulfur on an amino acid of the enzyme is oxidized and links up with a sulfur on glutathione. This study shows that glutathionylation occurs only on a specific amino acid (cysteine 645), which recent structural work indicates sits at the entrance to the active site. Attachment of the bulky glutathione subunit to this cysteine would be expected to block the active site, thus shutting down enzymatic activity. The results indicate that glutathionylation does indeed prevent activity of the enzyme, and furthermore, causes the enzyme to change its three-dimensional form. As the oxidative challenge abates, glutathionylation may be reversed, and the normal activity of the enzyme restored. Thus, glutathionylation of MetE may also serve to protect the active site from permanent oxidative damage. While glutathionylation is a common strategy in eukaryotes, MetE is so far one of the few proteins in bacteria known to be affected in this way. Shutting down MetE and limiting methionine production may play another important role, namely, communicating the bacterium's metabolic state to other nearby E. coli . Methionine is a precursor for the signaling molecule AI-2, which is released extracellularly and appears to serve as a key indicator of colony health and density. This information enables neighboring cells to better respond to changing and potentially hostile environments. Thus, the glutathionylation and inactivation of MetE may provide a simple mechanism by which a bacterium and its neighbors attempt to deal with oxidative stress.

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544939

The birth of Emerging Themes in Epidemiology: a tale of Valerie, causality and epidemiology

Emerging Themes in Epidemiology (ETE) is a new, online, Open Access peer-reviewed journal. The Journal is unique in that it was conceived and is managed by research degree students in epidemiology and related public health fields. The Journal's management is overseen by its Editor-in-Chief and Associate Faculty Editors, all of whom are senior members of faculty. ETE aims to encourage debate and discussion on the theoretical, methodological and practical aspects of epidemiologic research and practice. In addition, ETE is dedicated to the promotion of Open Access publication and the training of research students in the scientific publishing process. This editorial, to coincide with the launch of ETE , sets out the Journal's philosophy and aims. Epidemiology is a rich and innovative science that has much to gain from broader discussion of the causal frameworks that underpin it. ETE aims to be a major forum for such discussion.

" Causality. There is no escape from it, we are forever slaves to it. Our only hope, our only peace is to understand it, to understand the 'why' [ 1 ]." - The Merovingian As far as we know, the Wachowski brothers' Merovingian was not an epidemiologist, but his sentiments should resonate well with those in the field. Probably more than those in any other profession, epidemiologists are slaves to causality. Our professional lives are dedicated to the pursuit of pumphandles, spiderwebs and causal pies [ 2 , 3 ]. Adding to these colourful metaphors are ones proposing frameworks for epidemiologic research that involve Chinese boxes, computer-generated fractals and prison breaks [ 2 , 4 , 5 ]. But what is the student of epidemiology to make of all these curious abstractions? Lost in a sea of metaphors, they might very likely throw their arms up in the air and, in thorough confusion, decide to take a long and much-needed coffee break [ 6 - 8 ]. The epidemiologic literature on causality certainly makes for stimulating reading, but it would be interesting to know how many of us have causal pies and fractals on our minds as we reach for that red folder labelled "Logistic regression 101". Discursive articles on the usefulness of such metaphors are widely regarded as philosophical flights of fancy that we might eventually get around to reading after clearing that backlog of papers waiting to be written in the next two months. Yet what are our alternatives? The newly-qualified epidemiologist leaves their degree with a solid grasp of error, bias, confounding and statistical methodology, but with perhaps a single lecture on Koch-Henlé postulates and Bradford-Hill 'criteria' as the extent of their training into causal thinking. It is interesting to note that Last's Dictionary of Epidemiology does not include the term 'cause', opting instead to give a definition of 'causality' that involves a brief discussion of necessity and sufficiency [ 9 ]. Any reasoned assessment will quickly lead to the conclusion that guidelines for determining causal pathways as commonly taught in epidemiology courses are woefully inadequate, regardless of whether one decides to take an inductionist, refutationist, or hypothetico-deductivist view [ 10 , 11 ], or admits to not having a clue what any of these terms mean. The challenge for the modern epidemiologist is to put those newly-learned methods to use from within a causal limbo, with Robert and Austin as their guides and John Snow as spiritual counsellor. The anatomy of a cause James Wong's 2000 teen horror movie Final Destination [ 12 ] is unlikely to go down in history as a cinematic classic, but it is memorable for its clever, if rather gruesome, depictions of causal processes leading invariably to the death of a number of its unfortunate characters. The movie's motto is that "you can't cheat death's plan". Having narrowly avoided a fatal plane accident thanks to the protagonist's chilling premonition, a French teacher and five of her students are destined to fall victims to the Grim Reaper one by one- in the order they would have died had they been on the ill-fated plane with the rest of their class- unless they can find a way to break the cycle and cheat death. In the most elaborate death scene, Valerie, the French teacher, alone in her house and still visibly shaken by the loss of her colleague and students, becomes unnerved by noises outside. With John Denver's Rocky Mountain High ironically playing in the background, she tries to calm herself by making some tea. Moving to the kitchen sink, she fills the kettle with water, spilling some down the side. She wipes the kettle, turns towards the gas stove and tosses the towel carelessly behind her, which catches onto a knife block. With the kettle whistling, she picks up a school coffee mug, drops two tea bags inside and fills it with boiling water. Picking up the mug, she suddenly recognizes the school logo and, in shock, reflexively throws the mug's contents into the sink. Opting now for something stronger to calm her nerves, she takes some ice cubes from the freezer, drops them into the still-warm mug and re-fills it, this time with vodka. Oblivious to the crack that has appeared in the mug, she walks towards the living room, leaving a trail of vodka behind. As she stands by her computer monitor, vodka drips into the circuitry. An electrical surge creates a spark that ignites the alcohol, causing the monitor to explode and sending out flying shards of glass that slash her throat. Shocked and bleeding, she stumbles towards the kitchen sink, chased by a trail of burning alcohol. Reaching the stove, the trail of flames ignites the gas burners, lighting up her clothes and hair. Falling to the ground and still bleeding, she rolls around violently trying to put out the flames. In an act of desperation, she reaches up and grabs the dangling towel, tilting the knife block and sending a half dozen blades cascading into her stomach while flames catch the curtains and set the house on fire. Suppose you were the investigator arriving on the scene. There is a half burnt-down house, a blown-up computer, a broken mug and a corpse with third-degree burns, stab wounds and a cut throat, but no signs of struggle. What would you determine was the cause of this tragedy? The severe burns, the protruding knives and the neck wound would be pretty obvious choices. But perhaps it is more complicated than that. Perhaps there were extenuating circumstances without which this tragic outcome might not have occurred. The exploding computer maybe, or what about the vodka that burned leaving no trace, or the cracked mug? No, maybe it was the towel, complicitly catching onto the knife block. Or maybe we should blame the teacher's drinking habit. The point of this rather unsavoury story is that without the benefit of such extrinsic observation, detailed reconstructions of causal processes are unattainable. A reasoned observer might conclude that all of the above factors were in some way responsible. They all contributed to the process in their own small way, and had any one of them not been involved things may not have turned out the way they did. They were all what one might call 'component causes'. But is this enough to convince us of what the real cause was? Clearly not. Suppose now that you were an audience member and were somehow able to communicate directly with the characters in the movie. You might wish to warn Valerie of her impending ill fate. At which point in the whole sequence would you alert her so that her death could be prevented? Clearly you would not deny her a mug of tea and you would most likely have no way of knowing that the towel would land on the kitchen block with dire consequences just a few moments later. You might, of course, realize this at the last moment and warn her against reaching up and grabbing the towel, but she might still have died from her neck wound. You might have shouted for her to grab a fire extinguisher and put out the trail of flames, but it is unlikely she would have listened to you as she tried hopelessly to stop the bleeding from her neck. You might, with better foresight, have pointed out to her that vodka was dripping from her mug. Or perhaps with hindsight, you might have recognized that the best thing would have been to provide some moral support and consolation in her time of grief, with which the whole sorry incident might have been avoided altogether. One thing is now clear. There are steps within causal processes on which we can act to try and alter their course- the trail of flames might have been extinguished, and the consequences of the dripping vodka might have been avoided. There are other steps on which we can have no influence, eg. the tossed towel landing on the knife block. Another important thing is also apparent: causal processes have hierarchies. Depending on what happens at one stage, a number of alternative events may result at the next. Had Valerie been able to stop the trail of flames, she might have reached the kitchen sink, realized she could not stop the bleeding and called an ambulance. Or she might have run out of the house shouting for help and her neighbour, trained in first aid, might have saved her life. Saving victims of horror movies, however, is not an easy job. Knowing when best to step in is not necessarily that simple, as we have seen with Valerie. In some cases we may be given a number of opportunities and intervening at any of them might lead to a positive outcome. But in other cases, once certain factors are in place there will be an air of inevitability in everything that follows, and all our attempts to intervene thereafter may prove to be little more than an exercise in futility. Perhaps it is now time to admit that I have extended this fanciful analogy far beyond what is appropriate. I make no apology if in so doing I have in some way managed to convey the idea that epidemiology thrives on causal processes, on elucidating their complexities and identifying the most effective points for intervention no matter at what level of their elaborate hierarchies. If this is a worthwhile venture, then the discussion of how we conceptualize and study these causal processes surely is so too. Emerging Themes in Epidemiology (ETE) was born out of this ideal- that contrary to common belief, epidemiology is not merely a collection of standardized tools to be applied at will to any health-related situation, but that it is a rich and innovative science that aims to describe reality in all its complexity, spanning the molecular to the global, with the ultimate goal of improving the health of individuals and populations. And that in order for this to be achieved, we need to improve our understanding of how factors, at any level of biological or social organization, eventually lead to ill health. ETE is founded on three core principles: • That epidemiology and epidemiologists have much to gain from a broader and more fundamental discussion of the concepts and theoretical frameworks that underpin the practice of epidemiologic research • That Open Access publication has a crucial role to play in reducing the current inequities in access to scientific information, which should be a universal and freely-available resource for the benefit of the whole of society • That students of epidemiology and related fields can make substantial contributions to the introduction of new concepts and ideas into mainstream epidemiologic research, not only through writing, but also through having a direct influence over what is published In keeping with this philosophy, we recognize that epidemiology has much to gain from developments in other fields and we welcome contributions from all public health professionals. We will consider articles that comment critically on current epidemiologic theory and practice, either generally or within a specific specialty, including articles from other fields that have implications for the conduct of epidemiologic research. ETE will not generally publish research reports, although exceptions may be made in cases where the results can be placed within a broader public health context to present a new concept or theoretical framework. By making all of this material freely available online under the auspices of the Open Access publisher, BioMed Central, we aim to make Emerging Themes in Epidemiology a global forum for the discussion of new developments in epidemiologic thinking and practice that will benefit the global public health community. In doing so, we recognize that there is much that the scientific community can do to support Open Access publication. The health consequences of inequitable access to scientific information remain largely ignored, yet for years we have adhered to a system of publication that is restrictive and largely subsidized by institutions and libraries at great expense. Open Access is an important step towards making the publication process, and its associated costs, more transparent. We thus call on individuals, institutions, funding bodies and governments to engage in the Open Access movement by promoting and supporting Open Access publication. This will involve a major shift not just in publishing costs, but also in thinking. Our current measures for assessing the impact of academic research are inherently intertwined with our inequitable publication tradition. Developing new ways of assessing the quality of scientific research that are independent of journals' perceived 'impact' are imperative for the wider recognition of Open Access. In promoting the role of students in the publication process, we intend for ETE to be a training ground for postgraduate students, providing them with an opportunity to be involved at every stage of the publication process, including commissioning, reviewing and writing articles. Our editorial board is formed principally of research students, who operate the Journal with support from an international group of associate faculty. Our collaborations extend to a growing number of students from diverse institutions serving as article referees. We welcome suggestions for extending these collaborations in the future. The third millennium has brought with it exciting challenges for epidemiologists. An explosion in the availability of genetic and molecular information, the development of bioinformatics, the increasing application of sophisticated statistical analyses to model complex systems and the gradual incorporation of sociological approaches to understanding health inequalities have arrived together with sobering statistics on the state of global public health. Knowing when and how to apply these and other developments at a time of rapid political, social and biological change while maintaining clear sight of our public health goals will be the major challenge for current and future generations. It is our hope that Emerging Themes in Epidemiology will be a tool for epidemiologists confronting that challenge.

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539044

Picturing AIDS: Using Images to Raise Community Awareness

In Botswana, explicit color photos of people with AIDS have been used to spread knowledge, with the aim of saving lives

Southern Africa poses special problems for AIDS educators and health care workers. Because there is a strong tradition of oral communication in the region, written educational materials often do not have as much impact as the spoken word. We have found that using colour images of HIV/AIDS in a workshop setting to provoke discussion can be a useful alternative to more conventional, written materials. In this article, we discuss our experience of using such images to raise community awareness about the AIDS epidemic in Botswana. Who We Are Teaching-aids At Low Cost (TALC) is a nongovernmental organisation that supplies cheap teaching aids and books to raise standards of health care and standards of living—especially in poverty-stricken areas—worldwide ( http://www.talcuk.org/ ). The organisation has traditionally focused on developing countries, particularly sub-Saharan Africa and Asia. In recent years, TALC has become more global; it now distributes materials to more than 200 countries and sends educational materials on CD-ROM at no cost to health workers in developing nations. In 1964, TALC was founded at the London School of Hygiene and Tropical Medicine as a way of providing low-cost colour transparencies to help students from resource-poor countries to teach after they returned home. By the early 1980s, nearly half a million transparencies were being sold at cost each year. Those who used them came to appreciate how important colour images could be, particularly amongst people who have grown up in societies where knowledge is spread primarily through oral communication and less use has been made of the written word. Early on in the HIV/AIDS epidemic, we decided that our experience of distributing visual teaching materials could be used to spread information about this new pandemic, which was hitting African societies particularly hard. We produced four sets of 24 colour transparencies on HIV/AIDS, with a detailed accompanying text. Edwin Mapara: The Botswana Experience Today, there are an estimated 260,000 people in Botswana living with HIV. This—in a country with a total population of 1.6 million—gives Botswana a prevalence rate of 36.5%, the second highest in the world after Swaziland [1] . As a medical student in Zambia in 1985, I studied patients with Kaposi's sarcoma. The consultant in charge appreciated that this was due to HIV infection, but when she started to acknowledge this publicly, she was strongly censored by the existing authorities and was almost forced to leave the country. I realised that if this kind of denial persisted, the epidemic would spread more widely and would become an even greater disaster. I wanted to try to bring home to both the authorities and the African people the truth about the spread of the disease and the need for fundamental changes in sexual behaviour. In 1990, on qualifying, I took up a post at the Athlone Hospital, a 175-bed district hospital in the Lobatse region of Southern Botswana. I joined other health workers who shared my concerns. We started the Athlone Anti-AIDS Project to address HIV prevention and care both in the hospital and in the wider community. We began to have organised discussions with local people about HIV/AIDS. The response we heard was often, “You talk about this terrible disease, which may affect us, but show us a patient”. This is how we came to use a set of slides from TALC, in a teaching programme that the Ministry of Health in Botswana called “radical and insensitive”. We emphasised the essential messages about AIDS prevention by using coloured pictures of black Africans. These pictures included explicit images of ulcers on a penis and a vagina. The slides included clinical manifestations of HIV/AIDS (such as herpes zoster and Kaposi's sarcoma) and other sexually transmitted diseases, images that explained the basic virology and transmission of HIV ( Figure 1 ), and images about HIV prevention (such as condoms) and care (such as caring for orphans infected by HIV/ AIDS). Figure 1 Don't Worry—Only a Few Sticks This slide is used in workshops to show that while we only see a handful of patients with symptomatic HIV, many more of us are HIV positive and are infecting others; we do not know our HIV status, since we have not been tested and we have no symptoms. (Illustration: TALC) Showing these pictures to local people was hugely controversial. For example, some elderly participants walked out when they saw the explicit pictures. Some community members approached local political counsellors to voice their concerns about a “decay of culture” and a “lack of respect”. Some parents did not want their children to see the images at all, because “they would corrupt their morals and young minds” and would encourage children to “experiment with sex”. As the team leader, I was fined chickens on several occasions by local chiefs and elders for the “crime” of showing these explicit TALC slides. The government gave our teaching project very little support in the early days; we were even cautioned by the highest authorities at the Ministry of Health. The Church, too, wanted nothing to do with our programme of “loose morals”. Despite these obstacles, over the next ten years we held over a hundred workshops, which eventually involved all government departments and levels of society in Botswana. Today, we are still using the same pictures. In 2000, the United Nations Development Programme declared Athlone Hospital's initiative as one of the “best practices” in Botswana [2] , and it is being replicated nationwide. TALC slides have been shown from the pulpits of churches, and community members will ask for the colour pictures specifically when the team is invited to lead a workshop. Given the terrible impact that AIDS has had on the community, the same community members who once resisted our teaching project ask us angrily why doctors were not sufficiently aggressive in using pictures in the early days of AIDS. One telling statement made in a workshop was: “you doctors are to blame for what has happened to Africa, and particularly to our children. You should have done this ten years ago before one quarter of the population became infected. The blood of our children, who have died, rests on your heads”. Making the Best Use of Pictures In Botswana, I used a slide projector and occasionally a mobile electrical generator, but such equipment is not widely available in most African countries. As an alternative to using colour slides, TALC has developed a folded A4 (210 mm × 279 mm) sheet with 12 colour images as a way of presenting the important messages about HIV/AIDS. This leaflet is available on request; E-mail: info@talcuk.org (or mail TALC, P. O. Box 49, St. Albans, AL1 5TX, United Kingdom). In our experience, the slides or leaflet work best if you can get the participants to sit in small groups for discussion. Each group should have at least one set of pictures. In your introduction, mention that to talk about sex or death is not taboo in a world of AIDS. Encourage active participation by all. Show one picture at a time—”let the picture talk”—and do not initially look at the accompanying text. Ask the participants to describe what they see in English or in the vernacular. Encourage participants to work out for themselves what the message is in the picture. Discuss all the possible answers. Then look at the text that accompanies the picture. Provide an answer built from the participants' words. If appropriate, ask people about their own relevant experiences. Finally, ask the participants to pin the pictures to the wall, making sure that each picture is put up by a different participant. Revise the lessons learned at the end of the session. Revise again, weeks later, if possible. At the end of the day, the participants should be able to say, “we did it by ourselves”. The pictures show examples of how HIV/AIDS can affect people. They must not be thought of, or used, as a way to diagnose HIV/AIDS in participants or their relatives or friends. Emphasise to all participants that if they have any reason to suspect that they (or anyone they know) have HIV/ AIDS, they should attend a clinic where trained health workers can help them. Conclusion For people in Botswana, “seeing is believing”. Written descriptions are often not enough; showing pictures of herpes zoster, syphilis ulcers, or tuberculosis lymphadenopathy can be a powerful teaching tool. Once the initial shock is overcome, these colour pictures offer a straightforward way to demonstrate the realities of the disease far and wide.

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387283

The Elusive Baseline of Marine Disease: Are Diseases in Ocean Ecosystems Increasing?

Disease outbreaks alter the structure and function of marine ecosystems, directly affecting vertebrates (mammals, turtles, fish), invertebrates (corals, crustaceans, echinoderms), and plants (seagrasses). Previous studies suggest a recent increase in marine disease. However, lack of baseline data in most communities prevents a direct test of this hypothesis. We developed a proxy to evaluate a prediction of the increasing disease hypothesis: the proportion of scientific publications reporting disease increased in recent decades. This represents, to our knowledge, the first quantitative use of normalized trends in the literature to investigate an ecological hypothesis. We searched a literature database for reports of parasites and disease (hereafter “disease”) in nine marine taxonomic groups from 1970 to 2001. Reports, normalized for research effort, increased in turtles, corals, mammals, urchins, and molluscs. No significant trends were detected for seagrasses, decapods, or sharks/rays (though disease occurred in these groups). Counter to the prediction, disease reports decreased in fishes. Formulating effective resource management policy requires understanding the basis and timing of marine disease events. Why disease outbreaks increased in some groups but not in others should be a priority for future investigation. The increase in several groups lends urgency to understanding disease dynamics, particularly since few viable options currently exist to mitigate disease in the oceans.

Introduction Marine organisms serve as hosts for a diversity of parasites and pathogens. Mortalities affect not only the host population, but can cascade through ecosystems. Loss of biologically engineered habitats such as seagrass beds ( Lewis 1933 ; Taylor 1933 ) and cascading trophic effects due to removal of consumers ( Lessios 1988 ) can alter community structure. Understanding marine disease and the timing of outbreaks is increasingly important given escalating anthropogenic stressors affecting marine ecosystems. Humans directly affect community structure (e.g., overfishing [ Jackson et al. 2001 ; Myers and Worm 2003 ]) and facilitate introduction of terrestrial pathogens to marine organisms (e.g., canine distemper virus in Antarctic seals [ Bengtson and Boveng 1991 ]). Human-mediated climate change may also affect disease prevalence. A recent review predicts disease in both terrestrial and marine ecosystems could increase with future climate warming ( Harvell et al. 2002 ). Previous literature reviews suggesting a higher rate of disease outbreaks in the last three decades ( Epstein et al. 1998 ; Harvell et al. 1999 ), coupled with predictions of future increases due to climate change ( Harvell et al. 2002 ), lend new urgency to understanding causes of marine disease outbreaks. Evidence suggests the increase is real ( Harvell et al. 1999 ), yet lack of baseline data for most marine communities precludes a direct test of the hypothesis. We developed a proxy method to test a prediction of the increasing disease hypothesis: that reports of disease in the scientific literature, normalized to overall publication rates, increased since 1970. We searched an online literature database (ISI Web of Science) and quantified reports of disease in natural populations of marine organisms from 1970 to 2001. Nine marine taxonomic groups were searched: turtles, corals, mammals, urchins, molluscs, seagrasses, deca-pods, sharks/rays, and fishes. Previous analyses of ecological literature specifically assessed trends among scientists such as taxonomic bias ( Clark and May 2002 ) and taxonomic chauvinism ( Bonnet et al. 2002 ) in research. Our proxy method is to our knowledge the first quantitative use of normalized trends in the literature to investigate an ecological hypothesis. In the absence of baseline data, the literature proxy method detects important trends of disease in major groups of marine plants, invertebrates, and vertebrates. Results The largest confounding factor when using literature searches to correlate disease events with time is temporal change in the total number of publications (related to disease or not) on the taxonomic group. To control for changes in total publication, data were normalized using a yearly proportion of disease reports from natural populations relative to total literature inputs for each taxonomic group. Total disease reports, not normalized to literature inputs, increased in all groups ( Table 1 ). However, normalized results varied with taxonomic group. Normalized disease reports increased in turtles, corals, mammals, urchins, and molluscs. No significant trends were detected for seagrasses, decapods, and sharks/rays (though disease occurred in these groups). Counter to the hypothesis, disease reports decreased in fishes ( Figure 1 ). Figure 1 Percent of Literature Reporting Disease over Time in Each Taxonomic Group r s is Spearman's ρ. α is controlled for multiple comparisons with Holm's sequential Bonferroni adjustments. (A) Turtle. (B) Coral bleaching and disease (closed square); coral disease including infectious bleaching (open circle); coral bleaching (asterisk). (C) Mammal. (D) Urchin. (E) Mollusc. (F) Seagrass. (G) Decapod. (H) Shark/ray. (I) Fish. Table 1 Spearman's Rank Correlation Analysis The table shows total reports (not corrected for research effort), normalized reports, and normalized reports with most frequent author removed. r s is Spearman's ρ. α is controlled for multiple comparisons with Holm's sequential Bonferroni adjustments. Bold indicates significance The relevance of our approach hinges on the assumption that an actual change in disease over time is accompanied by a corresponding change in publication frequency by scientists. We evaluated this assumption by testing the protocols with a case in which the baseline was known (raccoon rabies [ Rupprecht and Smith 1994 ]). Normalized reports of raccoon rabies increased since 1970 (see Table 1 ) just as the disease increased from an index case in Virginia in 1977 to an epizootic affecting eight mid-Atlantic states and the District of Columbia by 1992 ( Rupprecht and Smith 1994 ). Despite improvements in search protocols, use of a literature proxy is limited by the inability to distinguish between an event that did not occur and an event that was not reported. We tested whether particular authors contributed disproportionate primary literature inputs that could bias results. Papers by the most prolific author in each taxonomic group were removed to determine whether there was an “author effect,” and none was observed in any taxonomic group (see Table 1 ). Multiple reports of a single disease event could also bias the data. Multiple reports were removed from the turtle, coral, urchin, mammal, shark/ray, and seagrass literature. Removal of the reports did not alter the significance of the results; thus, multiple reports in the mollusc, decapod, and fish literature were not removed, owing to the large volume of literature in these groups. Discussion We address an ecological hypothesis, that disease of marine organisms increased since 1970, using a quantitative literature proxy method. Although total reports of marine disease increased over time ( Epstein et al. 1998 ; see Table 1 ), a parallel increase in publication rates confounds interpretation of this pattern. Our approach normalizes data to overall publication within each group to circumvent this problem. While an increase in disease reports was detected in many taxa, our finding that disease did not increase in all taxa has two important implications. First, the increases were not exclusively the result of increased study of disease by marine biologists. Second, factors such as global change may have complex effects on disease. Although some aspects of global change, such as warming and pollution, are predicted to make hosts more susceptible to infection ( Scott 1988 ; Holmes 1996 ), some stressors may impact parasites more than their hosts ( Lafferty 1997 ). Signs of infection with coldwater disease in salmonids, for example, occur between 4°C and 10°C and disappear as water temperature increases ( Holt et al. 1989 ). In addition, stressors that depress host population density may reduce density-dependent transmission of host-specific infectious disease by reducing contact rates between infected and uninfected individuals ( Lafferty and Holt 2003 ). New or increasing stressors, such as global warming, could increase disease if stressed hosts are more susceptible to infection. Elevated sea surface temperature due to El Niño events is a common explanation for coral bleaching ( Williams and Bunkley-Williams 1990 ; Hoegh-Guldberg 1999 ) and may increase coral susceptibility to disease ( Harvell et al. 2001 ). Increases in turtle and mollusc disease also appear temperature-related. Green turtle fibropapilloma tumors are hypothesized to grow rapidly in summer and reach a debilitating size by winter, when cold water temperatures further stress turtles, resulting in winter strandings ( Herbst 1994 ). The geographic range of the oyster parasite Perkinsus marinus extended 500 km north owing to an increase in average winter low temperatures ( Ford 1996 ). Pollution is another ubiquitous and increasing stressor. Bioaccumulation of lipophillic toxins in marine mammals affects the immune system and increases susceptibility to disease ( Lafferty and Gerber 2002 ). Disease could also increase if transmission increases with host density. Some sea urchins experienced increased populations due to overfishing of their predators, and these high-density populations are associated with bacterial disease ( Lafferty and Kushner 2000 ). Regulations such as the United States Marine Mammal Protection Act of 1972 fully protect pinniped populations, and several species have increased in abundance to levels where transmission efficiency would be expected to increase. The decline in infectious diseases of wild fishes over time corresponds to documented reductions in fish populations through intense fishing ( Jackson et al. 2001 ; Myers and Worm 2003 ). Fisheries that reduce the abundance of a fished species should also reduce infectious disease transmission ( Dobson and May 1987 ). This has been documented in experiments ( Amundsen and Kristoffersen 1990 ) and in observations of parasite declines associated with overfishing ( Sanders and Lester 1981 ). Grouping diseases within taxa could obscure important patterns. For example, the trend for increasing coral disease was driven by coral bleaching ( r s = 0.87, p < 0.0001), while infectious coral diseases, including infectious bleaching, did not increase over time ( r s = 0.13, p = 0.4934; see Figure 1 B). The infectious bleaching literature includes several papers since 1996. To ensure the lack of a significant coral disease trend was not due to multiple papers published on this topic at the end of the time range surveyed, an additional analysis was conducted with all infectious bleaching papers excluded; r s and p values did not change ( Table 2 ). Table 2 Normalized Coral Disease Reports Original data include papers on infectious bleaching. r s and p values are the same for both analyses. Italics indicate changes in proportions after removal of infectious bleaching literature While we did not detect an increase in normalized coral disease reports over time, impacts of disease can be high. The recent shift of dominant corals ( Acropora to Agaricia ) on reefs due to white band disease was unprecedented in the last 3,000 y ( Aronson et al. 2002 ). Future research should take a finer-scale look at disease, particularly disease impacts, within each taxonomic group. Further investigation is also warranted to determine why some groups showed no temporal pattern in disease reports. We examined temporal trends in disease reports since 1970 to identify groups experiencing increased outbreaks. The strong pattern of increased reports in groups such as turtles, mammals, and urchins reflects perceived changes noted by scientists ( Harvell et al. 1999 ). Trends in other groups, such as seagrasses and fishes, suggest that an increase in disease did not occur across all taxa. Although this proxy approach does not directly test hypotheses of temporal changes in disease, a strong signal likely reflects an underlying pattern in nature. In the absence of baseline data, this is a useful approach for detecting quantitative trends in disease occurrence. Understanding disease dynamics, including trends in disease occurrence, is fundamental to conserve ecosystems faced with rising anthropogenic stresses. Materials and Methods We searched the Science Citation Index Expanded (5,900 journals, ISI Web of Science versions 1.1 and 1.2) for papers published from 1970 to 2001 with titles containing specific host taxonomic strings alone and in combination with a disease string ( Table 3 ). We excluded articles clearly about disease in nonnatural settings, such as hatcheries, aquaculture, and mariculture, or about experimental or laboratory infections. Searches for corals were performed twice to quantify reports of bleaching separately from infectious bleaching (e.g., Vibrio shiloi [ Israely et al. 2001 ]) and disease. Only titles were searched, as online abstracts are not available for many articles prior to 1990. Searching the complete citation would bias results after 1990 because more text of each publication would be searched. Table 3 Taxonomic Groups and Search Strings Abstracts (or entire manuscripts, when necessary) were obtained for articles within the turtle, coral, urchin, mammal, shark/ray, and seagrass literature that appeared to report the same disease event (e.g., multiple reports of the Caribbean Diadema urchin mortality). If more than one paper reported an event, only the earliest published report was included in the analysis. Because significance of results was not altered, multiple reports of disease were not removed from mollusc, decapod, and fish literature owing to the large number of publications returned for each group. Often, returned titles contained part of the search string, but were not relevant (e.g. “crab nebula” when searching “crab”). Modifications to search strings excluded most irrelevant articles, and titles were read to determine relevance. If more than 50 titles were returned, titles were randomly sorted and the greater of 20% (maximum of 200) or 50 returned titles were read. Total relevant articles were calculated as the proportion of relevant articles read times the total number returned. Protocols were tested using raccoon rabies, a disease for which baseline data are available ( Rupprecht and Smith 1994 ). Potential biases were considered and tested. Extensive descriptive or taxonomic work early in the study of a group could bias results against a large number of disease reports. If such a bias existed, one would expect both a large number of disease reports and a large number of nondisease publications in the beginning of the literature survey period. Neither prediction is true—the number of both disease reports and nondisease publications either remains relatively constant or increases through time in all groups. Frequent publishing by one author could bias results. Papers by the most published author in a taxonomic group were removed from the analysis to determine their effect. Papers on a particular “hot” topic could also bias results, particularly if that topic is disease and inflates normalized disease reports late in the survey period. For example, a recent mortality event could increase scientists' awareness of disease, resulting in increased publishing without a concomitant increase in the phenomenon. This likely does not affect our results because (a) disease is not the only “hot” topic experiencing increased publication rates and (b) while multiple papers on disease may be published, not all are reports of disease in natural populations. A 3-y running mean was used to reveal trends obscured by clustered reporting (e.g., a symposium volume on a topic) and time lags between observation and publication (approximately 3 y, determined by comparing event and publication dates). Data were analyzed with Spearman's rank correlation (JMP version 5.0) with α controlled for multiple comparisons using Holm's sequential Bonferroni adjustments.

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Comparative analysis of protein coding sequences from human, mouse and the domesticated pig

Background The availability of abundant sequence data from key model organisms has made large scale studies of molecular evolution an exciting possibility. Here we use full length cDNA alignments comprising more than 700,000 nucleotides from human, mouse, pig and the Japanese pufferfish Fugu rubrices in order to investigate 1) the relationships between three major lineages of mammals: rodents, artiodactyls and primates, and 2) the rate of evolution and the occurrence of positive Darwinian selection using codon based models of sequence evolution. Results We provide evidence that the evolutionary splits among primates, rodents and artiodactyls happened shortly after each other, with most gene trees favouring a topology with rodents as outgroup to primates and artiodactyls. Using an unrooted topology of the three mammalian species we show that since their diversification, the pig and mouse lineages have on average experienced 1.44 and 2.86 times as many synonymous substitutions as humans, respectively, whereas the rates of non-synonymous substitutions are more similar. The analysis shows the highest average dN/dS ratio in the human lineage, followed by the pig and then the mouse lineages. Using codon based models we detect signals of positive Darwinian selection in approximately 5.3%, 4.9% and 6.0% of the genes on the human, pig and mouse lineages respectively. Approximately 16.8% of all the genes studied here are not currently annotated as functional genes in humans. Our analyses indicate that a large fraction of these genes may have lost their function quite recently or may still be functional genes in some or all of the three mammalian species. Conclusions We present a comparative analysis of protein coding genes from three major mammalian lineages. Our study demonstrates the usefulness of codon-based likelihood models in detecting selection and it illustrates the value of sequencing organisms at different phylogenetic distances for comparative studies.

Background Large scale sequencing projects of many different species allow us to investigate phylogenetic issues in much more detail and to identify whether certain genes have had an extraordinary evolution in one or more species and thus gain insight into the actions of natural selection. Despite the sequencing of an increasing number of mammalian genomes and the implementation of more sophisticated evolutionary models using maximum likelihood and Bayesian methodology, the branching order within the mammalian phylum is still not completely resolved. The main reason for this uncertainty is that the diversification of these orders occurred over a short period of time, making the inference of branching order a difficult problem. One of the highly debated issues concerns the relative order of branching among primates, artiodactyls and rodents [ 1 - 9 ]. Here, the Japanese pufferfish Fugu rubrices is used as an outgroup to estimate the branching order of the three species relative to each other. Codon based models [ 10 , 11 ] allow for powerful analysis of protein coding nucleotide sequences. Evolutionary hypotheses may be tested using likelihood ratio tests between nested models. For an introduction to the practical use of these models see [ 12 ], for a more thorough review of the methodology see [ 13 ]. The parameter of primary interest is the ratio of nonsynonymous to synonymous substitutions (ω), also known as the dN/dS ratio. The dN/dS ratio measures the relative importance of evolutionary forces that have shaped a particular protein. A dN/dS ratio significantly larger than one strongly suggests that positive Darwinian selection has acted on the protein. Different extensions to the basic codon model exist, and these can be divided into three main categories: (1) Lineage-specific models that average ω over sites but differentiate between lineages [ 14 ]; (2) site-specific models that average ω over lineages but differentiate over sites [ 15 ]; (3) branch-site specific models that combine the two previous extensions by allowing ω to vary over sites in all background lineages, but allow for a different value of ω in one or more pre-specified lineages [ 16 ]. The models we use here and their relationships are shown in Table 1 . Numerous studies have shown the ability of the site-specific and the branch-site specific models to detect positive selection in cases where the branch-specific models did not, indicating that averaging over sites is generally a more serious problem than averaging over lineages and that in many cases using a branch-site specific model increases the power to detect positive selection [ 17 - 22 ]. In a recent study of cDNA trios of human, mouse and chimpanzee a codon based branch-site specific model was used to search for human genes that have undergone positive selection since our divergence from other primates [ 23 ]. Here, a similar search is done on a different phylogenetic level using a collection of porcine genes. While the study by Clark and colleagues concentrates on the divergence between humans and chimpanzees (branch a in Figure 1 ) our study searches for genes that have undergone positive selection since the divergence of primates, artiodactyls and rodents. Several recent studies have shown that some of the branch-site specific models under certain conditions might have a high false positive rate when used to detect positively selected sites [ 24 , 25 ]. This problem has recently been addressed by Yang and colleagues with the implementation of a new Bayes empirical Bayes (BEB) method for predicting positively selected sites. This new method is much better at avoiding false positives while still retaining a high sensitivity (Z. Yang, pers. comm.). Here we use the new and improved BEB version of the branch-site specific model originally presented in [ 23 ] to detect genes that may have been influenced by positive selection. Results The distribution of sequence lengths of the 1120 three-species alignments is shown in Figure 2 . Since the full length cDNAs were assembled from random ESTs, there is a bias towards assembling relatively short genes. Therefore the subset of genes used in this analysis is not a random sample from the pig genome. This decreases the power of our evolutionary tests, since short alignments have less power when testing for positive selection, but we do not anticipate any other systematic bias in our results. Mammalian phylogeny The relative branching order of the three mammalian species was investigated with the individual genes as well as with a concatenated super gene. Using the empirical amino acid substitution model of Whelan and Goldman [ 26 ] we maximized the likelihood under the three conflicting topologies shown in Figure 3a–c . In 123 of the 988 alignments all amino acids are identical in the three mammalian species giving us no information to discriminate between the three topologies. Of the remaining 865 alignments 245 favour topology A, while 440 and 180 favour topology B and topology C respectively. A concatenated super gene of all 988 alignments clearly favoured topology B over topology A, which again has a higher likelihood than topology C, consistent with the results from the individual gene comparisons (Table 2 .). We used the baseml program of PAML to compare the three topologies in a nucleotide based framework. Different nucleotide based substitution models were used to maximize the likelihood on the three topologies for each of the three codon positions separately. The results of using different models of nucleotide evolution were highly similar so here we only discuss the results obtained with the HKY85 model [ 27 ]. The results based on the third codon position shows that Fugu is too distantly related to the three mammals to be informative in placement of the root of the mammals (results not shown). The first and second codon positions do not show such saturation and should therefore be useful in comparing the three topologies. Consistent with the results based on the amino acid substitution model we see that topology B is favoured in most genes, followed by topology A and topology C, respectively. The actual numbers from the second codon position are 215, 386 and 179 in favour of topology A, topology B and topology C respectively and 208 alignments are uninformative. The corresponding numbers for the first codon position are 215, 545, 175 and 53 (Table 2 .). The internal branch is rather short in all cases. Therefore in the remaining analyses we treat the mouse, human, pig split as a trifurcation. Depending on which topology is actually the right one, the only bias introduced by treating the topology as a star tree, as shown in Figure 3d , is a minor overestimation of the branch length of the species that actually roots the other two. The rates of evolution The three-species alignments were used to estimate the synonymous and nonsynonymous substitution rates of the three branches under the free ratio model, see Table 3 . Figure 4a–f shows the distribution of the synonymous and nonsynonymous branch lengths for each gene in all three species. The synonymous rates are significantly different between the three species. The average synonymous substitution rate, estimated using the concatenated super gene, is approximately 2.86 times larger in mouse compared to pig, and approximately 1.44 times larger in pig than in human. The nonsynonymous rates are more similar among the three species. The corresponding values for the nonsynonymous rates are 2.08 and 1.17 respectively. Table 3 shows the mean, median and variance of both the synonymous and nonsynonymous rate distributions as well as the values obtained from the concatenated super gene. The average values from the individual genes are highly similar to the results obtained from the concatenated super gene. Positive Darwinian selection The dN/dS ratios on the three different lineages were estimated under the free ratio model (Figure 4g–i ). Most genes in all three species have an average dN/dS ratio very close to zero with the average dN/dS ratio higher in human than in pig, which again is higher than in the mouse lineage. The one ratio model averages over sites and lineages, which makes this an extremely conservative method of detecting positive selection. Only four of the 1120 three-species alignments have an average dN/dS ratio larger than one, see Table 4 , and of those only one is significantly larger than one (XM_165930). The free ratio model allows each lineage to have its own dN/dS ratio. This model has slightly more power than the one ratio model due to its ability to find lineage specific signals. The likelihood ratio test (LRT) of these two models should not be considered as a stringent test for positive selection, but more as a test for different selective forces among lineages. The LRT shows that 154 genes have significantly different dN/dS ratios among lineages at the 5% significance level, 73 at 1% and 41 at the 0.1% level of significance. Table 5 shows the 24 genes that have a dN/dS ratio larger than one in one or more lineages as well as the result from each gene of a LRT that tests whether the estimated value of ω is significantly larger than one. As with the one ratio model only one gene shows a result significantly larger than one. The gene is the same one as reported with the one ratio model (XM_165930) and the lineage with a dN/dS ratio significantly larger than one is the lineage leading to pig. Several studies have shown that averaging over sites is more conservative when searching for positive selection than is averaging over lineages. The branch-site specific model A and model B [ 16 ] were originally designed to search for genes where only a small fraction of codons in a specific foreground lineage has evolved under positive selection. Several studies have shown that the original models are prone to predicting false positives under certain conditions, and one should therefore be very careful drawing conclusions from studies based on those models. Here we use a new and improved version of a branch-site model developed for the analyses of human, chimpanzee and mouse gene trios [ 23 ]. The new model we use here is implemented in PAML v. 3.14 and uses the new and improved Bayes empirical Bayes approach to predict which sites have evolved under positive selection in the foreground lineage. Likelihood ratio tests were done separately with human, pig and mouse as the predefined foreground lineage. The LRT when contrasting the neutral model with the branch-site model has two degrees of freedom. By using the human lineage as foreground lineage we find 288 genes that show signals of positive selection (dN/dS in the foreground lineage is larger than one). In 58 of those genes the branch-site model fits the data significantly better than the neutral model at the 5% significance level. We find 34 and 15 genes at the 0.01 and 0.001 levels of significance respectively. The corresponding numbers of genes using pig as foreground lineage are 314, 55(0.05), 23(0.01) and 5(0.001). Using mouse as foreground lineage results in 352, 67(0.05), 25(0.01) and 4(0.001). The genes found to be under positive selection in any of the three species with a LRT significance level of 0.001 are shown in Table 6 . The molecular function of the genes predicted to be under positive selection was determined using the Panther server [ 28 ] and the NCBI server using the newest build of the human genome. Both annotation servers are updated on a regular basis when new information becomes available. During the course of this study the annotation of several genes changed. Of our 1120 alignments 188 are currently not annotated as functional genes indicating that they might possibly be pseudogenes in human; see the Discussion for more details on this subject. The proportion of genes that we report to have undergone positive selection in the human lineage at the 5% level of significance can therefore be viewed as either 58/1120 ~5.2% or 43/931 ~4.6%, indicating that possible pseudogenes are only slightly overrepresented in the genes predicted to have undergone adaptive evolution. The genes predicted to have been under positive selection in the pig and mouse lineage show a similar trend. Several different models have been developed that allow for heterogeneity of ω over sites in an alignment. We used the M4 model [ 15 ] which allows each codon to fall into one of 5 categories corresponding to ω equal to 0, 1/3, 2/3, 1 and 3. The first category represents the fraction of codons that have evolved under strong purifying selection allowing no nonsynonymous changes to occur. The next two categories represent different intensities of purifying selection. The category with ω = 1 represents neutrally evolving sites, while the last category with ω = 3 represents codons that have evolved under positive selection. The results of this analysis on the concatenated super gene can be seen in Table 7 . Only 1.6 % of all codons appear to have evolved under positive selection, and approximately 69 % have been under strong functional constraints. Codon usage bias The concatenated super gene was also used to investigate the patterns of codon usage in the three species; the results of this investigation are summarized in Table 8 . A test for equal codon distributions is rejected in all three pair wise comparisons (P < 0.0001, 60 d.f.). Using nucleotide frequencies to estimate the codon equilibrium frequencies fits the data poorly, so does the equal frequency model (Table 9 ). For a description of the codon equilibrium frequency models, see the Methods. The F3 × 4 model was extended with one extra parameter that accounts for CpG avoidance at the second and third codon position. Since all changes in the second position of a codon are nonsynonymous, the frequency of NCG codons is expected to be lower than under the F3 × 4 model. The extra parameter introduced improves the log likelihood by approximately 1236 units (~44%). This can be compared to the approximately 321 units per extra parameter introduced when going from the F3 × 4 model to the codon table model. When analysing the super gene it is still better to use the actual codon frequencies, but with individual genes the number of codons can sometimes be so small that the use of actual codon counts can be problematic. We also implemented a similar model that incorporated the avoidance of CG in first and second position by introducing an additional parameter but this does not improve the fit of the model significantly (results not shown). This is probably caused by the fact that all four codons with CG in the first and second position code for the same amino acid, Arginine. Arginine has six different codons and the two codons without a CG pair (AGA and AGG) are generally favoured over the other four (Table 8 ), but this tendency is apparently accounted for when modelling nucleotide frequencies at the three codon positions, so here we only present the model that accounts for CpG avoidance at the second and third codon position. Table 9 shows that the choice of codon equilibrium frequency model has detectable effects on the parameter estimates. Most striking is the apparent overestimation of the transition/transversion ratio and the dN/dS ratio when the model is less parameter-rich. Discussion The phylogeny of the early mammalian radiation has been extensively debated over the last two decades. The classical view based on fossil evidence states that all major orders of placental mammals first appear right after the Cretaceous-Tertiary (KT) boundary approximately 65 million years ago [ 29 ]. This sudden appearance of all major placental orders is known as the mammalian radiation. With the use of molecular data this late radiation has been challenged and it is now widely accepted that the radiation of the placental orders probably occurred many million years before the KT boundary [ 29 - 31 ]. Molecular data have also been used to investigate the relative branching orders of many of the larger clades of placental mammals [ 1 - 7 , 9 , 30 ]. One of the issues that have been debated extensively is the placement of Rodentia in the placental tree. Some studies favour a basal placement of the rodents [ 1 , 3 - 5 , 32 , 33 ] while other studies favour a sister relationship between primates and rodents [ 6 - 8 ]. Recently strong evidence based on insertions, deletions and ancient transposable elements in favour of a sister relationship of primates and rodents has been reported [ 2 , 34 ]. The incongruence of single gene phylogenies was investigated in a recent study of eight yeast species [ 35 ]. The phylogeny commonly believed to be correct is completely resolved when concatenating 20 or more randomly chosen genes to form a super gene. A concatenated multi gene approach was also shown to resolve single gene incongruences in a recent study on green algae [ 36 ]. Here we use 988 full cDNA alignments comprising 672,918 nucleotides to investigate the branching order of the three mammalian species. We present results based on both single gene phylogenies and a concatenated super gene. All genes including the concatenated super gene were analysed with both nucleotide and amino acid based substitution models. All methods favour a primate-artiodactyls clade with rodents as an outgroup but with a relatively short internal mammalian branch, indicating that the mammalian radiation happened within a short period of time. The different methods used in this study have very different assumptions but they all show the same general results. The HKY85 model takes into account differences in nucleotide frequencies and transition/transversion biases and allows for differences in substitution rates among the lineages. However, it is still possible that complexities unaccounted for such as non-stationarity and irreversibility of the substitution process have created biases that lead to long-branch attraction of Fugu and Mouse and an erroneous conclusion. Furthermore, the incongruence between our analysis and many recent studies is also affected by the following. (1) The choice of outgroup; bony fishes are believed to have diverged approximately 450 million years ago [ 31 ], making saturation effects in synonymous sites a real problem. We are therefore forced to only consider nonsynonymous sites or amino acid replacements in the phylogenetic analyses. The recently completed genome sequence of the chicken ( Gallus gallus ) shows that the average value of dS between human and chicken genes is approximately 1.66 [ 37 ], which indicates that many genes may still be too distantly related for synonymous sites to avoid problems with saturation. A marsupial species would provide a much better outgroup when available [ 3 , 32 ]. (2) Taxon sampling; by only using three species the variance of the parameter estimates can be quite high and the power to discriminate between two conflicting topologies quite low. The sequencing of more species will lessen this problem. (3) Overly simplistic evolutionary models; here we use only nucleotide and amino acid based models. If a more closely related outgroup was available the use of more complex codon based models could be beneficial in resolving the apparent conflict. Several extensions have been made to the codon models during the past few years. One obvious extension to the codon models is a model that incorporates CG avoidance within and over codon boundaries. This will clearly improve the fit of the data to the model and therefore give more accurate parameter estimates. Including context dependencies over codon boundaries and information about protein structure have also been shown to increase the fit of the models to protein coding data and therefore should result in better parameter estimates [ 38 , 39 ]. (4) Gene trees and species trees can be different; the split between the three groups probably occurred within a very short period of time, allowing for the possibility that different genes actually have different phylogenies due to ancient polymorphisms at the time of the speciation. Using even larger number of genes and a sufficiently sophisticated model should lessen this problem [ 35 , 36 ]. The rate of synonymous substitution was estimated to be almost three times higher in rodents than in other mammals, in agreement with previous investigations that also showed an elevated rate in rodents [ 40 - 42 ]. This has historically often been explained by a generation time effect. Species that have short generation times experience more generations in the time span we consider and consequently they will experience more neutral substitutions over time. The fact that the pig, which has a generation time intermediate between mouse and humans, has an intermediate rate of synonymous substitutions, seems to agree with this theory. For a more thorough discussion of the generation time hypothesis in mammals see [ 43 ]. The nearly neutral theory of molecular evolution predicts that the generation time effect should be smaller for non-synonymous substitutions [ 42 , 44 , 45 ]. The simple argument is that animals with short generation times such as rodents often have a very large effective population size. In a population with a large effective population size slightly deleterious mutations will be removed from the gene pool more effectively than in a population with a small effective population size, where genetic drift will reduce the efficiency of natural selection. Figure 4g–h shows the distribution of the dN/dS ratio in the three lineages. The average dN/dS ratio is highest in humans suggesting a small effective population size, while it is smallest in mouse suggesting a larger effective population size. Previous studies of the occurrence of positive selection based on pair wise comparisons have revealed a very low occurrence of positive selection. In a study of 3595 alignments only 17 genes showed evidence of positive selection [ 46 ]. The branch specific models used here only find one gene where the dN/dS ratio is significantly larger than one. The gene reported is XM_165930. XM_165930 was originally annotated as being similar to cold shock domain protein A, but it has recently been removed from Genbank as a result of standard genome annotation processes. Codon based branch-site models similar to the ones used here were used in a paper based on a three way comparison among chimpanzees, humans and mice [ 23 ]. They report that approximately 1.6 % of all the genes studied have been undergoing positive selection in the lineage leading to modern humans. Using a similar criterion our study indicates that approximately 3.0 % of the genes studied have been undergoing positive selection on the lineage leading to humans; the corresponding numbers for pig and mouse are 2.0 % and 2.2 % respectively. When comparing these two studies it is important to consider the following three things: (1) the relatively short average length of the genes studied here decreases the power of the models to detect positive selection; (2) the use of the new BEB method for detecting positively selected sites should reduce the number of false positives, making our estimates more conservative and more accurate; (3) our study deals with a completely different phylogenetic level, covering a much longer time span than the study by Clark and colleagues. The multiple testing and the small number of taxa used in a study like this imply that the results presented should not be taken as conclusive evidence for positive selection, but more as an approach to searching among the thousands of genes to look for genes that may have evolved in a biologically interesting manner. Comparative approaches such as the one we use here can only be a first step towards showing that positive Darwinian selection may be a key part in the evolution of many different gene families. Further experimental and computational analyses must then be used to investigate the suggested candidates more thoroughly. During the course of our investigation a large fraction of the genes were re-annotated as putative pseudogenes: 188/1120 ~16.8%. However, all these genes have uninterrupted reading frames in all three species; only a tiny fraction of all codons seems to have evolved in a neutral-like fashion (ω~1), and the distributions of the synonymous as well as the nonsynonymous rates of these putative pseudogenes are almost identical to the distributions of the remaining genes (results not shown). The only difference is a slight increase in the dN/dS ratio in the human lineage, which is actually due to a few genes that experience an unusually high dN/dS ratio. Omitting these genes from the analysis removes the observed differences completely. Thus, if all these genes are indeed pseudogenes in human, the loss of function must have occurred quite recently and they may not be pseudogenes in pig and mouse. Conclusions The collection of a large set of pig cDNA sequences has enabled us to study long term evolutionary trends in mammalian genes. Our results indicate that the codon models are able to detect evolutionary signals indicating adaptive evolution in several genes. Our phylogenetic investigation of the primate, rodent, artiodactyl split disagree with most recent findings in favouring a primate, artiodactyl clade with rodents as an outgroup. Our study indicates that several genes that are not classified as genes in the most recent human annotation might after all be real genes; or at least they have become pseudogenes very recently, and the orthologous genes in mouse and pig might still be functional. This shows the potential of comparative methods in identifying functional regions of the genome. Methods cDNA alignment Complete cDNA from the domesticated pig Sus scrofa was assembled at the Danish Institute of Agricultural Sciences (DIAS) from cDNA libraries from 100 different tissues constructed at DIAS and the Royal Veterinary and Agricultural University in the following way. Total RNA was purified from selected tissues using Rneasy (Qiagen) or Tri ReagentR and poly(A+) mRNA was selected using Oligotex (Qiagene) or PolyATract (Promega). Directional cloneable cDNA was synthezised from Poly(A+) mRNA using the cDNA Synthesis Kit (Stratagene) and was ligated into Eco RI/Xho I digested pTrueBlue (GenomicsOne) or pBluescript (Stratagene) followed by electrotransformation into E. coli XL1-Blue MRF' (Stratagene). 5'-EST sequencing was performed using standard protocols (Applied Biosystem). The sequences were trimmed to the longest open reading frame and the termination codons were removed. Homologues sequences from human, mouse and the Japanese pufferfish Fugu rubrices were obtained with the blastall program with default parameters; the E-score was set to 10 -8 . We constructed two different datasets, one with and one without Fugu rubrices . Individual alignments were made using ClustalW version 1.83 with default parameters [ 47 ]. We kept the pig reading frame intact in the alignments by removing any columns where the alignment gave rise to gaps in the pig sequence. Alignments that resulted in premature stop codons, or were shorter than 30 codons, were removed. We used the one ratio model to estimate the total branch length of the tree as well as the synonymous branch lengths. These distributions were used to detect peculiar genes where one or more sequences might not be a true orthologue, and all outliers were thereafter removed from the dataset. This analysis gave 1120 alignments of mouse, human and pig, and of these 988 also included Fugu. The 1120 original cDNAs from Sus scrofa have been deposited in Genbank with the following accession numbers: AY609387-AY610506. Phylogeny and rates of evolution Nine hundred and eighty-eight four-species alignments were concatenated into a super gene. The three topologies were compared using the super gene as well as each individual gene. Both nonsynonymous nucleotide substitutions and amino acid substitutions were investigated with PAML v. 3.14 [ 48 ]. The nonsynonymous substitutions were represented by the first and second codon positions of all codons, and the three different topologies were investigated with baseml using the HKY85[ 27 ] model (model = 4) of nucleotide substitutions. The likelihood was then maximized under the three different topologies using all the individual genes as well the concatenated super gene. The codeml program with the codons translated to amino acids (seqtype = 3) were also used to investigate the three topologies. We used different models of amino acid evolution to maximize the likelihood under the three topologies and since the results were highly similar we only present the results from the empirical method of Whelan and Goldman (model = 2, aaratefile = wag.dat)[ 26 ]. Using the 1120 three species alignments, the synonymous and nonsynonymous rates of evolution were estimated with the codeml program (seqtype = 1) using the free ratio model (model = 2) with the transition/transversion ratio estimated from the data (fix_kappa = 0). Investigation of selection The different tests for positive Darwinian selection are all based on extensions of the basic codon based likelihood model [ 11 ]. Likelihood ratio tests (LRTs) were used to compare nested models where one allows for positive selection and the other does not. The probability that a codon i substitutes into another codon j during the time interval t is determined by the rate matrix Q = ( q ij ) with entries for i ≠ j , with corresponding substitution probability matrix given by exp(Qt). Here π j is the equilibrium codon frequency of codon j , κ is the transition/transversion ratio and ω is the dN/dS ratio. All parameters are estimated independently for each gene. The star topology of the three species is used to estimate the branch lengths (τ human , τ pig , τ mouse ) for synonymous and non-synonymous substitutions. Positive selection was tested in two different ways. Test 1 averages over sites but differentiates among lineages. The LRT compares the free ratio model where all three lineages have a different value of ω estimated from the data with the one ratio model where all three lineages share a common value of ω [ 14 ]. We note that this test is more a test of variable dN/dS ratios among lineages than a test for positive selection. The free ratio model has three parameters for ω and the one ratio model only one. The LRT statistic is calculated as 2 times the differences in maximum log likelihood and is asymptotically distributed as a χ 2 distribution with 2 degrees of freedom. The genes found in one or more lineages evolving with a dN/dS ratio > 1 are compared to a nested model where the dN/dS ratio is fixed at 1 in the lineages shown to have a dN/dS ratio larger than one to see whether the result can be attributed to natural selection or just relaxation of selective pressures. Test 2 is based on a new and improved version of the branch-site method presented in [ 23 ]. We will refer to this model as model A. The LRT is based on a comparison of the neutral model and model A. The neutral model assumes two categories of sites, a proportion p 1 of sites where ω 1 are estimated from the data and is forced to lie between 0 and 1, and a proportion p 2 of neutrally evolving sites where ω 1 = 1 ( p 1 + p 2 = 1). Model A furthermore allows a pre-specified branch to have a proportion of sites that evolve with a different value of ω estimated from the data. This value cannot be smaller than 1. The LRT follows a χ 2 distribution with 2 degrees of freedom. If the value of ω in the foreground lineage is estimated to be equal to one the model collapses to the neutral model. PAML v. 3.14 [ 48 ] was used to estimate likelihood and parameters under each model. Codon equilibrium frequencies can be estimated from data using either simple proportions in the full data set (the CT model with 60 parameters), assuming equal frequencies (Fequal model), multiplying overall counts of nucleotide frequencies (F1 × 4 model, 3 parameters) or counts of nucleotide frequencies for each codon position (F3 × 4 model, 9 parameters). The codon table (CT) was used for analysis of the concatenated super gene and the F3 × 4 model was used on the individual genes. CpG Extension of the codon models A simple extension of the F3 × 4 codon equilibrium frequency model can incorporate CpG avoidance by adding an extra parameter that penalizes a C followed by a G in the second and third codon position. The new model is parameterised as follows Here π i 1 1 represents the frequency of nucleotide i 1 , at codon position 1, and ψ(0 < ψ < 1) is a CpG penalizing parameter. The scaling factor c ψ ensures that the codon frequencies sum to one. Authors' contributions FGJ carried out the analyses and was the primary writer of the text. AH and FGJ together implemented some of the analysis tools. FGJ, AH and MHS together developed the ideas and discussed the interpretation of the results. MF and CB gathered the EST data used. HHJ assembled the cDNAs and carried out Blast searches. All authors read and approved the final manuscript.

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Computational identification of developmental enhancers: conservation and function of transcription factor binding-site clusters in Drosophila melanogaster and Drosophila pseudoobscura

27 predicted gene-regulatory regions in the Drosophila melanogaster genome were analyzed in vivo , confirming 15 active enhancer regions. A comparison with Drosophila pseudoobscura sequences revealed that conservation of binding-site clusters accurately discriminates functional regions from non-functional ones.

Background The transcription of protein-coding genes in distinct temporal and spatial patterns plays a central role in the differentiation and development of animal embryos. Decoding how the unique expression pattern of every transcript is encoded in DNA is essential to understanding how genome sequences specify organismal form and function. Understanding gene regulation requires discovering the cis -acting sequences that control transcription, identifying which trans -acting factors act on each regulatory sequence, and determining how these interactions affect the timing and organization of transcription. The first step in this process is by no means straightforward. Regulatory regions are often large and complex. Functional cis -acting sequences are found 5' and 3' of transcripts and in introns, and can act over short or long distances. Most of the described animal regulatory sequences were identified by experimental dissection of a locus, and astonishingly few of these are well characterized. Despite the paucity of good examples, as multiple regulatory sequences from different organisms were identified and characterized, some common features became apparent [ 1 , 2 ]. Most animal regulatory sequences act as compact modular units, with regions of roughly a kilobase (kb) in size controlling specific aspects of a gene's transcription. These regulatory units - referred to here as cis -regulatory modules (CRMs) - tend to contain functional binding sites for several different transcription factors, often with multiple sites for each factor. As the first animal genome sequences were completed [ 3 - 6 ], researchers began to tackle the challenge of identifying regulatory sequences on a genomic scale. We and several other groups began to ask whether common characteristics of regulatory sequences - modularity and high binding-site density - might be distinguishing characteristics that would permit the computational identification of new regulatory sequences. A number of in silico methods to identify regulatory sequences on the basis of binding-site clustering have been developed and applied to animal genomes [ 7 - 10 ]. Some of the predictions have the expected in vivo regulatory activity [ 11 - 17 ], yet few of these predictions have been systematically evaluated. The transcriptional regulatory network governing early Drosophila development is perhaps the best system in which to apply and evaluate these methods. Development of the Drosophila embryo is arguably better understood than that of any other animal. Sophisticated genetic screens [ 18 , 19 ] have identified most of the key regulators of early development, and the molecular biology and biochemistry of these factors and their target sequences have received a great deal of attention. The spatial and temporal embryonic expression patterns of a large number of genes are known from microarray [ 20 ] and in situ expression studies [ 21 ]. Transcriptional regulation plays a uniquely important role in pre-gastrula patterning, as most of the key events occur in the absence of cell membranes and the cell-cell signaling systems that play a crucial role later in fly development and throughout the development of most other animals. In a previous study [ 11 ], we identified 37 regions of the Drosophila melanogaster genome with unusually high densities of predicted binding sites for the early-acting transcription factors Bicoid (BCD), Hunchback (HB), Krüppel (KR), Knirps (KNI) and Caudal (CAD). As nine of these regions overlapped previously known CRMs, we proposed the remaining 28 as predicted CRMs (pCRMs). We tested one of the previously untested pCRMs for enhancer activity in a standard reporter gene assay [ 22 , 23 ] and showed that it is responsible for directing a portion of the embryonic expression pattern of the gap transcription factor gene giant ( gt ) in a posterior stripe. Here, we report the systematic testing of the remaining 27 untested pCRMs for enhancer activity, resulting in collections of both bona fide positive and false-positive predictions, allowing us to develop and evaluate methods to improve the accuracy of methods for identifying functional cis -regulatory sequences. We were particularly interested in methods based on the comparison of genome sequences of related species. The genome sequence of D. pseudoobscura (which diverged from D. melanogaster approximately 46 million years ago [ 24 ]) was recently completed by the Baylor Human Genome Sequencing Center, and several other Drosophila species are currently being sequenced. The morphological and molecular events in early embryonic development are highly conserved among drosophilids, and we expect the activity of the transcriptional regulators and the architecture of regulatory networks to be highly conserved as well. Most D. melanogaster regulatory sequences should have functional orthologs in other Drosophila species [ 25 , 26 ], and a major rationale for sequencing other Drosophila species is the expectation that regulatory sequences have characteristic patterns of evolution that can be used to identify them and to better understand their function. Most methods used to identify regulatory sequences from interspecies sequence comparison are fairly simple. They identify 'conserved' non-coding sequences (CNSs), operationally defined as islands of non-coding sequence with relatively high conservation flanked by regions of low conservation, and assume that this conservation reflects regulatory function. Although crude, these methods have been remarkably effective in identifying mammalian regulatory sequences [ 27 , 28 ], and preliminary studies in Drosophila suggest that similar methods will be valuable in insects as well [ 29 ]. However, despite such successes, the extent of the efficacy of comparative sequence analysis in regulatory sequence discovery remains unclear. A systematic comparison of human-mouse sequence conservation in known regulatory regions and ancestral repeats (which provide a model for neutral evolution) suggests that regulatory regions cannot generally be distinguished on the basis of simple sequence conservation measures alone [ 30 , 31 ]. Similarly, a recent analysis of D. melanogaster and D. pseudoobscura showed that known regulatory regions are only slightly more conserved than the rest of the non-coding genome [ 32 ], highlighting the need for further study and the development of comparative methods that go beyond measures of sequence identity. Results Expression patterns of pCRM containing transgenes The 37 pCRMs are shown in Table 1 . Each has been assigned an identifier (of the form PCEXXXX). The first nine overlap previously known enhancers of runt ( run ), even-skipped ( eve ), hairy ( h ), knirps ( kni ) and hunchback ( hb ). To determine whether any of the remaining 28 pCRMs also function as enhancers, we generated P-element constructs containing the pCRM sequence with minimal flanking sequence on both sides fused to the eve basal promoter and a lacZ reporter gene (see Materials and methods). As the margins of the tested sequences do not precisely correspond to the margins of the clusters, we assigned a unique identifier (of the form CEXXXX) to each tested fragment (identical CE and PCE numbers correspond to the same pCRM). We successfully generated multiple independent transgenic fly lines for 27 of the 28 pCRMs. We repeatedly failed to generate transgenes containing CE8007. This sequence contains five copies of an approximately 358 base-pair (bp) degenerate repeat. One additional pCRM (CE8002) also contains tandem repeats. While we were able to generate transgenes for CE8002 and assay its expression, these two tandem repeat-containing pCRMs (CE8007 and CE8002) were excluded from subsequent analyses. We examined the expression of these constructs by in situ RNA hybridization to the lacZ transcript in embryos at different stages in at least three independent transformant lines. Nine of the 27 transgenes showed mRNA expression during embryogenesis (Figure 1 ), while the remaining 18 assayed transgenes showed no detectable expression at any stage during embryogenesis. To identify the genes regulated by the nine pCRMs with embryonic expression, we examined the expression patterns of genes containing the pCRM in an intron and genes with promoters within 20 kb of the CRM (see Figure 1 ). We used the embryonic microrarray and whole-mount in situ expression data available in the Berkeley Gene Expression Database [ 21 ], supplemented with additional whole-mount in situ experiments where necessary (data not shown; these new in situ 's will be included in the public expression database [ 33 ] at its next release). Six of the active pCRMs drive lacZ expression in patterns that recapitulate portions of the expression of a gene adjacent to or containing the pCRM. Four of these new enhancers act in the blastoderm and two during germ-band elongation. CE8001 is 5' of the gene for the gap transcription factor giant and recapitulates the posterior domain (65-85% egg length measuring from the anterior end of the embryo) of gt expression in the blastoderm as previously described [ 11 ]. CE8011 is 5' of the gene for the POU-homeobox transcription factor nubbin ( nub ). The CRM recapitulates the endogenous blastoderm expression pattern of nub , first detected as a broad band extending from 50 to 75% egg length. Although nub expression continues in later embryonic stages, CE8011 expression is limited to the blastoderm stage. CE8010 is 5' of the pair-rule gene odd-skipped ( odd ) and drives expression of two of its seven stripes: stripe 3 at 55% and stripe 6 at 75% egg length. This CRM also has the ability to drive later, more complex, patterns of expression. During stages 6 and 7, expression is detected in the procephalic ectoderm anlage and in the primordium of the posterior midgut. By stage 13, expression is also detected in the anterior cells of the midgut which will give rise to the proventriulus, the first midgut constriction, the posterior midgut and microtubule primordial as well as cells in the hindgut, all similar to portions of the pattern of wildtype odd protein expression previously described [ 34 ]. CE8024 is 3' of the pair-rule gene fushi-tarazu ( ftz ) and drives expression of two of its stripes: stripe 1 at 35% and stripe 5 at 65% egg length. Using a similar CRM reporter assay, this pattern of expression was also detected by [ 35 ]. CE8012 is in the third intron of POU domain protein 2 ( pdm2 ) and appears to completely recapitulate its stage-12 expression pattern, which is limited to a subset of the developing neuroblasts and ganglion mother cells of the developing central nervous system. A similar pattern of expression was previously described for the protein product of pdm2 [ 36 ]. It is worth noting that we do not detect expression of CE8012 in the blastoderm stage, whereas the endogenous gene exhibits a blastoderm expression pattern similar to nub . CE8027 is 3' of the gene for the Zn-finger transcription factor squeeze ( sqz ) and recapitulates the wild-type expression pattern of sqz RNA in a subset of cells in the neuroectoderm at stage 12. The wild-type sqz expression pattern was previously described [ 37 ]. The remaining three active pCRMs cannot be easily associated with a specific gene. CE8005 drives expression in the ventral region of the embryo. It is 3' of a gene encoding a ubiquitously expressed Zn-finger containing protein ( CG9650 ) that is maternally expressed and deposited in the embryo. This strong maternal expression potentially obscures a zygotic expression pattern. Two additional adjacent genes, CG32725 and CG1958 , showed no expression in whole-mount in situ hybridization of embryos. CE8016 drives a seven-stripe expression pattern in the blastoderm. It is in the first intron of CG14502 which shows very low level expression by microarrays in the blastoderm, and has no obvious detectable pattern of expression in whole-mount in situ hybridization of embryos. This pCRM is approximately 2 kb 5' of scribbler (sbb) , which is expressed maternally, possibly obscuring an early zygotic expression pattern (a few in situ images show a hint of striping). sbb is also expressed later in development in the ventral nervous system. An additional potential target, Otefin ( Ote ), is also expressed maternally and relatively ubiquitously through germ-band extension. All other nearby genes displayed in Figure 1 showed no embryonic expression in whole-mount in situ hybridization or by microarray. CE8020 drives an atypical four-stripe pattern in the blastoderm - two stripes at 7% and 26% that are anterior to the first ftz stripe and two stripes at 39% and 87%. It is in the first intron of ome ( CG32145 ), which is not expressed maternally and has no blastoderm expression, but is expressed late in salivary gland, trachea, hindgut and a subset of the epidermis. All other nearby genes displayed in Figure 1 showed no embryonic expression in whole-mount in situ hybridization or by microarray. With these results, and the nine previously known enhancers, at least 15 of the 37 highest density clusters of the five transcription factors used in our initial screen have early-embryonic enhancer activity. The remainder of this paper examines 35 of the original 37 clusters, with the two tandem repeat-containing clusters excluded. We divide these 35 into three categories - 15 positives (the nine overlapping previously known enhancers plus the six new enhancers identified here), three ambiguous (the three positives without a clear regulated gene), and 17 negatives (see Table 2 ). We largely focus on differences between the positives and negatives. Distinguishing active and inactive clusters All 15 positives are within 20 kb of the transcription start site (or, where the transcription start site is unknown, the start of the gene annotation) of transcripts expressed in spatiotemporal patterns consistent with regulation by the maternal and gap transcription factors used in our screen (that is, in anterior-posterior patterns in the blastoderm or in the developing neuroblasts of the central nervous system). Only one of the 17 negatives was located within 20 kb of a plausible target (PCE8021 is 7 kb upstream of reaper ), so out of 16 pCRMs located within 20 kb of a gene with appropriate expression, 15 (94%) are active enhancers. The positives are, on average, larger than the negatives (average cluster size of positive = 900 bp, while average cluster size of negatives was 711 bp), a difference that is significant by the Komogorov-Smirnov (KS) test ( p = 0.017). The positives have a slightly higher density of binding sites, but this difference was not significant. The binding site composition of the positives and negatives are similar (the positives contain more KR, and fewer BCD binding sites, but again these differences are not highly significant). Although others have reported that some factors have characteristic spacings with respect to themselves and other factors [ 38 ], we could not find evidence for such spacing or identify other differences that could distinguish positive pCRMs from negative (Figure 2 ). Use of D. pseudoobscura We assembled the D. pseudoobscura genome from traces deposited in the NCBI's TraceDB using the Celera assembler [ 39 , 40 ]. These assemblies were used to examine the conservation of our pCRMs and to assess whether conservation could be used instead of or in addition to binding site clustering as a way to identify CRMs. We first assessed whether positive pCRMs could be distinguished from their flanking sequences based on degree of conservation. In vertebrate comparative genomics, relatively simple methods (such as VISTA [ 41 ]) are commonly used to identify CNSs that are a surprisingly rich source of new cis -regulatory sequences. We evaluated the potential of using such methods with D. melanogaster and D. pseudoobscura in two ways. First, we constructed percent-identity plots for the regions containing all of the 37 pCRMs (Figure 3 ; similar plots for all pCRMs are available in the online supplement at [ 42 ]) with the location of pCRMs and other known regulatory sequences clearly indicated. Although it appears that some CRMs (that is, eve stripe 3/7) would have been successfully identified by such simple comparative methods, positive pCRMs do not collectively appear distinguishable from flanking sequence on the basis of conservation alone. Although positive pCRMs are almost all in highly conserved blocks, there is a surprisingly high amount of non-coding sequence conservation throughout these regions, and most negative pCRMs are also contained in highly conserved blocks. It remains to be seen whether this difference in the conservation landscape of Drosophila non-coding sequences compared to vertebrates reflects a significant difference in the functional organization of non-coding sequences, or simply indicates that there is too little divergence between D. melanogaster and D. pseudoobscura to detect useful differences in the rates of evolution (see Discussion). We next assessed whether positive pCRMs can be distinguished from negative pCRMs on the basis of their degree of similarity between D. melanogaster and D. pseudoobscura . For each pCRM-containing region, we identified orthologous contigs from the D. pseudoobscura assembly and aligned them using the alignment program LAGAN [ 43 ]. We were able to find orthologous regions for 32 pCRMs (see Table 2 ). Using the simple measure of percent identity, we find that positive pCRMs are, on average, more highly conserved than negative pCRMs (see Table 2 ). Although this difference is significant ( p = 0.002 by KS test), the distribution of conservation scores for positive and negative pCRMs overlap considerably, and thus conservation alone is not a useful way of distinguishing positive and negative pCRMs (see Figure 4b ). To get a genome-wide perspective on the degree of conservation in positive pCRMs, we analyzed the conservation of CRM-sized (1 kb) regions in randomly chosen sections of the genome (Figure 4b ). Positive pCRMs are, generally, more conserved than average CRM-sized sequences, and some positive pCRMs are among the most highly conserved non-coding sequences in the genome. However, a conservation cut-off necessary to select the majority of positive pCRMs would select roughly one third of the non-coding regions of the genome, and thus is not a practical method for prioritizing sequences for functional analysis. Conservation of binding sites and conservation of clustering We expect that most genes will have similar expression patterns in D. melanogaster and D. pseudoobscura , and that most D. melanogaster enhancers should have functional orthologs in D. pseudoobscura . For those enhancers we seek to identify here - namely those where binding site clustering reflects their function - we expect clustering to be found in both D. melanogaster and D. pseudoobscura . Conversely, clusters that simply occur by chance in either genome but do not reflect the function of the sequence (as, we believe, is the case for many of our false-positive predictions) should not be conserved. Thus, looking for conservation of binding-site clustering should provide a valuable way of distinguishing functional and non-functional binding-site clusters in the D. melanogaster genome. We used the alignments described above to examine the conservation of individual predicted binding sites in all of the pCRMs (Table 2 ). We refer to a predicted D. melanogaster binding site that overlaps a predicted D. pseudoobscura binding site for the same factor in an alignment as an 'aligned' site. We require overlap and not perfect alignment to compensate for alignment ambiguity; the overwhelming majority (85%) of aligned sites are perfectly aligned. Although there is only a subtle difference in the binding-site density in the positive and negative pCRMs in D. melanogaster (22.7 sites/kb compared to 22.2), the density of aligned binding sites in positive pCRMs (13.8 sites/kb) is nearly twice that in negative pCRMs (6.8 sites/kb). This is a highly significant difference ( p < 0.001 by KS test) and aligned site density better discriminates positive and negative pCRMs than sequence conservation (compare Figure 4c and 4b ). Sixty-one percent of the predicted binding sites in positive pCRMs are aligned, while only 30% of the sites in negative pCRMs are aligned. Across the genome, 22.3% of predicted binding sites are aligned meaning that there is a roughly fourfold increase over background in the probability that a binding site in a positive pCRM is conserved in place compared to a binding site in a negative pCRM. Sixty-one percent is almost certainly an underestimate of the fraction of pCRM sites that are functionally conserved. The D. melanogaster - D. pseudoobscura alignments were not always unambiguous (using simulations we have assessed the role of alignment algorithms in identifying conserved transcription factor binding sites, see [ 44 ]), and some orthologous binding sites may not have been properly aligned. More important, studies of the evolution of various Drosophila enhancers suggest that the positions of binding sites within an enhancer are somewhat plastic, and the functional conservation of a binding site does not necessarily require positional conservation [ 25 , 26 ]. To characterize the extent of binding site conservation independent of positional conservation, we computed a second measure of binding-site conservation. We consider an unaligned binding site in D. melanogaster to be 'preserved' if it can be matched to a corresponding site in the D. pseudoobscura pCRM (allowing each D. pseudoobscura site to match only one D. melanogaste r site). If we consider both aligned and preserved sites to be conserved, then roughly 80% of the sites in positive pCRMs are conserved compared with 40% in negative pCRMs. The density of preserved but not aligned sites in positive pCRMs (4.3/kb) is considerably higher than in negative pCRMs (2.2/kb) or random sequences (1.8/kb). Thus, in the D. pseudoobscura orthologs of active D. melanogaster CRMs we observe an increase in binding-site density that cannot be explained by the positional conservation of sites found in D. melanogaster or the random occurrence of sites in the genome. Several of the 15 positive CRMs have high densities of these preserved but unaligned sites, but two in particular, runt stripe 3 and hairy stripe 6, stand out from the rest. These two have almost as many preserved sites as strictly aligned sites. Aligned plus preserved (conserved) site density (Figure 4d ) almost perfectly separates positive from negative pCRMs. Only one of the positive pCRMs (PCE8012) has a conserved site density below 14 sites/kb, while only one of the negative pCRMs (PCE8021) has a conserved site density above 14 sites/kb. eCIS-ANALYST: a comparative enhancer finder As the conservation of binding sites and binding-site clusters between D. melanogaster and D. pseudoobscura successfully distinguishes positive and negative predictions made using the D. melanogaster sequence alone, we incorporated comparative sequence data into our enhancer-prediction algorithm CIS-ANALYST [ 11 ]. Instead of searching for clusters of predicted binding sites in a single genome, eCIS-ANALYST (the 'e' is for evolutionary) searches for conserved clusters of sites between the two genomes (see Materials and methods). eCIS-ANALYST is available at [ 45 ]. Using 17 negative pCRMs and an expanded set of 25 positive pCRMs (which included the 15 positive predictions discussed above and 10 functional enhancers known to respond to the five factors; these 10 additional enhancers were discussed and analyzed in [ 11 ] but had binding-site densities below the threshold used there), we compared the ability of CIS-ANALYST and eCIS-ANALYST to identify positive pCRMs and to distinguish positive and negative pCRMs at different binding-site density cutoffs (Figure 5 ). The incorporation of the conservation criteria greatly improves the algorithm's apparent performance. The expected fraction of false positives is markedly reduced, and it is possible to lower the binding site threshold to recover six of the ten previously missed positive enhancers without increasing the number of expected false-positive predictions. New predictions As eCIS-ANALYST has markedly better specificity than CIS-ANALYST, we sought to identify BCD, HB, KR, KNI and CAD targets that were missed with the relatively stringent criteria used in our previous analysis. Rather than use a stringent cutoff (15 binding sites per 700 bp) as we did in [ 11 ], we performed three separate runs with lower cutoffs (for example, 10 sites per 700 bp in one run) and applied a conservation threshold (see Materials and methods and Additional data file 3) to select 929 conserved binding-site clusters. There were 842 new pCRMs within 20 kb or in an intron of an annotated transcript (Additional data file 7) and 87 more than 20 kb (Additional data file 8). We ranked these new pCRMs by a simple scoring scheme that measures both the density and the total number of sites conserved (we evaluated several different scoring schemes, and selected one that optimally identified regions near genes with blastoderm expression patterns; see Materials and methods). The 75 highest-scoring pCRMs within 20 kb of an annotated transcript are shown in Table 3 . Thirteen of the 15 positive pCRMs described above are in the top 75 ( ftz stripe 1/5 is number 107 and the pdm2 neurogenic enhancer is number 418) as are five other known enhancers. One of our negative pCRMs, CE8021, is ranked number 12. To focus our search for new enhancers on genes likely to be regulated by BCD, HB, KR, KNI and/or CAD, we searched FlyBase [ 46 ] and a database of Drosophila embryonic expression patterns [ 21 ] and identified 278 genes with anterior-posterior patterns in the blastoderm (AP genes; Figure 6 and see also Additional data files 2 and 9). Thirty-one of the 75 highest-scoring new predictions are adjacent to or within 20 kb of one or more of these genes, including 11 pCRMs that do not overlap previously described enhancers. The 75 highest-scoring predictions within 20 kb of an AP gene but not in Table 3 , are shown in Table 4 . In Tables 3 and 4 together, there are 106 high-scoring conserved binding-site clusters near AP genes, 90 of which do not overlap known enhancers. Discussion We performed a large and comprehensive evaluation of the efficacy of computational methods for the identification of functional cis -regulatory modules in Drosophila . Analysis of the in vivo activity of 36 high-density clusters of predicted BCD, HB, KR, KNI and CAD binding sites identified in our previous study [ 11 ] offers compelling support for the use of transcription factor binding-site clustering as a method to identify regulatory sequences, as at least 15 of these sequences function as early developmental enhancers in vivo . An evolutionary analysis of these sequences - based on comparisons of the D. melanogaster and D. pseudoobscura genomes - shows that sequence conservation alone can not reliably discriminate cluster-containing regions that function in vivo from those that do not. However, a new method that combines binding-site clustering and comparative sequence analysis to search for binding-site clusters that are present in multiple species does reliably discriminate active and inactive clusters. Using this method, we make several hundred predictions of new CRMs, a large number of which are located near likely target genes. Binding-site clustering The success of relatively simple binding-site clustering methods here and in other work is remarkable given the crudeness of these methods. As our negative predictions demonstrate, the mere presence of a cluster of binding sites is not sufficient to make an active embryonically expressed CRM. Although these 17 sequences have binding-site densities and compositions indistinguishable from their functional cousins, they do not function as enhancers in a simple transgene assay. It is possible that some of these negative pCRMs may be functional enhancers that respond to the factors used in our screen, perhaps requiring a different promoter or other flanking sequences not used in the transgene. While further experiments could address this possibility, we felt these were a low priority, as few of the D. pseudoobscura orthologs of these negative pCRMs have binding-site clusters, and few are near genes with appropriate expression patterns. Thus it is unlikely that many function in their endogenous locations in vivo . Both the general activity and, more important, the specific regulatory output of a CRM are a complex, and still poorly understood, function of the specific architecture of its sites. The emerging picture of the ordered multiprotein complexes that mediate enhancer activity suggests constraints on enhancer composition and architecture [ 1 , 2 , 47 ] whose elucidation will form a critical part of the future dissection of the function of cis -regulatory sequences. It is intriguing that three of the clusters we tested direct expression patterns that bear no obvious relationship to the expression of a neighboring gene despite our extensive efforts to identify such genes. We cannot yet exclude the possibility that these pCRMs have an in vivo function related to their observed expression patterns. However, the poor conservation of these elements in D. pseudoobscura suggest that they do not have a regulatory function, and raises the possibility that some 'random' clusters of binding sites (that occur by chance or perhaps through selection on some functionally unrelated sequence feature) have the necessary characteristics to be active enhancers in the proper genomic environment (that is, near a promoter and not silenced by trans -acting chromatin mechanisms). That any such sequences exist suggests that the compositional and architectural constraints on binding sites in enhancers may be fairly weak. Whatever the nature of these constraints, it is clear that binding-site density is not the sole defining characteristic of functional enhancers. However, it is a surprisingly effective distinguishing one, and the usefulness of this and related methods [ 48 ] suggests that the broader application of such methods to different collections of transcription factors will be extremely valuable in annotating the regulatory content of animal genomes. New enhancers We identified double-stripe enhancers for ftz and odd . ftz and odd are generally classified as 'secondary' pair-rule genes whose expression is governed by other pair-rule genes rather than by the maternal and gap transcription factors that govern the so-called 'primary' pair-rule genes ( eve , h and runt ) ([ 49 ]; also reviewed in [ 50 ]). However, the ftz and odd enhancers described here were identified on the basis of binding sites for maternal and gap transcription factors, and function like the enhancers of primary pair-rule genes in directing expression in specific stripes. It has been suggested that the ftz enhancer is an evolutionary relic of the homeotic role played by ftz in primitive insects [ 51 ], a view supported by the apparently normal expression and activity of ftz when this element is missing. However, given our observation that non-functional binding sites clusters are not conserved, even over the relatively short evolutionary distance separating D. melanogaster and D. pseudoobscura , it seems unlikely that this element is purely vestigial. In fact, Yu and Pick [ 52 ] examined the expression pattern of the endogenous ftz gene and show that stripes 1 and 5 appear before other ftz stripes and they postulate the existence of stripe-specific regulatory elements that may exist outside of the characterized zebra and upstream elements such as the one identified and characterized in this study. The conservation of binding sites in both the ftz and odd enhancers suggest that they play an important role in development, and further call into question the distinction between primary and secondary pair-rule genes. Two of the new enhancers (CE8011 and CE8012) are adjacent to and apparently regulate two linked genes with very similar patterns of embryonic expression. Both nub (also known as pdm1 ) and pdm2 are expressed in the anterior and posterior midgut primordium and in neuroblasts. CE8011, found immediately upstream of nub , regulates its early expression, and not its later neuroblast expression. In contrast, CE8012, found in an intron of pdm2 regulates its expression only in neuroblasts and not earlier. While we did not detect a neuroblast enhancer for nub or a blastoderm enhancer for pdm2 in our single-species binding-site cluster search, a number of interesting pdm2 regions were discovered in our eCIS-ANALYST search (two are listed in Table 4 ). Regulatory models and improving the accuracy of CRM prediction The accuracy of our enhancer predictions would almost certainly be improved if we restricted our search space to genomic regions adjacent to genes known to be regulated by particular transcription factors. Drosophila enhancers have been known to work at distances of up to 100 kb, but most are within 10 kb of their target gene. All of our true-positive predictions were within 10 kb of the known or predicted transcription start site of a gene with a pattern that was known, or plausibly could have been, regulated by the five regulators used in our screen (anterior-posterior patterns in the blastoderm; expression in neuroblasts). In contrast, only one of the negative predictions was this close to such a gene - an additional four were within 50 kb. As the comprehensive atlas of embryonic expression patterns is completed [ 21 , 53 ] it will be possible to restrict searches for CRMs to regions of the genome near genes with expression patterns that could arise from the regulators being considered, or to prioritize the results of whole-genome screens on the basis of whether they are near plausible targets. Comprehensive methods for inferring regulatory interactions where they are not already known will be critical for the widespread application of binding-site clustering methods. In addition to allowing less stringent focused screens, they will also help overcome the combinatorial challenge raised by the existence of up to 700 sequence-specific transcription factors in Drosophila . Even assuming the availability of binding data for all of these factors, it will not be possible to search for targets of all combinations of these factors - there are too many possibilities. This is not just a practical problem - it is a fundamental statistical problem. While the false-positive rate for a single combination of factors is low, if we tried even all pairs of factors, it is likely that every region of the genome would have a high binding-site density for some collection of factors. Sequence data from other Drosophila species may allow us to determine which of these collections are conserved and therefore likely to be functional, but it is unlikely that all aspects of regulation can be inferred from comparative analyses and therefore it is essential that we continue to dissect the regulatory network by traditional means. A greater current limitation in the widespread application of binding-site clustering methods is the absence of high-quality binding data for most Drosophila transcription factors. The initial success of methods that use in vitro binding data to predict regulatory targets has prompted the characterization of binding specificities for many additional factors. However, the heterogeneity of approaches used makes it difficult to combine these data in an optimal manner. In addition, most of the available transcription factor binding data consists of a few to several dozen high-affinity sites. While these data are very useful, they do not fully represent the binding capacity of a factor and thus do not permit the identification of intermediate or low-affinity sites which are known to be important in some regulatory systems [ 54 ]. We have begun to apply high-throughput methods [ 55 ] to characterize a broad spectrum of target sites for all of the transcription factors involved in early embryogenesis. The results will ultimately allow us to estimate the binding affinity of each factor for any target sequence. Comparative genomics in CRM predictions The extent of non-coding sequence conservation between D. melanogaster and D. pseudoobscura was surprising. A major motivation for the National Human Genome Research Institute (NHGRI) support of the D. pseudoobscura genome sequencing was the identification of conserved regions that would guide the annotation of functional sequences in D. melanogaster . D. pseudoobscura was chosen as the second member of this genus to be sequenced in part because it was felt that it had separated from D. melanogaster sufficiently long ago that non-functional sequences would exhibit substantial divergence. However, despite an evolutionary separation that is greater than human and mouse (an average synonymous substitution rate of 1.8-2.6 substitutions/site [ 29 ] compared to 0.6 substitutions/site [ 30 ]), and despite some variation in conservation in non-coding sequences, we were not able to use standard measures of sequence conservation to differentiate active pCRMs from their flanking sequence or from inactive pCRMs, reinforcing other recent observations [ 32 ]. One reason for the limited efficacy of these methods is that they do not recognize the specific patterns of conservation characteristic of different classes of functional sequences. For example, coding sequences can be easily recognized from the characteristic triplet pattern in evolutionary rates where the third (and often synonymous) position of codons tends to evolve at a greater rate than the first two positions [ 56 , 57 ]. Similarly, RNAs that form conserved secondary structures can be recognized by patterns of co-substitution ([ 58 ] and references cited within). The early developmental enhancers we are studying here are made up of large collections of transcription factor-binding sites, and it is expected that both individual functional binding sites and the overall composition of functional CRMs will be conserved [ 25 , 26 ]. Conservation of binding-site clustering is a specific evolutionary signature of this class of functional regulatory sequences, and, like the evolutionary signatures of protein-coding and RNA genes, can be used to specifically identify these sequences from comparative sequence data. Contrast PCE8010 (the odd stripe enhancer) and PCE8015 (Figure 3 ). Both have the same overall amount of sequence conservation, indicating that they are under some functional constraint. However, 80% of the predicted binding sites in PCE8001 are conserved, compared to 20% for PCE8015. The conservation of binding sites (both number and location) in PCE8001 makes it highly unlikely that the cluster was found by chance in D. melanogaster , and suggests (correctly) that this sequence is actively responding to the presence of these binding sites. The poor conservation of binding sites in PCE8015 (no greater than is found in random regions of genome) suggests either that the BCD, HB, KR, KNI and CAD sites in this region are not functional or that the region is undergoing rapid functional diversification. Of course the absence of binding site conservation does not suggest that the sequence is non-functional, merely that these sequences are unlikely to have the particular function we are studying here. From the data shown in Figure 4 , we expect the incorporation of binding-site conservation into the CRM search process to greatly reduce the number of false-positive predictions. We anticipate that a significant number of the new predictions from our genome-wide screen and screen targeted at genes with early anterior-posterior patterns to be active CRMs, and we have begun testing these predictions. The pattern of binding-site conservation in positive pCRMs sheds additional light on the processes that govern CRM evolution. We find that predicted binding sites in positive D. melanogaster pCRMs are roughly three times more likely to be aligned to predicted sites in the D. pseudoobscura compared to predicted binding sites in negative pCRMs, in the sequences flanking pCRMs, or in random regions of the genome. The demonstration that this strictest form of binding-site conservation is strengthened in functional CRMs contrasts with an earlier study that concluded that binding sites in functional CRMs had only a slightly elevated probability of falling in conserved sequence [ 32 ]. Their methodology differed from ours in that they used randomly shuffled binding-site positions within functional CRMs as the background, while we used actual predicted binding-site positions in randomly picked regions of the genome. In addition to this colinear conservation, we also observe that there is an overall enrichment for binding sites in positive pCRMs independent of the conservation of individual sites. Specifically, the presence of a binding site for a factor in a positive D. melanogaster pCRM increases (relative to negative pCRMs and random genomic fragments) the probability of finding a site for the same factor in the orthologous region of D. pseudoobscura , even if the site is not in the same (aligned) position. Thus, in this set of positive pCRMs, there appears to be selection to maintain binding site composition, but not always the specific order and orientation of sites. This is consistent with models of enhancer plasticity that have been proposed and discussed elsewhere [ 25 , 59 - 61 ]. The relative importance of binding-site architecture and binding-site composition to maintaining the function of an enhancer over evolutionary time remains unclear. Over relatively short evolutionary distances (as between D. melanogaster and D. pseudoobscura ) most binding sites are conserved and found in the same place. Over longer evolutionary distances, individual binding sites are often poorly conserved even as the overall composition and function of a CRM is conserved. From a practical perspective, this requires adjusting how conservation is incorporated into searches for clusters of binding sites that are likely to be CRMs. For relatively short evolutionary distances, searches for clusters of aligned sites will be less sensitive to noise and will focus on functional binding sites. For longer distances, where binding site turnover will likely preclude searching for clusters of conserved sites, searches for conserved binding site clusters should still work well. In fact, this latter method can work - with some modification - among species whose sequences can no longer be aligned. Anopheles gambiae diverged from its common ancestor with D. melanogaster roughly 220 million years ago, and there is little or no detectable non-coding sequence similarity between these two species. Nonetheless, we find clusters of HB, KR and KNI binding sites in the vicinity of gap and pair-rule genes and suggest that many of these are functional orthologs of D. melanogaster CRMs. Despite strong selection to maintain function, enough binding-site turnover has occurred in these CRM during their 220 million years of independent evolution to eliminate detectable sequence similarity. But they remain functionally similar and we can detect this functional similarity through its evolutionary signature. With methods like the one we have presented here, aided by new and better binding data on Drosophila transcription factors and an impending wealth of comparative sequence data, we anticipate rapid progress on the identification and functional characterization of regulatory sequences. We will then be able to turn our attention to the next great challenge - understanding the precise relationship between the binding-site composition and architecture of regulatory sequences and the expression patterns they specify. Materials and methods Collection of CRMs The collection of CRM sequences was previously described [ 11 ] Transgenics DNA fragments identified as candidate CRMs were amplified from either bacterial artifical chromosome (BAC) or y; cn bw sp fly genomic DNA by PCR using two primers containing unique sequence and synthetic Asc I and Not I restriction sites (Additional data file 5). The PCR product was digested with Asc I and Not I, and inserted in its native orientation into the Asc I- Not I site of a modified CaSpeR-AUG-bgal transformation vector [ 62 ] containing the eve basal promoter, starting at -42 bp and continuing through codon 22 fused in-frame with lacZ [ 63 ]. The P-element transformation vectors were injected into w 1118 embryos, as described previously [ 63 , 64 ]. Transgenic fly lines containing CRMs CE8005 (7A), CE8016 (55C) and CE8020 (70EF) were verified by generating genomic DNA [ 65 ] from each line for PCR. PCR products were amplified using primers designed from the CaSpeR-AUG-bgal vector - forward primer 5' CGCTTGGAGCTTCGTCAC and reverse primer 5' GAGTAACAACCCGTCGGATTC and 35 cycles (Gene Amp 9700, Perkin-Elmer). The resulting PCR products were sequenced using standard conditions with BigDye version 3.0 and electrophoresed on a 3730 capillary sequencer (ABI). Whole-mount in situ hybridizations Embryonic whole-mount in situ RNA hybridizations were performed as previously described [ 21 ]. RNA probes were generated using cDNA clones RE29225 ( gt ), RE14252 ( odd ), RE34782 ( nub ), RE49429 ( pdm2 ), and RE47384 ( sqz ). Exon 1 of the ftz gene was amplified from genomic DNA using forward primer 5' GCGTTGCGTGCACATC and reverse primer 5' ATTCTTCAGCTTCTGCGTCTG. The PCR product was cloned into the TA vector (Invitrogen) and used to generate ftz RNA probe. Double-labeling RNA probes, using cDNAs or genomic DNA as templates, were labeled with fluorescein-12-UTP while lacZ RNA probes were labeled with digoxigenin-11-UTP (Roche). Hybridizations were performed as described above with the following modifications: (1) 2 μl of each probe were added to give a final concentration of 1:50; (2) sequential alkaline phosphatase staining was performed first with Sigma Fast red to detect endogenous transcripts, stopped by washing for 30 min in 0.1 M glycine-HCl pH 2.2, 0.1% Tween-20 at room temperature, and then continued as described to detect lacZ expression. Assembly The input to the genome assembly was the set of whole-genome shotgun reads from the Baylor Genome Sequencing Center retrieved from the National Center for Biotechnology Information (NCBI) Trace Archive, consisting of 2,607,525 total sequences. After trimming the sequences to remove vector and low-quality regions, the average read length was 607 bp. Approximately 75% of the reads were from short insert (approximately 2.5-3.0 kb) libraries, with another 25% from longer (6-7 kb) libraries. Another 46,040 reads came from the ends of 40-kb fosmids. We ran the Celera Assembler several times, and found that by adjusting one parameter in particular we could produce considerably better assemblies. In particular, the assembler has an arrival rate statistic j , which measures the probability that a contig is repetitive on the basis of its depth of coverage. The default setting is very conservative: if a contig has more than 50% likelihood of being repetitive, it is marked as such and is set aside during most of the assembly process. For large highly repetitive mammalian genomes this setting may be appropriate, but for D. pseudoobscura we found that setting it to 90% or higher produced considerably better contigs, while apparently causing few if any misassemblies. The overall assembly contained 10,089 scaffolds and 10,329 contigs, containing 165,864,212 bp. The estimated span of the scaffolds, using the gap sizes estimated from clone insert sizes, is 172,362,884. The largest scaffold was 3.05 million base-pairs (Mbp) and the scaffold N50 size was 418,046. (The N50 size is the size of the smallest scaffold such that the total length of all scaffolds greater than this size is at least one half the total genome size, where genome size here is 172 Mbp.) There are 308 scaffolds larger than 100,000 bp, whose total span is 129.5 Mbp. The N50 contig size, using 166 Mbp as the genome size (not counting gaps), was 43,555. Another measure of assembly quality is the number of large contigs: if we define 'large' as 10 kbp, then the assembly contains 3177 large contigs whose total length is 131,067,828 bp. (For reference, the assembly produced by the Baylor Human Genome Sequencing Center contains 129.4 Mbp in all contigs, including small ones, and the span of all scaffolds is 139.3 Mbp.) All of our contigs and scaffolds are freely available by anonymous ftp at [ 66 ]. Alignment and conservation of pCRMs The extent and pattern of conservation between D. melanogaster and D. pseudoobscura in regions containing pCRMs were determined as follows. The D. melanogaster genomic sequence of the region of interest (with known repetitive elements masked) was extracted from a BioPerl genome database [ 67 ] containing Release 3.1 sequence and annotations from the Berkeley Drosophila Genome Project [ 68 ]. Potentially orthologous D. pseudoobscura contigs/scaffolds were identified using WU-BLAST 2.0 [ 69 ] using default parameters except for (-span1 -spsepqmax = 5000 -hspsepsmax = 5000 -gapsepmax = 5000 -gapsepsmax = 5000). High-scoring pairs (HSPs) with E-values less than 1e-20 were flagged as potential homologous regions. HSPs located more than 5,000 bp from each other in the D. melanogaster sequence were treated as separate hits. After examining dot-plots of the hits, we noticed a large number of small, local inversions that were found in both our assembly and the assemblies released by the Baylor Human Genome Sequencing Center. We used BLASTZ [ 70 ]) to automatically identify inversions, and when necessary inverted the corresponding D. pseudoobscura sequence. Each D. pseudoobscura sequence was aligned to the D. melanogaster corresponding sequence using LAGAN 1.2 [ 43 ] with default settings. A total of 31 genomic loci of approximately 50 kb were examined; these regions contain 36 pCRMs (the eve and h loci contain three pCRMs each, and PCE8003 and PCE8004 are within 20 kb of each other). Twenty-eight regions had aligned D. pseudoobscura sequence that spanned all or most of the region. For three regions (PCE8002, PCE8003/8004 and PCE8009) we were not able to identify large regions of orthologous sequence; these were excluded from subsequent comparative analyses. Dot-plots of the alignments from all 30 regions are available at [ 42 ]. Scoring gross conservation of pCRMs The conservation of a specific genomic segment was scored as the fraction of D. melanogaster bases aligned to the identical base in aligned regions (percent identity). Scoring binding-site conservation of pCRMs We used two definitions of binding-site conservation. A binding site was considered 'aligned' if it overlaps a predicted D. pseudoobscura binding site for the same factor in the LAGAN alignment. Only overlap, and not strict alignment, was required to compensate for small errors in the alignment. A non-aligned binding site was considered 'preserved' if it could be matched to a D. pseudoobscura site for the same factor within the bounds of the pCRM, allowing each D. pseudoobscura site to be the match for only a single D. melanogaster site. The number of aligned plus preserved sites for each factor in a region is thus equal to the minimum number of sites for that factor in the two species. Generating an orthology map for genome searches To develop an orthology map for genome-wide searches, we used NUCmer [ 71 ] to align the Release 3 D. melanogaster genome (with annotated repetitive elements and transposable elements masked) and the D. pseudoobscura scaffolds described above. NUCmer was run with the command line parameters (-c 36 -g 10 --mum -d 0.3 -l 9). NUCmer generated a collection of short, highly conserved regions of homology ('anchors') spaced on average every 1 kb throughout the D. melanogaster genome. Anchors flanking either side of a D. melanogaster region of interest were used to pull out the corresponding D. pseudoobscura region, and additional flanking anchors were examined to ensure that the region was unambiguously orthologous. The region identified was re-aligned to the melanogaster region with LAGAN 1.2 using default settings. Random sampling of non-coding genome To characterize properties of non-coding sequences across the genome, we picked 4,000 1-kb segments of the D. melanogaster genome, sampled uniformly from all non-coding sequence. For 3,300 of these, we could find orthologous regions in D. pseudoobscura , and these were used to calculate the properties of random non-coding sequence shown in Figure 4 and discussed in the text. Properties determined using this data are considered properties of only the portion of the genome that is detectably orthologous under our conditions. The regions themselves are available as supplemental material at [ 42 ]. eCIS-ANALYST genome searches Binding-site clusters in the D. melanogaster genome were determined as described in [ 11 ], where the minimum number of sites (min_sites) and the window size (wind_size) are variable. Release 3 genomic sequence with exons masked was searched with PATSER [ 72 ] using the following command line options: -c -d2 -l4. An 'alphabet' file (specified with the command line parameter '-a') was used to provide the following background frequencies: A/T = 0.297, G/C = 0.203. Position weight matrix (PWM) models were identical to those used in [ 11 ]. In the online version of eCIS-ANALYST, the minimum PWM match threshold site_p is also variable, but in the current study it was held constant at 0.0003 for all factors. Tests using alternate values for this variable did not lead to significant improvement in prediction efficacy. For each potential D. melanogaster cluster, we identified the corresponding D. pseudoobscura region using the homology anchors described above. A pairwise alignment was made using LAGAN 1.2 (default parameters), and the number of aligned and preserved binding sites were determined as described above. The 2-kb flanking either side of the pCRM was included in the alignment to avoid edge effects, and was subsequently removed when calculating pCRM properties. We examined our functional (positive) and non-functional (negative) pCRMs and noticed that in the positives, the lower bound for the number of conserved sites as a function of D. melanogaster sites followed an approximately logarithmic curve (Additional data file 3). From this observation, we classified a D. melanogaster binding site cluster as conserved if: where NS m is the number of binding sites in the D. melanogaster pCRM and NS c is the number of conserved binding sites. Different values of the logarithmic base b give different behavior. The data shown in Additional data file 3 support values of b between 1.15 and 1.4. We defined a more intuitive parameter, CF (conservation factor), which can range from 0 to 1 where 0 is the least stringent threshold ( b = 1.4) and 1 is the most stringent ( b = 1.15) b = 1.4 - ( CF * (1.4 - 1.15))     (2) We performed genome searches with CF values of 0.25, 0.5, 0.55 and 0.75 and manually inspected the results with respect to false-negative and false-positive rates based on our 15 positive and 17 negative pCRMs (Additional data file 3). While we did not strictly optimize a single metric, we picked the values that gave a reasonable balance between false positives and false negatives, b = 0.25 for aligned sites alone, and b = 0.55 for aligned plus preserved sits. Genome-wide predictions eCIS-ANALYST genome searches were run with the following parameters: min_sites = 10, wind_size = 700 (run #1), and min_sites = 13, wind_size = 1,100 (run #2). All conserved clusters (with conservation defined as described in Equations 1 and 2 above) were combined. In order to capture weaker clusters, we performed an additional run (run number 3) using min_sites = 9, wind_size = 700. For this low stringency run, we used a non-standard conservation threshold different from the one described above, accepting all clusters with at least four aligned plus preserved sites, independent of the number of sites in D. melanogaster . We merged overlapping clusters from runs 1-3, yielding 929 non-overlapping clusters as described in Results. Four metrics were then used to rank these 929 pCRMs: the number of aligned binding sites; the density of aligned binding sites; the number of aligned plus preserved binding sites; and the density of aligned plus preserved binding sites. All values were normalized according to background distribution of random non-coding sequences. The four normalized values were then summed to compute an overall score, which was then renormalized to arrive at a final z-score used to rank pCRMs in Tables 3 and 4 and Additional data files 7, 8, 10, and 11. Additional data files The following additional data files are available with the online version of this article. Additional data file 1 shows the binding site densities (column 1), aligned site densities (column 2), and aligned plus preserved site densities (column 3) for individual transcription factors. The top portion of each panel contains a histogram of the values for randomly chosen 1,000 bp regions of the D. melanogaster genome. The blue line plots the cumulative distribution. The colored asterisks show the average values for each class of pCRM. The panel below the histogram shows the values for each pCRM (each dot represents one pCRM, with positives in blue, negatives in red, ambiguous in green). Additional data file 2 shows expression patterns of 65 genes adjacent to 122 pCRMs identified by eCIS-ANALYST. The images were obtained from the BDGP Embryonic Expression Pattern Database [ 33 ], and include all pCRMs from Additional data files 7,8,10,11 for which an adjacent gene had an early segmentation pattern. Additional data file 3 shows discrimination of positive and negative pCRMs. Comparisons of the number of predicted binding sites in D. melanogaster pCRMs to the number of aligned sites (top panel) and aligned plus preserved sites (bottom panel). Blue dots represent the 15 positive pCRMs from the text; green dots the ten known CRMs that were below the threshold used in [ 11 ]; red dots negative pCRMs; pink dots ambiguous pCRMs. Gray boxes represent the distribution of values for random 1,000 bp non-coding regions. The blue line shows the discrimination function (see Materials and methods). Additional data file 4 shows new pCRMs. Three 30 kb regions were chosen to illustrate new predictions: (A) the argos locus, (B) the CG4702 locus (note that CG31361 is not expressed in blastoderm embryos and PCE8494 is a low-scoring pCRM), and (C) the SoxN locus. Exons are shows as blue boxes, introns are represented with horizontal lines, and the direction of transcription is indicated by the arrow. New pCRMs are shown as gray ovals. The green graphs show average (in 300 bp windows) percent identity and fraction of bases in conserved blocks. Below the percent identity plots are shown insertions (gray boxes) and deletions (orange boxes) in the D. melanogaster sequence relative to their D. pseudoobscura ortholog. The location of binding sites in D. melanogaster , binding sites in D. pseudoobscura and aligned binding sites along with the density of sites averaged over 700 bp are shown in the bottom three panels for each region. Additional data file 5 gives the primers used to amplify pCRMs for transgenics. Additional data file 6 gives additional information from Table 2 . Additional data file 7 gives all new pCRMs from genome-wide eCIS-ANALYST located within 20 kb of annotated transcript. Additional data file 8 gives all new pCRMs from genome-wide eCIS-ANALYST located more than 20 kb from annotated transcript. Additional data file 9 lists genes with anterior-posterior patterns and the source of the information. Additional data file 10 gives all new pCRMs from genome-wide eCIS-ANALYST located within 20 kb of gene with anterior-posterior pattern. And, finally, Additional data file 11 gives all new pCRMs from genome-wide eCIS-ANALYST located between 20 kb and 50 kb from gene with anterior-posterior pattern. Supplementary Material Additional data file 1 The binding site densities (column 1), aligned site densities (column 2), and aligned plus preserved site densities (column 3) for individual transcription factors Click here for additional data file Additional data file 2 Expression patterns of 65 genes adjacent to 122 pCRMs identified by eCIS-ANALYST Click here for additional data file Additional data file 3 Discrimination of positive and negative pCRMs. Comparisons of the number of predicted binding sites in D. melanogaster pCRMs to the number of aligned sites (top panel) and aligned plus preserved sites (bottom panel) Click here for additional data file Additional data file 4 New pCRMs Click here for additional data file Additional data file 5 The primers used to amplify pCRMs for transgenics Click here for additional data file Additional data file 6 Additional information from Table 2 Click here for additional data file Additional data file 7 All new pCRMs from genome-wide eCIS-ANALYST located within 20 kb of annotated transcript Click here for additional data file Additional data file 8 All new pCRMs from genome-wide eCIS-ANALYST located more than 20 kb from annotated transcript Click here for additional data file Additional data file 9 Genes with anterior-posterior patterns and the source of the information Click here for additional data file Additional data file 10 All new pCRMs from genome-wide eCIS-ANALYST located within 20 kb of gene with anterior-posterior pattern Click here for additional data file Additional data file 11 All new pCRMs from genome-wide eCIS-ANALYST located between 20 kb and 50 kb from gene with anterior-posterior pattern Click here for additional data file

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548695

DNA repeat arrays in chicken and human genomes and the adaptive evolution of avian genome size

Background Birds have smaller average genome sizes than other tetrapod classes, and it has been proposed that a relatively low frequency of repeating DNA is one factor in reduction of avian genome sizes. Results DNA repeat arrays in the sequenced portion of the chicken ( Gallus gallus ) autosomes were quantified and compared with those in human autosomes. In the chicken 10.3% of the genome was occupied by DNA repeats, in contrast to 44.9% in human. In the chicken, the percentage of a chromosome occupied by repeats was positively correlated with chromosome length, but even the largest chicken chromosomes had repeat densities much lower than those in human, indicating that avoidance of repeats in the chicken is not confined to minichromosomes. When 294 simple sequence repeat types shared between chicken and human genomes were compared, mean repeat array length and maximum repeat array length were significantly lower in the chicken than in human. Conclusions The fact that the chicken simple sequence repeat arrays were consistently smaller than arrays of the same type in human is evidence that the reduction in repeat array length in the chicken has involved numerous independent evolutionary events. This implies that reduction of DNA repeats in birds is the result of adaptive evolution. Reduction of DNA repeats on minichromosomes may be an adaptation to permit chiasma formation and alignment of small chromosomes. However, the fact that repeat array lengths are consistently reduced on the largest chicken chromosomes supports the hypothesis that other selective factors are at work, presumably related to the reduction of cell size and consequent advantages for the energetic demands of flight.

Background Genomes sizes (as measured by the DNA mass per diploid nucleus) are smaller on average in birds than in other tetrapod classes, and genome sizes within the class Aves show less variation than those of other tetrapod classes [ 1 , 2 ]. It has been proposed that reduced genome size in birds represents an adaptation to the high rate of oxidative metabolism in birds, which results primarily from the demands of flight [ 1 - 4 ]. Cell size and nuclear genome mass are correlated in vertebrates, and cell sizes of birds are smaller than those of mammals [ 1 ]. Smaller cells are advantageous in an animal with a high rate of oxidative metabolism because a smaller cell has a greater surface area per volume of cytoplasm, thus facilitating gas exchange. An alternative to the hypothesis that the reduced genome size is adaptive is the hypothesis that it resulted from an event of genomic DNA loss that was fixed in the ancestor of all birds due to genetic drift. The fixation of even a deleterious mutation is possible if the population undergoes an extreme bottleneck [ 5 ]. Some authors have argued that such a bottleneck may have occurred in the ancestor of birds at the end of the Cretaceous period [ 6 ], although this conclusion is not consistent with recent molecular evidence placing the radiation of the avian orders well prior to that time [ 7 ]. In order to decide whether genome reduction in birds was adaptive or due to a random event, Hughes and Hughes [ 8 ] compared the lengths of corresponding introns of orthologous chicken ( Gallus gallus ) and human ( Homo sapiens ) genes. They found that corresponding introns were significantly shorter in chickens, indicating that numerous independent deletions have occurred in the introns of birds. These results support the hypothesis that genome size reduction in birds is adaptive, since it is unlikely that such a large number of independent deletion events were due to chance alone. Additional evidence in support of the adaptive hypothesis is provided by the observation that a secondary increase in genome size has occurred in avian lineages which have become flightless or have reduced flying ability [ 9 ]. It has been suggested that an important factor in genome size reduction in birds has been that birds have lower levels of repetitive DNA than other vertebrates [ 10 ]. Genomes of mammals and reptiles are estimated to consist of about 30–50% repeats, while those of birds have been estimated to consist of only 15–20% repeats [ 10 - 12 ]. In birds chromosomes are of two types: a minority of macrochomosomes (3–6 μm in length) and a larger number of microchromosomes (0.5–2.5 μm in length). In the chicken, there are six pairs of macrochromosomes, and thirty-three pairs of microchromosomes [ 13 ]. There is a high rate of chiasma formation on avian microchromosomes, and this may be an adaptation that ensures correct pairing of these chromosomes during meiosis and mitosis [ 14 ]. Burt [ 10 ] proposed that the avoidance of repeats in the avian genome may in turn be an adaptation that enhances the probability of chiasma formation between homologous microchromosomes. This hypothesis and the hypothesis that genome size reduction represents an adaptation to flight are not mutually exclusive, since both factors may be at work simultaneously. Consistent with Burt's hypothesis, Wicker et al. [ 15 ] reported that in the chicken genome the ratio of repeats to protein-coding genes is higher on macrochromosomes than on minochromosomes. The sequencing of a substantial portion of the chicken genome has made it possible to examine quantitatively the distribution of repeating sequences on different chromosomes in the genome. Here we compare the distribution of repeats on 28 sequenced autosomes of chicken with that on the 22 human autosomes in order to test the hypothesis that reduction in repeat density in the avian genome has occurred as a result of adaptive evolution. Results The characteristics of repeat arrays on the 28 sequenced chicken chromosomes are summarized in Table 1 . The number of repeat arrays varied from 319 on chromosome 16 to 283,761 on chromosome 1; and the percent of the chromosome occupied by repeats varied from 4.1% on chromosome 32 to 14.9% on chromosome 1. In spite of the considerable variation among chicken chromosomes with respect to the percent of the chromosome occupied by repeats, the overall percentage of the chicken genome occupied by repeats (10.3%) was less than one quarter the percentage of the human genome occupied by repeats (44.9%) (Table 2 ). Even the most repeat-rich chicken chromosome, chromosome 1, had a repeat density less than one third that of the human genome (Tables 1 and 2 ). The range of repeat array lengths was much narrower Table 1 DNA sequence repeats on the assembled portion of the chicken genome. Chromosome Chromosome length (bp) No. repeat arrays Total repeat length (bp) (% of sequence) 1 188,239,860 283,761 27,978,835 (14.9%) 2 147,590,765 214,512 19,430,497 (13.2%) 3 108,638,738 151,571 12,198,434 (11.2%) 4 90,634,903 121,663 8,905,732 (9.8%) 5 56,310,377 69,048 4,638,645 (8.2%) 6 33,893,787 38,873 2,468,824 (7.3%) 7 37,338,262 41,189 2,397,200 (6.4%) 8 30,024,636 33,974 2,086,343 (6.6%) 9 23,409,228 24,255 1,384,475 (5.9%) 10 20,909,726 19,914 1,075,555 (5.1%) 11 19,020054 20,514 1,095,858 (5.8%) 12 19,821,895 19,419 1,116,593 (5.6%) 13 17,279,963 16,894 1,015,160 (5.9%) 14 20,603,938 21,588 1,417,684 (6.9%) 15 12,438,626 11,830 640,595 (5.2%) 16 239,457 319 18,614 (7.8%) 17 10,632,206 9,508 554,602 (5.2%) 18 8,919,268 8,312 574,276 (6.4%) 19 9,463,882 8,635 491,763 (5.2%) 20 13,506,680 12,826 766,482 (5.7%) 21 6,202,554 6,001 359,040 (5.8%) 22 2,228,820 2,636 183,334 (8.2%) 23 5,666,127 4,932 234,823 (5.7%) 24 5,910,111 5,435 356,373 (6.0%) 26 4,255,270 3,385 188,003 (4.4%) 27 2,668,888 2,833 252,335 (9.5%) 28 4,731,479 5,183 446,256 (9.4%) 32 1,018,878 806 42,242 (4.1%) Table 2 Features of DNA sequence repeats on human and chicken autosomes. Human Chicken No. chromosomes analyzed 22 28 Total sequence length (bp) 2,864,255,932 901,598,378 No. repeat arrays 4,698,717 1,160,319 Total repeat length (bp) (% of sequence) 1,287,381,310 (44.9%) 92,440,122 (10.3%) Mean repeat array length (bp) [median] (range) 274.0 [188.0] (7–160,603) 79.7 [25.0] (6–7,096) Mean no. repeat arrays per chromosome [median] (range) 213,578 [219,247] (57,109–409,783) 41,440 [14,860] (319–283,761) In the chicken genome, there was a significant positive correlation (r = 0.847; P < 0.001) between chromosome length and the percentage of the chromosome occupied by repeats (% repeats)(Figure 1 ). The four largest chicken chromosomes (chromosomes 1–4) contributed strongly to this positive correlation. In the case of the four largest chromosomes, there was a clear linear relationship between chromosome length and % repeats (Figure 1 ). In the human genome, there was also a positive, but non-significant correlation (r = 0.412; n.s.) between chromosome length and % repeats (Figure 1 ). Figure 1 The percentage of the chromosome occupied by repeats (% repeats) as a function of chromosome length in human ( blue dots ) and chicken ( red dots ). As illustrated in Figure 1 , % repeats values for human chromosomes were considerably higher than those for chicken chromosomes, even when the chromosome length were similar. An analysis of covariance was used to compare % repeats between the two species, with chromosome length as a covariate. There was a significant difference between species (P < 0.001) and a significant effect of chromosome length (P < 0.001), but there was not a significant interaction between species and chromosome length. These results imply that there is a similar slope to the linear relationship between chromosome length and % repeats in the two species, but that the % repeats values for human are significantly higher than those for chicken. Comparison of summary statistics human and chicken genomes showed that both mean and median repeat array lengths were considerably greater in the former species than in the latter (Table 2 ). In order to provide a statistical test of this difference that was not biased by the presence of different array types in the two genomes, we conducted paired tests on the 294 simple sequence repeat types shared by the two genomes (Table 3 ). Both the mean array length and the maximum array length were significantly greater in human than in chicken (Table 3 ). By contrast, the minimum array length did not differ significantly between chicken and human (Table 3 ). In order to test whether these differences between the two species were due mainly to the influence of the smaller chicken chromosomes, we repeated the analysis using only the four largest chicken chromosomes (chromosomes 1–4). In this case also, both the mean array length and the maximum array length were again significantly greater in human than in chicken, while the minimum array length was not significantly different between species (Table 3 ). Table 3 Mean (± S.E.) of variables describing simple sequence repeat types shared between human and chicken. Human Chicken P (paired-sample t-test) All chicken chromosomes (294 repeat types): Mean array length (bp) 83.6 ± 2.8 58.1 ± 1.5 < 0.001 Minimum array length (bp) 22.1 ± 1.6 22.1 ± 0.7 n.s. Maximum array length (bp) 457.9 ± 25.0 193.5 ± 8.1 < 0.001 Chicken chromosomes 1–4 (286 repeat types): Mean array length (bp) 83.8 ± 2.9 56.4 ± 1.7 < 0.001 Minimum array length (bp) 21.7 ± 1.6 24.8 ± 1.1 n.s. Maximum array length (bp) 466.7 ± 25.5 153.3 ± 6.9 < 0.001 Discussion Tabulation of DNA repeat arrays in the assembled portion of the chicken autosomes showed the overall percentage of repeats to be 10.3%. This value is similar to, but slightly lower than, previously published estimates (about 15%) based on reassociation kinetics [ 11 , 12 ]. By contrast, a similar tabulation in the human autosomes showed the overall percentage of repeats to be 44.9%. Because the value for chicken is substantially lower than the mammalian value, the results support the hypothesis that a relative scarcity of repeating DNA is a major factor in causing the relatively compact size of the avian genome [ 15 ]. Moreover, when simple sequence repeat array types shared between chicken and human genomes were compared, mean repeat array length and maximum repeat array length were significantly lower in the chicken than in human. The fact that these differences occurred consistently in nearly 300 distinct array types is evidence that the reduction in repeat arrays in the chicken has involved numerous independent evolutionary events. Mutational changes to simple sequence repeat arrays typically involve slippage events that either decrease or increase the number of repeat units [ 16 ]. The fact that simple sequence repeat arrays are shorter in the chicken than in the human implies that mutational events increasing array length have been eliminated by selection in the chicken to a greater extent than in human. Such long arrays might have included some that were inherited from the ancestors of birds and others that arose due to mutational events within Aves. In either case, the evidence for numerous, independent events of elimination of long arrays implies that reduction of DNA repeat length and thus of overall genome size in birds has occurred as a result of adaptive evolution. There were substantial differences among chicken chromosomes with respect to the percentage of the chromosome occupied by repeats, and % repeats increased significantly as a function of chromosome length. This trend implies that the avian genome is characterized by an especially pronounced avoidance of longer repeats on the smaller chromosomes. This finding is consistent with the hypothesis of Burt [ 10 ] that the reduction of repeating DNA in avian genomes is adaptive in permitting chiasma formation and alignment of microchromosomes. However, even the largest chicken chromosomes had repeat densities much lower than human chromosomes of similar length (Figure 1 ). This implies that avoidance of repeats on microchromosomes cannot be the sole factor at work in repeat avoidance in avian genomes. This interpretation is further supported by the fact that mean repeat array length and maximum repeat array length of repeat types shared between chicken and human genomes were significantly lower on the largest four chicken chromosomes than in human. Thus, the largest chicken chromosomes, like the rest of the chicken genome, showed a pattern indicating adaptive reduction of repeat array length. Our results imply that some other selective factor besides the need for alignment of minichromosomes contributes to genome size reduction in birds. Together with previous evidence [ 9 ], the results are thus consistent with the hypothesis that genome size reduction in birds is adaptive in that it leads to reduction of cell size and thus is advantageous in view of the energetic demands of flight. Methods The chicken ( Gallus gallus ) genome assembly (May 2004 freeze, release 25.1b.1) was downloaded from Ensembl web site at . Only autosomes were used in the analyses; data were available for chromosomes 1 through 24, 26, 27, 28 and 32. We extracted Ensembl annotations of the features of repeat arrays (including repeat name, start and end positions on the chromosome, and orientation). The human genome assembly (May 2004 freeze, build 35 (hg17)) was downloaded via the UCSC Genome Browser . Repeat information based on the RepeatMasker annotations (repeat name, start and end positions on the chromosome and orientation) was extracted from the UCSC genome annotation database. Only autosomes (chromosomes 1 through 22) were used. For both chicken and human, repeats tallied included simple sequence repeats, class I elements, class II elements, low-complexity regions, and satellite regions. In addition, we compared between genomes a set of 294 simple sequence repeats which were present in both genomes; i.e., repeats of the same short nucleotide motif were present in both genomes. For these 294 repeat types, mean, minimum and maximum length of repeat arrays were compared in pairwise fashion between human and chicken. Authors' contributions HP gathered and summarized the data. ALH conducted statistical analyses and wrote the manuscript. Both authors read and approved the final manuscript.

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548695.xml

539050

Randomized, Controlled Trial of Therapy Interruption in Chronic HIV-1 Infection

Background Approaches to limiting exposure to antiretroviral therapy (ART) drugs are an active area of HIV therapy research. Here we present longitudinal follow-up of a randomized, open-label, single-center study of the immune, viral, and safety outcomes of structured therapy interruptions (TIs) in patients with chronically suppressed HIV-1 infection as compared to equal follow-up of patients on continuous therapy and including a final therapy interruption in both arms. Methods and Findings Forty-two chronically HIV-infected patients on suppressive ART with CD4 counts higher than 400 were randomized 1:1 to either (1) three successive fixed TIs of 2, 4, and 6 wk, with intervening resumption of therapy with resuppression for 4 wk before subsequent interruption, or (2) 40 wk of continuous therapy, with a final open-ended TI in both treatment groups. Main outcome was analysis of the time to viral rebound (>5,000 copies/ml) during the open-ended TI. Secondary outcomes included study-defined safety criteria, viral resistance, therapy failure, and retention of immune reconstitution. There was no difference between the groups in time to viral rebound during the open-ended TI (continuous therapy/single TI, median [interquartile range] = 4 [ 1 – 8 ] wk, n = 21; repeated TI, median [interquartile range] = 5 [ 4 – 8 ] wk, n = 21; p = 0.36). No differences in study-related adverse events, viral set point at 12 or 20 wk of open-ended interruption, viral resistance or therapy failure, retention of CD4 T cell numbers on ART, or retention of lymphoproliferative recall antigen responses were noted between groups. Importantly, resistance detected shortly after initial viremia following the open-ended TI did not result in a lack of resuppression to less than 50 copies/ml after reinitiation of the same drug regimen. Conclusion Cycles of 2- to 6-wk time-fixed TIs in patients with suppressed HIV infection failed to confer a clinically significant benefit with regard to viral suppression off ART. Also, secondary analysis showed no difference between the two strategies in terms of safety, retention of immune reconstitution, and clinical therapy failure. Based on these findings, we suggest that further clinical research on the long-term consequences of TI strategies to decrease drug exposure is warranted.

Introduction Antiretroviral therapy (ART) has been a milestone in the treatment of HIV infection. Current treatment guidelines for HIV-1 infection in the United States recommend the initiation of ART in patients with CD4 T cell counts of less than 350 cells/μl [ 1 ]. In implementing these guidelines, health-care providers face the ongoing challenge of developing treatment strategies that minimize drug-related toxicity and adverse effects while retaining effective control of viral replication. Furthermore, treatment-associated costs (particularly in resource-poor areas), difficulty in maintaining long-term optimal adherence [ 2 ], and the emergence of viral resistance [ 3 , 4 , 5 ] have limited the feasibility of life-long ART-mediated viral suppression, increasing the need for alternative treatment strategies. Intermittent therapy strategies, consisting of alternating cycles on and off ART, have increasingly emerged as a potential intervention to address limitations of continuous ART [ 6 , 7 , 8 , 9 ]. Therapy interruption (TI) studies in ART-treated patients with suppressed HIV infection [ 10 ] have addressed the general questions as to whether such strategies can achieve greater viral control through increased antiviral responses (autoimmunization hypothesis) or simply serve as a strategy to reduce cost of long-term therapy and drug-associated toxicity. While pilot studies and uncontrolled (or incomplete) trials in patients with chronic HIV infection have addressed viral and immune outcomes of fixed-length TI and fixed on-drug cycles [ 11 , 12 , 13 , 14 , 15 , 16 ], no completed randomized, controlled trial has yet addressed by intent-to-treat analysis the outcome during an open-ended TI of sequential TIs versus continuous treatment in patients with confirmed suppression. The largest study to date in this area is the prospective single-arm Swiss–Spanish Intermittent Trial (SSITT) conducted in 133 recruited patients undergoing sequential 2-wk TIs and showing a lack of impact of this strategy on achieving sustained viral loads of less than 5,000 copies/ml off therapy in those that completed the study [ 11 ]. However, the lack of a control arm in this study has left unanswered questions about the impact of multiple TIs on time to rebound, immune reconstitution, therapy failure, and viral resistance when analyzed against a randomized control arm of continuous treatment followed for equal time before a single open-ended interruption. We completed a randomized, controlled trial on the outcome of repeated 2- to 6-wk TIs in patients with chronic infection in which the comparator group maintained continuous therapy and then an open-ended interruption period was applied in both treatment groups. The study addressed the potential for repeated interruptions of therapy to delay time to viral rebound as a primary outcome and analyzed secondary outcomes regarding study-defined safety criteria, viral suppression and resistance, and retention of immune reconstitution. Methods Participants Between August 2000 and December 2003, we enrolled 42 patients infected with HIV who were older than 18 y and on ART; eligibility criteria included CD4 counts of greater than 400 cells/μl on ART with a nadir of no less than 100 cells/μl, ART-mediated suppression (< 500 copies/ml) for more than 6 mo and less than 50 copies/ml at recruitment on any antiretroviral regimen. Approval of the study protocol was obtained from the institutional review board (IRB) of the Philadelphia Field Initiating Group for HIV Trials (Philadelphia, Pennsylvania, United States). Written informed consent was obtained from all patients. Human experimentation guidelines of the United States Department of Health and Human Services and of the authors' institutions were followed. The study protocol, including the patient consent form, the CONSORT form, and the IRB approval, can be found in Protocols S1–S4 . Randomization and Study Design Forty-two eligible patients from the Jonathan Lax Immune Disorder Clinic in Philadelphia, Pennsylvania, were randomized via sealed envelopes in a 1:1 fashion to a first phase (phase I) of either (1) three successive TIs of 2, 4, and 6 wk, respectively, or (2) maintenance of ART for 40 wk before a final interruption of therapy in both arms (phase II) subject to therapy reinitiation criteria as described below. Phase II consisted of an open-ended interruption to allow for virological and immunological comparisons between the groups off therapy. Study visits were every 2 wk for the repeated interruptions group and every 4 wk for the continuous ART group during phase I. Both groups were followed every 2 wk during phase II. We followed a study design with step-wise increases in the length of TI cycles to address potential safety concerns (resuppression was confirmed after shorter TIs before longer interruptions were initiated) and the hypothesis that sequential viral replication intervals would stimulate viral control and a delay in time to viral rebound. Phase I procedures for the repeated interruptions group included the following. (1) Interruption of therapy was individually timed to occur after two HIV RNA measurements of less than 50 copies/ml without any viral load measurements greater than 400 copies/ml in between; these interruptions increased from 2 to 4 to 6 wk sequentially. (2) If a 0.5-log or greater reduction in viral load did not occur by 6 wk of reinitiated therapy or less than 50 copies/ml was not achieved within 20 wk of reinitiated therapy, patients were withdrawn as therapy failures and a resistance test was performed. (3) Patients were also withdrawn as therapy failures if (a) the CD4 cell number declined by more than 45% of the baseline CD4 count, (b) participants developed an opportunistic infection, even if retaining required CD4 count levels, or (c) a viral load of greater than 500,000 copies/ml occurred once, with or without development of acute retroviral syndrome as defined by fever, skin lesions, and pharyngitis. Phase I procedures for the continuous therapy arm included the following: (1) patient monitoring if detected viremia was between 50 and 999 copies/ml, with the patient withdrawn if their viral load did not return to less than 50 copies/ml immediately prior to phase II, and (2) patient study withdrawal as therapy failure if during the 40-wk ART period viral load rebounded to more than 1,000 copies/ml at two consecutive time points. Phase II procedures for both arms included the following: (1) monitoring for patient study withdrawal criteria as described in phase I, (2) determining time to primary end point of a viral load greater than 5,000 copies/ml, (3) monitoring until the time of therapy reinitiation at a viral load greater than 30,000 copies/ml for three consecutive time points, and (4) after reinitiation of therapy, follow-up on therapy to confirm resuppression to less than 50 copies/ml at 6, 10, and 14 wk on therapy. Clinical and laboratory parameters (CD4 count and viral load) were monitored at each visit, and venous blood was collected for additional secondary outcomes during selected study visits. In both phase I and II, participants taking non-nucleoside reverse-transcriptase inhibitors (NNRTIs) were instructed to stop them a day earlier than the remaining drugs in the regimen. Primary and Secondary Outcomes The primary outcome was time to confirmed virological rebound during phase II. Rebound was defined as first time point with greater than 5,000 copies/ml. Viral replication magnitude as defined by mean HIV-1 plasma RNA area under the curve (AUC HIV RNA ) was measured as a secondary outcome at weeks 12 and 20 of phase II based on reinitiation-of-therapy criteria outlined above. Additional secondary outcomes included (1) safety outcomes (serious adverse events [SAEs] and patient withdrawal based on criteria defined above), (2) retention of ART-mediated immune reconstitution, and (3) detection of viral resistance. Retention of immune reconstitution was analyzed by (1) same-day whole blood flow-cytometry-based analysis of CD4 and CD8 T cells, including total and naïve (CD62 l/CD45RA) and memory (CD45RO) subsets as described [ 17 ], and (2) same-day recall response analysis of peripheral blood mononuclear cell lymphoproliferative responses to Candida albicans as described [ 17 ]. Viral resistance mutations were retrospectively analyzed on cryopreserved plasma samples by genotyping of first available sample with viral load greater than 100 copies/ml following each interruption using the TruGene Assay (Visible Genetics, Toronto, Canada) at the Gladstone Institute of Virology and Immunology (San Francisco, California, United States) as previously described [ 18 , 19 ]. Sample Size The sample size required was calculated using PS [ 20 ] software, and based on a type I error of 0.05, with 90% power, to detect a difference of 4 wk or more in time to viral rebound between arms. Eighteen patients per group resulted in sufficient power (18 for 90%, 13 for 80%) to determine a difference of 4 wk or greater between groups in time to rebound of virus during the open-ended interruption. Assuming a loss to follow-up of 15%, we targeted 21 patients per group, or 42 total. Statistical Analysis The primary analysis was an intent-to-treat analysis in which dropouts were assigned a week 0 rebound time (e.g., maximum failure to delay rebound). In secondary analyses, these dropouts were excluded. The log-rank test was used to test the null hypothesis of no difference between arms in the number of weeks from initiation of the open-ended TI to reaching viral rebound as defined. Patients not reaching end point at 26 wk after the beginning of the open-ended TI were censored. Wilcoxon rank sum tests were used to compare baseline and week 0 of the open-ended interruption between groups. Wilcoxon signed rank tests were used to test for no change from baseline to week 0 of phase II. Finally, Wilcoxon rank sum tests were employed to test between groups for equality of the mean AUC HIV RNA up to 12 and 20 wk. In all cases, a two-sided alpha level of 0.05 was used to define statistical significance. Unless otherwise stated, results are presented as median (interquartile range) in text and tables. Results Patient Flow and Discontinuations Trial patient flow is summarized in Figure 1 . Between August 2000 and December 2003, 42 patients at the Jonathan Lax Immune Disorder Clinic at the Philadelphia Field Initiating Group for HIV Trials were enrolled, randomized, and followed as shown in Figure 2 . In the continuous therapy/single interruption arm, 16 of 21 patients reached the open-ended interruption. Reasons for study discontinuation in this arm were loss to follow-up ( n = 1; patient moved away) and virological failure during continuous therapy ( n = 4; further discussed below). In the repeated interruptions arm, 18 of 21 patients reached the open-ended interruption following three TIs of 2, 4, and 6 wk duration, with median peak rises in viral loads of 136 (50–2,590), 13,651 (180–222,589), and 18,887 (3,893–96,101) copies/ml, respectively. Median time to less than 50 copies/ml after resumption of therapy was 2 (0–4), 3 (1.8–12), and 9.5 (2–12) wk, respectively, with 9, 18, and 20 wk as the maximum time needed to achieve suppression in 100% of patients before reaching the open-ended interruption. Study discontinuation in the repeated interruptions arm was due to protocol violation ( n = 1; patient restarted therapy during interruption out of protocol), loss to follow-up ( n = 1; patient imprisoned), and virological failure during on-therapy period ( n = 1; further discussed below). Figure 1 Study Flow Figure 2 Study Design (Phases I and II) Baseline Criteria and Follow-Up The demographic and clinical characteristics of the two groups at baseline are summarized in Table 1 . Seventy-five percent of participants were on their second to fourth regimen while 25% were in their first regimen . No significant difference was found in baseline parameters between arms, with 33%–47% of patients on protease-inhibitor-containing and 61%–71% on NNRTI-containing regimens. Owing to the high participation of patients on NNRTI-based regimens and concerns about TI and safety in general, patient outcomes and treatment failure were reviewed monthly by the IRB of this study during the first 8 mo of study, quarterly for the following 4 mo, and semi-annually thereafter. Figure 2 shows study design for both arms, with a median follow-up of 41 (41–42) wk during phase I for the continuous therapy/single interruption arm and 42 (30–51) wk for the repeated interruptions arm. Follow-up during phase II had a median duration of 27 wk in both arms (continuous therapy/single interruption arm, 27 [8.75–47]; repeated interruptions arm, 27 [16.5–35]). Following reinitiation of therapy after phase II, patients suppressed viral replication to less than 50 copies/ml by a median time of 10 (6–12) wk in both arms, excluding for two patients in the continuous therapy/single interruption arm who elected to stay off ART indefinitely and one patient from the repeated interruptions arm who reported nonadherence following regimen reinitiation yet reached 52 copies/ml before withdrawing from additional follow-up. Table 1 Baseline Demographic and Clinical Characteristics per Study Arm a Numbers include cases of PI/NNRTI combined use at study entry AA, African American; C, Caucasian; H, Hispanic, IV, intravenous drug usage; PI, protease inhibitor; S, sexual transmission Primary Outcome An intent-to-treat analysis of the time to viral rebound (>5,000 copies/ml) in the open-ended interruption showed no difference between groups (continuous therapy/single TI, median = 4 [ 1 – 8 ] wk, n = 21; repeated TI, median = 5 [ 4 – 8 ] wk, n = 21; p = 0.36). Figure 3 (top panel) shows the probability of plasma HIV-1 RNA remaining less than 5,000 copies/ml for the two groups ( n = 21 per group). Exclusion of drop-outs in an as-treated analysis did not alter conclusions (single TI, median = 5 [ 4 – 9 ] wk, n = 18; repeated TI, median = 6 [ 4 – 8 ] wk, n = 16; p > 0.05). Additional secondary analysis of the magnitude of viral load as shown in Figure 3 (second panel) showed similar viral replication as determined by mean AUC HIV RNA analysis at week 12 (single TI, median = 124,621 [ 23 ,326–262,348] AUC HIV RNA ; repeated TI, median = 100,400 [47,221–365,731] AUC HIV RNA ; p > 0.05) or week 20 (single TI, median = 114,550 [ 31 ,829–362,628] AUC HIV RNA ; repeated TI, median = 153,097 [67,427–515,421] AUC HIV RNA ; p > 0.05). Figure 3 Lack of a Difference between Groups in Plasma HIV-1 RNA during Phase II Top panel shows Kaplan-Meyer plot summarizing time to a viral load of more than 5,000 copies/ml in both arms. Second panel shows viral load (mean ± standard error) per arm during 27 wk of TI (median time of phase II). Bottom table shows number of patients at time points shown for viral load in the second panel; the decrease in viral load over time is due to the reinitiation of therapy in patients with higher viral loads. Secondary Outcomes SAEs and patient discontinuation No patient discontinuation in either group was due to study-defined changes in CD4 cell count (reviewed further below) or due to study-associated SAEs (disease progression or acute retroviral syndrome). However, four non-study-related SAEs occurred: two patients from the continuous therapy/single interruption arm were hospitalized, one for a cholecystectomy and one for acute rectal bleeding, during the 40-wk ART period; a patient from the repeated interruptions arm died of liver cancer during week 26 of the open-ended interruption after previously reaching a viral load greater than 5,000 copies/ml yet electing to stay off ART; and a patient from the repeated interruptions arm developed a transient ileitis. Immune reconstitution No significant difference was observed between groups in CD4 T cell counts at the start of phase II, as illustrated in Figure 4 . In addition, no difference in the percentage of naïve CD4 cells or decrease of recall response to C. albicans was observed, confirming the absence of significant differences in the retention of baseline immune reconstitution correlates between arms. However, a significant decrease in the abundance of CD4 cells relative to other T cell types as summarized in CD4% (but not in absolute CD4 count ) was present in the repeated TI arm, corresponding to a significant increase in CD8 T cell count. In spite of fluctuations in CD4 T cell count levels between the start and end of each monitored TI, a recovery of CD4 count levels was achieved upon resuppression following each TI in conjunction with a retention of lymphoproliferative responses against C. albicans before, during, and after each TI, as illustrated in Figure 5 . Figure 4 T Cell Subsets and Recall Lymphoproliferative Response at the End of Phase I End of phase I values for each arm are summarized (median and first and third quartiles) in the stacked figures showing from top to bottom: CD4 T cells/μl, CD4%, CD4 − CD45RA + CD62L + % (naïve phenotype), CD8 T cells/μl, CD8%, and C. albicans lymphoproliferative response (shown as stimulation Index, SI). Unpaired p values for each variable are shown above corresponding bracket. Figure 5 CD4 T Cells/μl and T Cell Recall Lymphoproliferative Response during Sequential TIs in Phase I Shown are data from the repeated interruptions arm. Panels show the TI initiation visit and TI end visit of each sequential TI inclusive of the initiation visit for phase II (open-ended TI). Viral resistance mutations and therapy failure An intent-to-treat analysis of the combined number of patients per arm with detected resistance mutations irrespective of therapy failure in phase I and during the final TI in phase II showed no significant difference between arms (continuous therapy/single TI, 7/21; repeated TI, 10/21; p > 0.05). Study-defined criteria for therapy failure of a previously suppressive regimen were met by 4/21 patients in the continuous therapy/single interruption arm (patients S37, S47, S52, and S59) in association with self-reported nonadherence to therapy and detection of resistance mutations in phase I, as listed in Table 2 . One patient in the repeated interruptions arm (1/21; patient S56) failed therapy after 20 wk following the third TI by maintaining a viral load between 50 and 999 copies/ml in the presence of previously undetected resistance mutations. Table 2 Therapy Failures with Plasma HIV-1 Protease and Reverse Transcriptase Inhibitor–Associated Resistance Patterns during on Therapy Periods (Study Phase I) Bold identifies drugs for which mutations were detected in plasma 3TC, lamivudine; ABV, abacavir; ddI, didanosine; d4T, stavudine; EFZ, efavirenz; NVP, nevirapine; RT, reverse transcriptase a Mutations associated with patient's regimen In patients who reached phase II in the absence of therapy failure, a total of 12 patients were identified to have resistance mutations at the first viremic time point (continuous therapy/single TI, 3/16; repeated TI, 9/18; p = 0.06). A greater number of resistance mutations was detected in the repeated interruption arm, as summarized in Table 3 . In ten out of these 12 patients, a change in resistance patterns was observed when comparing the first viremic time point to the last. All 11 of 12 patients in Table 3 who reinitiated therapy retained suppressive ability of their respective regimens, as did all other patients who did not show resistance mutations in phase II. In the repeated interruptions arm, analysis of newly detected resistance mutations in phase II, as defined by a lack of detection during viremic time points in phase I, identified 3/18 patients (patients S4, S22, and S43) with this pattern (see notations in Table 3 ). Table 3 Non-Therapy Failures with Resistance Detected off ART at First and Last Viremic Time Point in Comparator Open-Ended TI (Phase II) Bold identifies drugs against which mutations were detected a Mutations associated with patient's regimen b Patient/physician changed regimen after open-ended interruption for reasons not related to suppression activity on previous regimen: patient S7 changed to 3TC, TNV, EFZ, NVP; patient S40 changed to LOP, RTV, ddI, TNV; and patient S35 changed to LOP, RTV, ABV, TNV c Mutations not detected at the first plasma HIV-1 RNA tested during prior TIs d Resistance shown for patient S35 is last available, at week 2 of the third TI e Patient S45 was lost to follow-up after the end of the third TI. Resistance shown is last available, at week 6 of the third TI. Resuppression noted after completion of the third TI 3TC, Lamivudine; ABV, Abacavir; d4T, Stavudine; ddI, Didanosine; EFZ, Efavirenz; LOP, Lopinavir; NLF, Nelfinavir; NVP, Nevirapine; TNV, Tenofovir; ZDV, Zidovudine Discussion Earlier reports on TI strategies in patients with chronic HIV infection include multiple pilot or single-arm study designs centered on the effects on viral control by comparison with pre-therapy periods, detection of resistance mutations without parallel follow-up of a continuously treated arm, and inclusion of variable criteria regarding viral resuppression before proceeding with repeated TIs [ 11 , 12 , 14 , 16 ]. In contrast, our strategy mandated resuppression of viral replication to less than 50 copies/ml before each TI and presents the first comparison of viral replication during a final open-ended interruption of therapy between patients randomized to complete three sequential TIs or stay under continuous therapy. Our data, based on intent-to-treat analysis, did not show that repeated TIs resulted in a clinically significant virological benefit as measured by the time to viral rebound to more than 5,000 copies/ml (see Figure 3 ). Secondary as-treated analysis on viral replication magnitude also indicated a lack of difference between arms. Consistent with the findings of SSITT [ 11 ], analysis of our data by the categorical classification of a “responder” as a patient with viral load less than 5,000 copies/ml at week 12 off therapy showed no significant difference in this frequency between arms (single TI, 5/18; repeated TI, 5/16), suggesting the presence of “responders” irrespective of previous protocol-mandated TIs. Based on secondary outcome measures, the incidence of adverse events (SAEs, therapy failure, and patient discontinuation) or clinical disease progression (as indicated by CD4 count on therapy or opportunistic infections) was not observed to be different between arms. Prospective safety outcomes in our study are in accordance with reports from a retrospective analysis of 1,290 patients who interrupted treatment at least once (< 3 mo) without an increased risk of HIV-associated morbidity or mortality (with the exception of patients in Center for Disease Control and Prevention stage C during first interruption only) [ 21 ]. In regards to immunological outcomes, a concern associated with interruption of suppressive therapy is the potential for irreversible, viral-mediated CD4 T cell loss leading to disease progression [ 6 , 22 ]. We did not observe a decrease in CD4 cell numbers or lymphoproliferative responses against C. albicans when measured between arms before the open-ended TI (see Figure 4 ), nor following resuppression after monitored TI reinitiation cycles in the repeated interruptions arm (see Figure 5 ). The latter is consistent with observations by others and does not support an immediate immunological “cost” to short-term TIs [ 12 , 14 , 15 , 16 , 23 ]. However, we do show that monitoring CD4 cell numbers by percentage could lead to misinterpreting a significant loss of CD4 cells as a result of a significant increase in CD8 count following TIs, even though absolute CD4 count numbers remained unchanged (see Figure 4 ). Interestingly, the increase in CD8 T cell number also corresponded with an increase in HIV-specific responses as measured by interferon-gamma expression (data not shown), which in light of an absence of effect on viral load between arms further supports that TI strategies alone may not significantly alter the pre-existing balance between viral replication and host antiviral responses [ 14 , 16 , 23 , 24 ]. Importantly, no evidence for an increase of viral resistance in association with therapy failure was present in the repeated interruptions arm (See Table 2 ). We did not observe a greater clinical failure of NNRTI-based regimens in the repeated interruption arm due to “single drug” periods as predicted by recently redefined drug half-life estimates and the presence of viral replication during each interruption [ 25 , 26 , 27 ]. However, the percentage of patients with resistance mutations detected in this study in the repeated interruption arm (47%) is higher than the 17% observed in the SSITT cohort [ 11 ], in which patients with prior treatment failures were excluded [ 28 ]. We interpret this difference to mean that the resistance detected off drug in both our and their cohorts is likely associated with the greater number of drug-experienced patients in our cohort (75%) and the detection of prior archived resistance mutations as supported by Metzner et al. [ 29 ], who documented in 14/25 (56%) SSITT patients the presence of minor populations of M184V occurring at least once off drug during interruption of therapy. In spite of the lack of difference in the total number of patients with resistant mutations detected on therapy during phase I and off therapy in phase II (7/21 [33%] versus 10/21 [47%], respectively) in both arms, we do report in similarity to others a greater detection of resistance mutations in the TI arm when restricting analysis to the last off-drug period only [ 29 , 30 ] as three of 16 (18%) had mutations detected off drug in the continuous therapy/single interruption arm compared to nine of 18 (50%) in the repeated interruption arm. However, based on the lack of association between viral resistance detected off-drug shortly after TI and resuppression by the same regimen in all patients, it remains undetermined to what extent TIs favor the detection of archived mutations in chronically suppressed patients and to what extent these mutations are a signal for a future therapy failure. The latter is best exemplified by the data we collected on patients on NNRTI-based regimens in the repeated interruptions arm where two patients (S19 and S43) showed K103N detection (only during the off-drug periods) in the absence of therapy failure while maintaining the same regimen after each TI, including post-study follow-up ( Table S1 ). On the other hand, virological failure in the continued presence of an NNRTI-based regimen in phase I was associated with detection of K103N, as observed in one patient (S56) in the repeated interruption arm and three patients (S37, S52, and S59) in the continuous therapy arm with self-reported non-adherence. Drug resistance that occurs during virological drug failure predicts virological responses to salvage treatment [ 31 , 32 , 33 ]. In contrast, the clinical implications of drug resistance mutations that appear shortly after TI in chronically suppressed patients are not clear. Case reports in this cohort of patients have demonstrated that drug-resistant variants that appeared during TIs may not persist in subsequent time points even after repeated use of the same antiretroviral regimen [ 19 , 34 ]. We now observe that drug resistance appearing during TIs can be transient since 50% and 33% of patients listed in Table 3 showed complete and partial reversion to wild type, respectively, when comparing to resistance at the last available viremic time point in phase II (See Table 3 ). Further, we observed durable resuppression of plasma viral RNA level in many patients who had drug-resistance mutations off therapy that would otherwise be expected to affect part of their treatment regimen when reinitiated (see Table S1 ). Virus populations that expand shortly after TI may lack all of the adaptations required to achieve high levels of plasma viremia in the presence of drug during continuous treatment. These adaptations may include the resistance-associated mutations, which were detected, as well as secondary mutations that may increase the viral replication capacity [ 35 , 36 ] or envelope adaptations required to escape concurrent humoral immune responses [ 37 , 38 ]. It is of interest to note that despite the large amount of research activity on TIs in patients with suppressed chronic infection and the hundreds of monitored interruptions studied to date, only limited cases of development of clinical resistance (as evidenced by a lack of viral resuppression following therapy reinitiation) have emerged, in contrast to the multiple reports of detection of viral sequences off ART associated with resistance as shown in this study and others [ 11 , 19 , 29 , 30 , 39 , 40 ]. Taken together, while our data show no clinically significant benefit for repeated TIs of less than 1.5 mo in patients with CD4 counts greater than 400 on therapy with regard to viral control as defined by time to rebound, secondary outcomes document no significant difference in levels of retention of immune reconstitution between arms and no increased incidence of virological failure as a consequence of TIs. While our data indicate that this TI strategy should not be pursued outside of a clinical trial setting, we argue that it will be important to collect additional data on the potential benefits of drug-sparing regimens (such as reduced long-term toxicity and reduced cost) and to define long-term outcomes in comparison with continuous therapy. Supporting Information Registration of randomized trial at clinicaltrials.gov under identifier NCT00051818. Protocol S1 Protocol Text: Effects of Sequential TI (614 KB DOC). Click here for additional data file. Protocol S2 Study IRB Approval Current IRB approval for study at clinical site. (179 KB PDF). Click here for additional data file. Protocol S3 Wistar IRB Approval IRB approval to receive study biological material at the Wistar Institute for research. (201 KB PDF). Click here for additional data file. Protocol S4 CONSORT Checklist (50 KB DOC). Click here for additional data file. Table S1 Patients with Detected Resistance during Phase II: Regimen at Initiation of Phase II and Subsequent Post-Study Follow-Up to August 2004 (36 KB DOC). Click here for additional data file. Patient Summary Why Was This Study Done? Highly active antiretroviral therapy has revolutionized HIV treatment for patients who have access to the medications. But the drugs are expensive, have side effects, and can become ineffective when the virus develops resistance. Structured treatment interruptions (STIs), also known as “drug holidays” (because patients take a holiday from their drugs), have been suggested as possible alternatives to continuous therapy. Initially, there was fear that patients who went back on therapy after an interruption would not be able to control the virus again, but there was also hope that STIs might actually strengthen the immune system. In addition, STIs might alleviate some side effects, and they would certainly reduce costs. This study uses a particular design to examine the risks and benefits of STIs. What Did the Researchers Do? The researchers studied 42 patients who received either continuous therapy for 40 weeks or three successive treatment interruptions of two, four, and six weeks, followed by a final open-ended interruption for both groups. The researchers then recorded how long patients were able to control the virus before their viral load reached a certain threshold and they had to restart therapy. They also examined CD4 counts and therapy failure, and looked for resistant viruses on and off therapy. What Did They Find? In terms of being able to control the virus, it made no difference whether patients were on continuous therapy or had three STIs. In other words, when both groups stopped treatment at 40 weeks, the length of time that the patients could control the virus was the same in both groups. Eventually, all patients (except two who elected to stay off antiretroviral therapy) re-initiated therapy because of a rising viral load, and the patients once on therapy all regained control over the virus. Resistant viruses were found in patients from both groups, but during the final interruption they were more common in the group that had received the three STIs. What Does This Mean? The study confirms that STIs do not help with viral control, consistent with other studies that found that STIs had no clinical benefit. On the other hand, no short-term adverse events were present, as all patients were able to regain control over the virus after they went back on treatment (without a drop in CD4 count), even after several rounds of interruptions and tests to detect of resistant viruses. There remains concern about whether recurrent cycles of viral replication and suppression might in themselves be harmful, and whether the presence of resistant virus is a signal for future treatment failure. Given these unanswered questions, STIs should only be undertaken within clinical trials. What Next? Possible risks and benefits of STIs in the management of therapy remain an active area of research. Evidence so far has not shown clinical benefits. Ongoing studies need to clarify whether there are long-term risks (and what they are), so that we can weigh these against the benefits of reducing costs and side effects. Additional Online Information The Body information Web page on STIs: http://www.thebody.com/treat/sti.html Information on “continuing antriretroviral treatment” from AVERT, an international HIV and AIDS charity based in the United Kingdom: http://www.avert.org/conttrt.htm Information on STIs from NAM, a United Kingdom registered charity: http://www.aidsmap.com/en/docs/7980314C-97B5–412F-93B1-AD8B64F51F73.asp Factsheet on HIV treatments from the United States National Institute for Allergy and Infectious Diseases: http://www.niaid.nih.gov/factsheets/treat-hiv.htm Search results from Clinicaltrials.gov when searching for “HIV” and “treatment interruption” combined terms: http://www.clinicaltrials.gov/search/term=%22Treatment+Interruption%22%5BCONDITION%5D+AND+HIV+%5BCONDITION%5D

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549548

The evolution of the Sin1 gene product, a little known protein implicated in stress responses and type I interferon signaling in vertebrates

Background In yeast, birds and mammals, the SAPK-interacting protein 1 (Sin1) gene product has been implicated as a component of the stress-activated protein kinase (SAPK) signal transduction pathway. Recently, Sin1 has also been shown to interact with the carboxyl terminal end of the cytoplasmic domain of the ovine type I interferon receptor subunit 2 (IFNAR2). However, the function of Sin1 remains unknown. Since SAPK pathways are ancient and the IFN system is confined to vertebrates, the organization of the Sin1 gene and the sequences of the Sin1 protein have been compared across a wide taxonomic range of species. Results Sin1 is represented, apparently as a single gene, in all metazoan species and fungi but is not detectable in protozoa, prokaryotes, or plants. Sin1 is highly conserved in vertebrates (79–99% identity at amino acid level), which possess an interferon system, suggesting that it has been subjected to powerful evolutionary constraint that has limited its diversification. Sin1 possesses at least two unique sequences in its IFNAR2-interacting region that are not represented in insects and other invertebrates. Sequence alignment between vertebrates and insects revealed five Sin1 strongly conserved domains (SCDs I-V), but an analysis of any of these domains failed to identify known functional protein motifs. SCD III, which is approximately 129 amino acids in length, is particularly highly conserved and is present in all the species examined, suggesting a conserved function from fungi to mammals. The coding region of the vertebrate Sin1 gene encompasses 11 exon and 10 introns, while in C. elegans the gene consists of 10 exons and 9 introns organized distinctly from those of vertebrates. In yeast and insects, Sin1 is intronless. Conclusions The study reveals the phylogeny of a little studied gene which has recently been implicated in two important signal transduction pathways, one ancient (stress response), one relatively new (interferon signaling).

Background Sin1 was originally described as a human protein that modulated Ras function in Saccharomyces cerevisiae [ 1 ]. Strains of yeast that expressed the constitutively activated RAS2 Val 19 mutation had elevated levels of cyclic AMP, impaired growth control and were acutely sensitive to heat shock. This phenotype was reversed when the yeast strain was transfected with a cDNA (clone JC310) that encoded a then unknown protein. Although the authors suggested that the inferred interaction between the JC310 product and RAS might be fortuitous, they favored the possibility that that the unknown protein either was a true inhibitor of RAS or that it was a RAS target protein, which when over-expressed, had a protective action. A S. cerevisiae protein encoded by the AVO1 gene showed distant similarity the human JC310 product [ 2 , 3 ]. Approximately eight years after the identification of JC310 it was again identified, on this occasion in a yeast two-hybrid screen of a Schizosaccharomyces pombe cDNA library as a 665 amino acid protein that bound via polypeptide sequences in its C-terminal 244 amino acids to the Sty1/Spc1, stress activated MAP kinase (SAPK) [ 2 ]. A fission yeast strain lacking the Sin1 gene was sterile, sensitive to multiple types of stress, including heat shock, and had delayed cell cycles compared to a parental strain. Sin1 acted downstream of activated Sty1/Spc1 and appeared to be necessary for normal function of the transcription factor Atf1, a homolog of human ATF2. Wilkinson et al. [ 2 ] found that an apparent full length homolog of Sin1 from chicken allowed the heat sensitive strain of S. pombe to grow at 37°C, albeit very poorly. Moreover, fusion of the first 486 amino acids of yeast Sin1 (which does not restore growth) with the C-terminal 182 amino acids of the chicken Sin1 sequence protected against heat shock. Together, these data showed that Sin1 functions as a component of the stress-activated Sty1/Spc1 MAP kinase pathway in S. pombe and that a functional homolog of Sin1 exists in vertebrates. No further information concerning Sin1 appeared since the paper of Wilkinson et al. [ 2 ] until our discovery that the ovine (ov) Sin1 associated via its C-terminus to the cytoplasmic domain of IFNAR2, a subunit of the type I IFN receptor [ 4 ], and Schroder et al. [ 5 ] described transcripts for Sin1 in human tissue and provided an analysis of the human gene. The latter study confirmed that Sin1 was relatively well conserved across Metazoa and fungi (Ascomyctes and Basidiomycetes) and was also represented in amoebae, but not in other protozoan species. Ovine Sin1, which is 88% identical in sequence to chicken Sin1, can be co-immunoprecipated with the IFN receptor subunit IFNAR2 and shows a similar subcellular distribution to the receptor protein when co-expressed in mammalian cells [ 4 ]. Although ovSin1 was identified from a cDNA present in ovine endometrium and was initially considered to have a role in reproduction associated with the action of IFN-τ on the uterus during early pregnancy in the sheep, it became clear that the Sin1 gene was expressed in tissues other than endometrium and might have a general role in the action of type 1 IFN. In particular, it seemed possible that Sin1 might link the action of IFN to the stress activated SAPK signal transduction pathways. Such a linkage has been inferred from earlier studies in which early activation of p38 MAPK had been noted following exposure of a variety of cell lines to IFN-α, -β, or -τ [ 6 - 12 ]. Although the SAPK pathway is itself ancient and is found in all the species in which the Sin1 gene exists, the IFN system of receptors and ligands is restricted to vertebrates. We reasoned, therefore, that an analysis of Sin1 gene sequences might not only provide insight into the function of Sin1, but indicate how the protein evolved to interact with IFNAR2. The fact that the Sin1 gene appears to be expressed ubiquitously, that it is highly conserved across a wide range of taxa, and that it is a likely participant in several important signaling pathways, makes it an intriguing candidate for a functional/evolutionary analysis. Results Conservation of the Sin1 gene from yeast to mammals A combination of searching methods was employed to locate Sin1 genes in available cDNA and genome data bases (Table 1 ). Sin1 sequences were found in two yeast species ( Schizosaccharomyces pombe and Saccharomyces cerevisiae ), the red bread mold ( Neurospora crassa ) and a number of other fungal species (not shown here), Caenorhabditis elegans , a mosquito species ( Anopheles gambiae ), fruit fly ( D. melanogaster ), frog ( Xenopus laevis ), two fish species ( Fugu rubripes and Danio rerio ), chicken ( Gallus gallus ), mouse ( Mus musculus ), rat ( Rattus norvegicus ), human ( Homo sapiens ), sheep ( Ovis aries ), cattle ( Bos taurus ), and pig ( Sus scrofa ) (Table 1 ). No apparent ortholog could be detected in the plant Arabidopsis thaliana . Nor could sequences corresponding to Sin1 be found in protozoa other than amoebae and prokaryotic species. Table 1 Sin1 genes and their GenBank accession numbers Organism GenBank Accession No. Comments Yeast ( Saccharomyces cerevisiae NP_014563 Blastp the yeast protein database with fission yeast Sin1 protein. Yeast ( Schizosaccharomyces pombe NP_594703 Wilkinson et al. 1999. Red bread mold ( Neurospora crassa XP_322410 Blastp protein databases with budding yeast Sin1 protein. Worm ( Caenorhabditis elegans ) NM_064195 Blastp the worm protein database with ovSin1 protein. Fly ( Drosophila melanogaster ) AE003814 Blastp the fly protein database with ovSin1 protein. Mosquito ( Anopheles gambiae ) XM_319576 Blastp the mosquito protein database with ovSin1 protein. Fish ( Fugu rubripes ) N.A. Blastp the fugu protein database with ovSin1 protein. Frog ( Xenopus lavis ) BC043789 Search EST databases with chicken Sin1 cDNA. Chicken ( Gallus gallus ) AF153127 Wilkinson et al. 1999. Mouse ( Mus musculus ) BQ713136, BF781677, BU152256 Search mouse EST and genome databases with sheep Sin1 cDNA Rat ( Rattus norvegicus ) CK476507, BE127132, BF553331, BU759329, AW141364 Search EST and genome databases with sheep Sin1 cDNA. Pig ( Sus scrofa ) CF791532, CF178115, BP459453, CF177341 Search EST databases with sheep Sin1 cDNA. Cattle ( Bos taurus ) BF230134, AV603930, CB433957, BM480500 Search EST databases with sheep Sin1 cDNA. Sheep ( Ovis aries ) AY547378 Wang oberts, 2004 Human ( Homo sapiens ) NM_024117, BC002326 Search human EST and genome database with sheep Sin1 cDNA "Comments" briefly describe the methods used to obtain the sequences. The marked dissimilarity in inferred amino acid sequence between Sin1 from vertebrates and C. elegans (25% identity, Table 2 ), between the two yeast species (29% identity, Table 2 ; see Additional file: 1 ) and between S. pombe and N. crassa (28% identity, Table 2 , see Additional file: 2 ) in the approximately 500 aa of overlap suggests that even if homologs existed in plants and prokaryotes they would likely be overlooked by the search methods employed. Table 2 Pairwise comparisons of Sin1 cDNA and amino acid sequences from various species S. pomb e (665 aa) N. crassa (798 aa) C. elegans (684 aa) D. melanogaster (569 aa) A. gambiae (548 aa) F. Rubripes (530 aa) X. lavis (520 aa) G. gallus (522 aa) M. musculus (522 aa) R. norvegicus (522 aa) O. aries (522 aa) B. Taurus (522 aa) S. scrofa (522 aa) H. sapiens (522 aa) S. pombe (665 aa) - 28.2 34.1* 28.8* 35.6* 21.7 28.2 21.1 24.6 26.1 25.5 25.4 24.8 25.3 N. crassa (798 aa) NA - 32.6* 28.3* 30.7* 22.6 31.9* 20.3 36.2* 20.5 20.9 20.9 20.9 20.5 C. elegans (684 aa) NA NA - 27.5* 29.4* 22.3 22.6 21.8 23.0 23.0 23.5 23.2 22.8 23.2 D. melanogaster (569 aa) NA NA NA - 46.0 31.8 34.5 32.9 35.2 35.3 31.1 31.7 32.0 31.9 A. gambiae (548 aa) NA NA NA NA - 34.9 33.4 35.3 33.8 34.0 33.2 33.0 33.2 33.4 F. rubripes (530 aa) NA NA NA NA NA - 80.0 82.9 78.7 79.2 79.8 80.2 79.8 80.4 X. lavis (520 aa) NA NA NA NA NA 71.8 - 88.5 84.4 85.0 84.6 85.2 85.2 85.6 G. gallus (522 aa) NA NA NA NA NA 74.7 78.0 - 88.0 88.3 88.3 89.3 89.3 90.0 M. musculus (522 aa) NA NA NA NA NA 73.6 76.1 80.9 - 99.2 91.7 96.9 97.3 96.9 R. norvegicus (522 aa) NA NA NA NA NA 73.6 76.3 80.5 96.2 - 91.3 96.9 96.9 96.9 O. aries (522 aa) NA NA NA NA NA 73.3 75.3 81.7 96.0 96.0 - 98.7 97.9 98.1 B. taurus (522 aa) NA NA NA NA NA 73.4 75.6 81.7 91.9 91.8 98.2 - 98.9 99.0 S. scrofa (522 aa) NA NA NA NA NA 73.1 75.8 82.3 92.5 92.2 94.7 95.7 - 98.7 H. sapiens (522 aa) NA NA NA NA NA 73.9 75.9 82.8 92.4 92.5 94.1 94.9 95.7 - Notes: 1. Numbers in the upper-right half above the diagonal are identity percentages for amino acid sequences. 2. Numbers in the lower-left half below the diagonal are identity percentage for DNA sequences. 3. Numbers below the species names are the lengths of the Sin1 protein. 4. NA, Not applicable, i.e. no significant similarity was found. 5. Astericks, significant similarity occurs only in one region of the protein. For details, see the notes below: S. pombe-C. elegans: significant similarity occurs in one region (170 aa: 252–391). S. pombe- D. melanogaster : significant similarity occurs in one region (120 aa: 278–407). S. pombe- A. gambiae: significant similarity occurs in one region (94 aa: 282–375). N.crassa-C. elegans: significant similarity occurs in one region (90 aa: 376–465). N.crassa-D. melanogaster: significant similarity occurs in one region (58 aa: 407–464). N.crassa-A. gambiae: significant similarity occurs in one region (207 aa: 250–456). N.crassa-Xenopus: significant similarity occurs in one region (127 aa: 338–464). N.crassa-M. musculus: significant similarity occurs in one region (127 aa: 338–464). C. elegans-D. melanogaster: significant similarity occurs in one region (169 aa: 198–366). C. elegans-A. gambiae: significant similarity occurs in one region (178aa: 198–375). 6. Sequences and their GenBank accession numbers are: O. aries (AY547378), B. taurus (BF230134, AV603930, CB433957, BM480500), H. sapiens (NM_024117, BC002326), S. scrofa (CF791532, CF178115, BP459453, CF177341), M. muscus (BQ713136, BF781677, BU152256), R. norvegicus (CK476507, BE127132, BF553331, BU759329, AW141364), G. gallus (AF153127), X. laevis (BC043789), F. rubripes (Sequence accessible at ), D. melanogaster (AE003814), A. gambiae (XM_319576); S. pombe (AL136521, NP_594703, CAB66311); N. crassa (XP_322410). Figure 1 Alignment of Sin1 proteins from the fission yeast and sheep. The GAP program was used to align the two sequences. Black shading shows identical residues. Abbreviations: S. pombe, Schizosaccharomyces pombe (fission yeast. GenBank accession No. AL136521). O. aries, Ovis aries (sheep. GenBank accession No. AY547378). Figure 2 A phylogenetic tree for Sin1 primary sequences from various species. Sin1 polypeptide sequences were aligned by the program ClustalW, and the alignment output used by the program MEGA to generate a neighbor joining phylogenetic tree for the regions of alignment. GeneBank accession numbers for Sin1 sequences are listed in Table 1. Numbers beside branch points indicate the confidence levels for the relationship of the paired sequences as determined by bootstrap statistical analysis (1000 replicates). The lengths of the arms represent the extent of amino acid differences between the paired sequences, with the scale bar equivalent to 50 residues. Sin1 from the yeast species, S. cerevisiae and S. pombe which consist of 1172 aa and 665 aa, respectively, and also from the red bread mold, N. crassa (798 aa) are much longer than Sin1 from vertebrate and insect species, which are ~520 aa long. The regions of similarity among these three fungal proteins are confined entirely to the carboxyl termini of these molecules, although several gaps have to be introduced to align them. No similarities are detectable in the amino terminal extensions, which, in the case of S. cerevisiae , is 370 aa long. It is the carboxyl regions of the fungal proteins that can also be aligned with the Sin1 sequences from C. elegans , insects, and vertebrate species, including Ovis aries , the sheep (Fig. 1 ). A phylogenetic tree reconstructed from an alignment of amino acid sequences of Sin1 is shown in Fig. 2 . As anticipated, the sequences from the three fungi, C. elegans , the two insect species, and vertebrate species fell into distinct branches of the tree. The sequences for the mammalian species were tightly clustered, with identities ranging from 99% (humans and cattle) to 91.3% (sheep and rat) (Table 2 ). All the vertebrate cDNA encoded polypeptides of 522 aa (Table 2 ). There is considerable conservation of Sin1 from mammals to birds (~90%), amphibians (~85%), and fish (~80%) (Table 2 ). The insect sequences are rather longer than the ones from vertebrates, and several gaps have to be introduced to provide alignments (Fig. 3 , 4 , 5 ). Nevertheless, the insect amino acid sequences are approximately 33% identical to those of the mammals (Table 2 ). Five blocks of sequence (SCD I-V) are significantly more conserved than others when two insects, a fish, an amphibian, a bird and several mammals are compared (Figs. 3 , 4 , 5 & 6 ). Three of these regions are located towards the N-terminus and two additional regions towards the C-terminus. The most diverse region is located centrally. Figure 3 The alignment of Sin1 polypeptide sequences from insects and vertebrates. The ClustalW program was used to align all the protein sequences. Symbols (*, :, and .) show residues that are either identical(*), strongly similar (:), or weakly similar (.), respectively. Five Sin1 conserved domains (SCD) are highlighted as SCD I-V. The GenBank accession numbers for the sequences are: O. aries (AY547378), B. taurus (BF230134, AV603930, CB433957, BM480500), H. sapiens (NM_024117, BC002326), S. scrofa (CF791532, CF178115, BP459453, CF177341), M. musculus (BQ713136, BF781677, BU152256), R. norvegicus (CK476507, BE127132, BF553331, BU759329, AW141364), G. gallus (AF153127), X. laevis (BC043789), F. rubripes , D. melanogaster (AE003814), A. gambiae (XM_319576). Figure 4 The alignment of Sin1 polypeptide sequences from insects and vertebrates. The ClustalW program was used to align all the protein sequences. Symbols (*, :, and .) show residues that are either identical(*), strongly similar (:), or weakly similar (.), respectively. Five Sin1 conserved domains (SCD) are highlighted as SCD I-V. The GenBank accession numbers for the sequences are: O. aries (AY547378), B. taurus (BF230134, AV603930, CB433957, BM480500), H. sapiens (NM_024117, BC002326), S. scrofa (CF791532, CF178115, BP459453, CF177341), M. musculus (BQ713136, BF781677, BU152256), R. norvegicus (CK476507, BE127132, BF553331, BU759329, AW141364), G. gallus (AF153127), X. laevis (BC043789), F. rubripes , D. melanogaster (AE003814), A. gambiae (XM_319576). Figure 5 The alignment of Sin1 polypeptide sequences from insects and vertebrates. The ClustalW program was used to align all the protein sequences. Symbols (*, :, and .) show residues that are either identical(*), strongly similar (:), or weakly similar (.), respectively. Five Sin1 conserved domains (SCD) are highlighted as SCD I-V. The GenBank accession numbers for the sequences are: O. aries (AY547378), B. taurus (BF230134, AV603930, CB433957, BM480500), H. sapiens (NM_024117, BC002326), S. scrofa (CF791532, CF178115, BP459453, CF177341), M. musculus (BQ713136, BF781677, BU152256), R. norvegicus (CK476507, BE127132, BF553331, BU759329, AW141364), G. gallus (AF153127), X. laevis (BC043789), F. rubripes , D. melanogaster (AE003814), A. gambiae (XM_319576). Figure 6 The five highly conserved domains of Sin1 proteins. Sin1 primary sequences from various species were aligned by using the ClustalW program, and the five most conserved domains identified from the alignment in Fig. 3 and the sequences listed in Table 1. Conserved domains are shown as boxes with remaining regions as solid lines. SCD, S in1 c onserved d omain. Numbers beneath the species names are the lengths of the Sin1 proteins. Values in the boxes are the number of amino acid residues within a conserved domain. Numbers on the lines reflect the lengths of that region. The region of the greatest identity between these divergent insect and vertebrate sequences is an acidic region placed in conserved SCD III (Fig. 3 , 4 , 5 ). In mammals, this region is completely conserved and corresponds to residues L232-K267 (LHIAEDDGEVDTDFPPLDSNEPIHKFGFSTLALVEK; Figs. 3 , 4 , 5 ; Fig. 7 ). However, an analysis of this sequence reveals no known functional motifs and no strong similarity to sequences represented in other known proteins. Schroder et al. [ 5 ] have also noted this conserved sequence in their analyses of Sin1 sequences and have named it CRIM for c onserved r egion i n the m iddle. Figure 7 Alignment of Sin1 conserved domain III from various species. Sequences have been aligned by using the GCG PILEUP and GeneDoc programs. Degree of conservation is illustrated by intensity of shading (black, complete identity; light gray with black letters, complete identity across some but not all species; dark gray with white letters, high conservation but with conservative differences). The GenBank accession numbers for the sequences are: mm, M. musculus (BQ713136, BF781677, BU152256); rn, R. norvegicus (CK476507, BE127132, BF553331, BU759329, AW141364); bt, B. taurus (BF230134, AV603930, CB433957, BM480500); oa, O. aries (AY547378); ss, S. scrofa (CF791532, CF178115, BP459453, CF177341); hs, H. sapiens (NM_024117, BC002326); gg, G. gallus (AF153127); xl, X. laevis (BC043789); fr, F. rubripes ); dm, D. melanogaster (AE003814); ag, A. gambiae (XM_319576); ce, Caenorhabditis elegans (NM_064195); sp, Schizosaccharomyces pombe (AL136521, NP_594703, CAB66311); nc, Neurospora crassa (XP_322410). Sin1 from C. elegans retains the highly conserved 36 amino acid SCD I and the 127 amino acid Domain III (Fig. 6 & 7 ). SCD III is also retained in the fission yeast and the red bread mold. Vertebrates possess several unique sequences not present in insects and yeast, and, therefore, potentially implicated in the IFN signal transduction pathway including a carboxyl terminal region (KLSRRTSFSFQKDKK) immediately following the end of SCD V. Functional motifs in the Sin1 primary sequence When the ovine Sin1 sequence is scanned for functional motifs [ 13 ], the structure appears unusually barren. Two weak bipartite nuclear localization signals (NLS) [ 14 ] can be detected. One (residues 82–98, RRSNTAQRLERLRKERQ) is present in the SCDII domain, and the other (residues 503–519, RKLNRRTSFSFQKEKKS) is almost at the C-terminus within conserved domain V (Fig. 3 ). Nevertheless, data from the subcellular localization experiment showed that Sin1 is excluded from the nucleus when transfected in COS1 or L929 cells [ 4 ], suggesting these NLS are probably not functional. There are numerous motifs that are recognized as potential but weak sites for phosphorylation by either casein kinase II (CK2), protein kinase C, or protein kinase A (data not shown). None of the 17 CK2 sites, the 12 protein kinase C, or the 5 protein kinase A sites present in the ovine Sin1 primary sequence are conserved from mammals to fission yeast, although many are retained across all vertebrates. A weak site for myristylation (ovine residues 170–175, GTTATK; Figs. 1 & 3 , 4 , 5 ), and hence for membrane association, is retained in all the vertebrate species examined, but is absent in insects and yeast. In absence of any data on the functional significance of these sites, they will not be discussed further. Gene structure of Sin1 from various species The genomic sequence encompassing the transcribed region of the gene could be retrieved from the genome data bases for S. pombe , S. cerevisiae , C. elegans , D. melanogaster , A. gambiae , F. rubripes , R. norvegicus , M. Musculus , H. sapiens [ 15 , 16 ]. Sin1 exists as a single copy gene in all these species. For example, the human Sin1 gene is located on chromosome 9 (9q34.11-9q34.12) (data not shown) with the transcribed region composed of 11 exons and 10 introns and spanning a region of about 240 kb (Fig. 8 ). Exon 7 is spliced out of the shorter form of Sin1 [ 4 , 5 ]. The lack of exon 7 does not cause a frame shift because the intron phases of the two introns on both sides of exon 7 are identical (data not shown). Schroder et al. [ 5 ] have also demonstrated or predicted other minor splice variants for Sin1 in the human. The 11 exons account for only 0.9% of the gene sequence. It is, of course, unclear how many additional exons and introns are associated with the 5' UTR beyond the transcription start site(s), whose location has not been determined. Figure 8 A comparison of the Sin1 gene structure across species. The gene structure for all species was retrieved from the genome database of the species by using the BLASTn program to analyze the open reading frame of each Sin1 cDNA sequence. Only the regions of the gene containing the open reading frame are shown in the diagram. All sequences begin with start codons and end with stop codons. The numbers under species names are the protein length. Currently, the sheep and bovine genome sequences are not available, but it is likely that the Sin1 gene organization will be similar to that in the human. The current comparative synteny maps between human, sheep and cattle [ 17 - 19 ] predict that the Sin1 gene is located on sheep chromosome 3 (3p1.7-3p2.6) and bovine chromosome 11 (11q2.3-11q2.8), respectively. A comparative map for all the genes is shown in Fig. 8 . In fission yeast and insects, the Sin1 gene consists of a single exon. In worm, fish, rat, mice, and human, Sin1 has multiple exons. The exon/intron pattern, consisting of 11 exons, is observed in all vertebrates, including the two fish species (Fig. 8 ). It is noteworthy that although the genomic sequences of sheep and cattle are not available, the exon/intron pattern of their Sin1 genes is similar to that of other vertebrates based on the comparison between sheep or cattle Sin1 cDNA and human genomic sequence of Sin1 (data not shown). The lengths of these 11 exons are also remarkably conserved and fall within the normal range (50–200bp for most internal exons) (International Human Genome Sequencing Consortium, 2001). As expected, the sizes of the introns differ across species, and some are extremely long. Intron sizes generally decrease in the order human > mouse > rat > fish (Fig. 8 ). As expected, intron sizes were quite similar between rodents and human. The Sin1 gene from C. elegans is organized quite differently from that in mammals. It consists of 10 exons interrupted by nine relatively short introns. The region of the C. elegans gene that contains regions of similarity with the mammalian protein sequences consists of exon 1 (SCD I) and exon 5 (SCD III). As noted above and in Figure 8 , the Sin1 gene from insects and S. pombe is comprised of only a single exon. Discussion Sin1 is a little studied gene product of unclear function found in species ranging from mammals to fungi. Although the S. pombe gene product is longer than that of mammals, with an extension at its N-terminus, human Sin1 can rescue the stress sensitivity noted in the phenotype of a S. pombe strain that expressed a constitutively active form of RAS, indicating that function, as well as structure, has been conserved over hundreds of millions of years. Two facts should be considered when attempting to infer a role for Sin1 in vertebrates. The first, as discussed in the Background, is the known ability of type 1 IFN to activate MAPK/SAPK in mammalian cells. The second is the proven involvement of Sin1 in the yeast SAPK (Sty1/Spc1) pathway and its involvement in controlling transcription of stress-activated genes [ 2 ]. The present analysis was conducted in an attempt to gain more detailed information about Sin1 function from a phylogenetic analysis and comparison of Sin1 genes and gene products in different taxonomic groups. The Sin1 gene is remarkably divergent in both length and sequence identity within the fungi S. pombe , S. cerevisiae , and N. crassa , emphasizing the evolutionary distance between these three species. The regions of similarity are confined to the ~600 amino acid C-terminal regions of the three sequences (data not shown), and it is this region that is also conserved in insects and vertebrates (see Additional file: 1 & 2 ). This diversity in structure within the fungi is probably reflected in divergence of function. AVO1, the apparent Sin1 ortholog of S. cerevisiae , forms a membrane-associated complex with TOR2 and other protein components (AVO2, AVO3 and LST8), which control cell growth in response to nutrients [ 3 , 32 ]. Cells with deletion of AVO1 are unable to organize their actin cytoskeleton [ 3 ]. In contrast, the Sin1 ortholog of S. pombe is involved in a stress response signaling pathway by interacting with Sty1 [ 2 ]. A cross-species comparison of all the Sin1 sequences available, indicates five regions of greatest conservation, only one of which, a ~127 amino acid central region (SCD III), was easily defined in all taxa (Figs. 3 , 4 , 5 & 6 ). Even this region is poorly conserved in the budding yeast, S. cerevisiae , although certain landmark amino acids are retained (data not shown). Interestingly, Sin1 from insects and vertebrates, despite having only about 35% identity, are of similar length and possess the five regions of high identity. Conceivably, the SCD III domain is functionally essential in all the species, while SCDs I, II, IV, and V have evolved conserved function within the Metazoa. A not unreasonable assumption is that that Sin1 plays an evolutionarily conserved role in SAPK signaling across a broad range of taxa, including all metazoan and fungal species [ 5 ] but has assumed an additional function in vertebrates in mediating crosstalk with the IFN-signal transduction pathway. In vertebrates Sin1 falls into a class of highly conserved gene products. Its conservation is lower than that of two structural proteins, histone H3 and β-actin, but is comparable to that of CDK1 (Table 3 ). However, while CDK1 in yeast and insects retains considerable sequence identity with the vertebrate orthologs, much of the conservation of Sin1 is lost. It is tempting to speculate that Sin1 has been subjected to powerful evolutionary constraint that has limited its amino acid sequence divergence within vertebrates. It should be noted that our analyses cannot exclude the possibility that conservation of Sin1 among vertebrates reflects recent divergence of the sampled vertebrates relative to the other taxa examined. Once data become available, it will be instructive to compare Sin1 gene sequences from the invertebrate chordates (Tunicata and Cephalochordata) with those of the other metazoan taxa. Table 3 Comparison across species of the amino acid sequence conservation of Sin1 with some other conserved genes Yeast Drosophila Frog Chicken Mouse Cattle Human Histone H3 91.2% 98.5% 94.9% 95.6% 97.8% 98.5% 100% β-actin 90.4% 97.9% 99.5% 100% 100% 98.1% 100% CDK1 64.9% 71.7% 88.5% 93.3% 97.0% 98.7% 100% Sin1 25.3% 31.9% 85.6% 90.0% 96.9% 99.0% 100% Values for percentage identities were obtained by aligning amino acid sequences from various species with their human counterparts. CDK, cyclin-dependent kinase. The GenBank accession numbers for sequences are as follows. Human ( Homo sapiens ): histone H3 (AAH66884), β-actin (NP_001092), CDK1 (P06493), Sin1 (NM_024117, BC002326). Cattle ( Bos taurus ): histone H3 (P16105), β-actin (AAM98378), CDK1 (P48734), Sin1 (BF230134, AV603930, CB433957, BM480500). Mouse ( Mus musculus ): histone H3 (NP_062342), β-actin (NP_031419), CDK1 (NP_031685), Sin1 (BQ713136, BF781677, BU152256). Chicken ( Gallus gallus ): histone H3 (I50245), β-actin (NP_990849), CDK1 (P13863); Sin1 (AF153127). Frog ( Xenopus laevis ): histone H3 (P02302), β-actin (AAC27796), CDK1 (P35567, Sin1 (BC043789). Fly ( Drosophila melanogaster ): histone H3 (NP_724345), β-actin (NP_511052), CDK1 (NP_476797), Sin1 (AE003814). Yeast ( Schizosaccharomyces pombe ): histone H3 (NP_595567), β-actin (NP_595618), CDK1 (NP_595629), Sin1 (NP_014563). Sin1 was shown to be associated with the cytoplasmic domain of IFNAR2, a subunit of the type I IFN receptor [ 4 ]. Since insects appear to lack genes for type I IFN and their receptors (R. M. Roberts, unpublished observations), whereas vertebrates utilize this system primarily as an anti-viral response [ 20 - 22 ], it should be theoretically possible to define a sequence in silico unique to vertebrates but clearly absent in both D. melanogaster and A. gambiae that might account for the association of Sin1 with IFNAR2. Sin1 binds to the carboxyl end of the cytoplasmic domain of IFNAR2 via its own carboxyl 114 amino acids [ 4 ]. At least two candidate sequences exist in that part of Sin1. One is the rather basic carboxyl terminus (aa 510–522), another a HDYKHLYFESDA (aa 458–469) sequence, both of which are absent in the insect proteins (Figs. 3 , 4 , 5 ). Whether these sequences are participants in the interaction of Sin1 with IFNAR2 in vertebrates has not been examined experimentally. Of course, it is quite possible that insect Sin1 can bind vertebrate IFNAR2 or that amino acid substitutions elsewhere in the carboxyl end of the vertebrate sequence have evolved to promote the interaction. These possibilities have also not been tested. In this regard, IFNAR2, with which Sin1 interacts, has evolved much more rapidly than Sin1 itself. The sequence of human IFNAR2, for example, shows only about 58% and 29% identity to those of ovine and chicken IFNAR2, respectively [ 21 , 23 ], while orthologs have yet to be defined for IFNAR2 in frogs and fish, even though these animals are believed to have a functional IFN system, which includes the production of Type I IFN and downstream components in response to double stranded RNA [ 20 , 22 ]. Interestingly, the only highly conserved continuous sequence of chick and mammalian IFNAR2 within the Sin1 binding region is an acidic region (aa 493–515; human IFNAR2 numbering) at the very carboxyl terminus of the molecule ([ 23 ]; R.M. Roberts, unpublished observations). It seems possible that this conserved sequence provides the scaffold for Sin1 binding. As also observed by Schroder et al. [ 5 ], Sin1 is represented by a single gene in all species where it exists. In both insects and the two yeast species, the gene is intronless, while in C. elegans and in vertebrate species introns are present (Fig. 8 ). In budding yeast, only a small number (3.8%) of genes have introns [ 24 ], whereas in most other eukaryotes, including Drosophila , intronic sequences are a feature of the majority of genes and must be excised to produce a functional mRNA [ 25 ]. For D. melanogaster , for example, there is an average of 3 introns per gene [ 26 ]. These introns are short, averaging 240 bp in Drosophila [ 27 ]. Why the Sin1 genes are intronless in these species is unclear, but there is considerable evidence that retrotransposition occurs in yeast, Drosophila [ 28 ] and mammals [ 29 ]. In this process, reverse transcription of mRNA from a parental gene creates an intronless copy of the parental gene at a new position in the genome. If this mechanism created the Sin1 gene, a remnant or evolved version of the parental gene might be anticipated to exist, particularly if the transposition event occurred in recent evolutionary time [ 28 ]. It is unclear whether the intronless Sin1 gene in Drosophila resulted from such a retrotransposition event since there is not a detectable intronic copy elsewhere in the genome. The Sin1 gene from C. elegans has introns, but is organized very differently from that of vertebrates, where the intron/exon organization is highly conserved (Fig. 8 ). Unfortunately, the function of Sin1 is unknown. Its structural conservation from vertebrates to yeast [ 30 ] and its expression in most, if not all tissues of mammals [ 4 ] suggest a central, if elusive, role in life processes. Conclusions SAPK-interacting protein 1 (Sin1), a little-studied but widely expressed gene product, is encoded by a single gene in fungi, nematodes, insects, and all vertebrates analyzed and shows modest conservation of amino acid sequence that is consistent with some degree of conserved function in stress-activated signal transduction pathways. Sin1 is highly conserved in vertebrates where it has been implicated in linking interferon responses to the SAPK pathway. Methods Databases Sin1 genomic sequences from human, mouse, rat, fruit fly, mosquito, C. elegans , S. pombe , and S. cerevisiae , were retrieved from at NCBI Genome databases [ 18 ]. Sin1 cDNA sequences from human, mouse, rat, cattle and pig, and other Sin1 ESTs were retrieved from GenBank EST database after BLASTn analysis at NCBI [ 18 ]. For fish Sin1 genomic sequences, the incomplete puffer fish ( Fugu rubripes ) and zebrafish ( Danio rerio ) genome databases at the Ensembl site [ 16 ] were used. The budding yeast ( Saccharomyces cerevisiae ) ORF (open reading frame) database [ 33 ] was used to retrieve budding yeast Sin1. Software programs used to analyze sequences Pairwise global sequence alignment was performed by using either the BESTFIT or the GAP program from GCG (Madison, WI). Multiple global sequence alignment was performed by using either the PILEUP program (GCG, Madison, WI) and GeneDoc [ 34 ] or ClustalW program [ 35 ]. The phylogenetic tree for Sin1 was generated by using the ClustalW program and the MEGA program [ 36 ]. Motif search was performed by using the ScanProsite program [ 13 ]. Methods for obtaining Sin1 sequences from various species Fission yeast ( Schizosaccharomyces pombe ) and chicken ( Gallus gallus ): The two Sin1 sequences were published by Wilkinson et al. [ 2 ]. Budding yeast ( Saccharomyces cerevisiae ): The BLASTp program was used to search the budding yeast ORF database for any protein sequence that had significant similarity to the fission yeast Sin1 protein. The obtained budding yeast Sin1 protein sequence had a GenBank link where its cDNA was available. The cDNA sequence was used to analyze its genomic structure at the NCBI yeast genome site. Red bread mold ( Neurospora crassa ): Sin1 protein was retrieved from the Neurospora crossa protein data base by searching (BLASTp) with the budding yeast Sin1 protein. Worm ( Caenorhabditis elegans ): The Sin1 protein sequence was obtained from the C. elegans protein database by searching with ovine Sin1 protein. The cDNA sequence was then obtained from the GenBank link and used to determine the structure of the Sin1 gene. Fly ( Drosophila melanogaster ): The fruit fly Sin1 protein sequence was retrieved from the D. melanogaster protein database as above. The cDNA sequence was obtained from the GenBank link. Unexpectedly, querying the Drosophila genomic sequence with the C. elegans Sin1 sequence and vice-versa failed to yield a match in either case. Mosquito ( Anopheles gambiae ): The mosquito Sin1 protein sequence was retrieved from the Anopheles gambiae str. PEST protein database as above. The cDNA sequence was then obtained from the GenBank link. Puffer fish ( Fugu rubripes ) and Zebrafish ( Danio rerio ): Both Fugu rubripes and Danio rerio genome databases, which are accessible at two websites, NCBI and ENSEMBL, were queried with Sin1 cDNA sequences from sheep, chicken, and frog. For both species, only the Ensembl site provided the complete genomic sequence. Although the Fugu rubripes genome sequence is incomplete, the exons of Sin1 cDNA can be retrieved and successfully assembled into the full length structure by alignment with other Sin1 cDNA and gene sequences. No GenBank entry was available for the Fugu rubripes Sin1 gene. When a similar method was used to retrieve the Zebrafish Sin1 cDNA sequence, the full length sequence could not be obtained because the region (~20 kb) covering one exon (exon 4) was incomplete. Therefore, the fish Sin1 protein sequence used here is from Fugu rubripes . Frog ( Xenopus lavis ): The full-length cDNA sequence of Sin1 reported here was from African clawed frog, and was obtained by blasting the Xenopus EST database [ 37 ] with the chicken Sin1 sequence. The protein sequence was deduced from this cDNA sequence. Mouse ( Mus musculus ): The mouse Sin1 cDNA sequence was obtained by editing several ESTs, after performing a BLASTn search of the Mus Musculus EST database with the ovine Sin1 cDNA sequence. Searching the mouse genome database with the mouse Sin1 cDNA coding region then allowed the gene, down stream of its transcription start site to be located and its structure to be inferred. Rat ( Rattus norvegicus ): The rat Sin1 cDNA sequence was retrieved from several overlapping ESTs, which were obtained by searching the Rattus norvegicus EST database with the ovine Sin1 cDNA sequence. The coding region of the rat Sin1 cDNA was then used to search the rat genome database at the NCBI website for the genomic structure of the gene. Cattle ( Bos taurus ): The full length bovine Sin1 cDNA sequence was obtained from overlapping ESTs, which were obtained by searching the NCBI EST database with the ovine Sin1 cDNA sequence. Pig ( Sus scrofa ): The swine Sin1 cDNA sequence was obtained as above by searching the NCBI EST database with the ovine Sin1 cDNA sequence. Human ( Homo sapiens ): The sequence published by Colicelli et al. [ 31 ] was confirmed by performing a BLASTn search on human EST data bases with the ovine Sin1 cDNA sequence. Since the previously published sequence was not full-length, an additional human Sin1 EST (GenBank Acc. No. BC002326) was used to assembly the full length Sin1 cDNA sequence. The location of the gene and its structure downstream of its transcription start site were determined by searching the full human genome database with the Sin1 open reading frame. Sheep ( Ovis aries ): The sheep Sin1 cDNA sequence was cloned from a sheep endometrial cDNA library in a yeast two-hybrid screen [ 4 ]. GenBank accession numbers are summarized in Table 1 . Authors' contributions SW carried out the majority of the computational analyses under the direction of RMR, and wrote the first draft of the manuscript. RMR conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript. Supplementary Material Additional file 1 Alignment of Sin1 proteins from the fission yeast and the budding yeast. The Bestfit program was used to align the two sequences. Black shading shows identical residues. A conserved region (SCD III; see Fig. 4) is highlighted by a line above the sequence, and appears not so well conserved in the budding yeast as in other species. Abbreviations: S. pombe, Schizosaccharomyces pombe (fission yeast. GenBank accession No. AL136521). S. cerevisae, Saccharomyces cerevisae (budding yeast. GenBank accession No. NP_014563). Click here for file Additional file 2 Alignment of Sin1 proteins from the fission yeast and the red bread mold. The Bestfit program was used to align the two sequences. Black shading shows identical residues. Abbreviations: S. pombe, Schizosaccharomyces pombe (fission yeast. GenBank accession No. AL136521). N. crassa, Neurospora crassa (red bread mold. GenBank accession No. XP_322410). Click here for file

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554096

Theories of schizophrenia: a genetic-inflammatory-vascular synthesis

Background Schizophrenia, a relatively common psychiatric syndrome, affects virtually all brain functions yet has eluded explanation for more than 100 years. Whether by developmental and/or degenerative processes, abnormalities of neurons and their synaptic connections have been the recent focus of attention. However, our inability to fathom the pathophysiology of schizophrenia forces us to challenge our theoretical models and beliefs. A search for a more satisfying model to explain aspects of schizophrenia uncovers clues pointing to genetically mediated CNS microvascular inflammatory disease. Discussion A vascular component to a theory of schizophrenia posits that the physiologic abnormalities leading to illness involve disruption of the exquisitely precise regulation of the delivery of energy and oxygen required for normal brain function. The theory further proposes that abnormalities of CNS metabolism arise because genetically modulated inflammatory reactions damage the microvascular system of the brain in reaction to environmental agents, including infections, hypoxia, and physical trauma. Damage may accumulate with repeated exposure to triggering agents resulting in exacerbation and deterioration, or healing with their removal. There are clear examples of genetic polymorphisms in inflammatory regulators leading to exaggerated inflammatory responses. There is also ample evidence that inflammatory vascular disease of the brain can lead to psychosis, often waxing and waning, and exhibiting a fluctuating course, as seen in schizophrenia. Disturbances of CNS blood flow have repeatedly been observed in people with schizophrenia using old and new technologies. To account for the myriad of behavioral and other curious findings in schizophrenia such as minor physical anomalies, or reported decreased rates of rheumatoid arthritis and highly visible nail fold capillaries, we would have to evoke a process that is systemic such as the vascular and immune/inflammatory systems. Summary A vascular-inflammatory theory of schizophrenia brings together environmental and genetic factors in a way that can explain the diversity of symptoms and outcomes observed. If these ideas are confirmed, they would lead in new directions for treatments or preventions by avoiding inducers of inflammation or by way of inflammatory modulating agents, thus preventing exaggerated inflammation and consequent triggering of a psychotic episode in genetically predisposed persons.

Background When the solution to a clinical or scientific puzzle eludes us for more than a century, as with schizophrenia (formerly dementia praecox), we need new ways of thinking about the problem [ 1 , 2 ]. Efforts to understand schizophrenia have focused on neurons and, especially, the role of presumed excess dopamine neurotransmission. We believe that genetic, environmental, and stochastic factors combine with epigenetic factors to create episodes of the illness [ 3 - 5 ]. Thus, the syndrome of schizophrenia is viewed as an endpoint in a dynamic process variously conceptualized as degenerative or developmental or alternating at different points in the process [ 6 - 10 ]. Degenerative models imply that after a period of normal development, the organism, or one of its parts, takes a wrongful turn in its trajectory and begins to malfunction. This describes the eventual outcome for all life forms and is a biological restatement of the second law of thermodynamics. Since degeneration is universal, stating that an illness is degenerative is not particularly helpful. What would be helpful is to determine when in the life course the degeneration begins and how the degeneration is initiated and proceeds. Answers to the "when?" and "how?" questions would then describe the degenerative process in developmental terms. Developmental models of schizophrenia implicate abnormalities of early brain development predisposing to future schizophrenia. The proponents of the model further argue that the perturbations of development are limited to the early times of development and are discontinuous. Without this qualifier, developmental models are indistinguishable from degenerative models where the degeneration commences early in the life span. The early abnormalities are not necessarily the cause of schizophrenia, but, instead, create a state of risk for a future episode of schizophrenia. That is, a diathesis or predisposition is not a disease. Consequently, there must be factors later in life that convert the vulnerability to an illness. These additional factors are presumed to damage development in such a way that a predisposition becomes actualized. To gain a complete understanding of the syndrome, we must again return to the question of " what happens?" Following this line of reasoning, the distinction between degenerative and developmental models blurs. In fact, a medical-behavioral condition can be both developmental and degenerative as exemplified by Down syndrome [ 11 - 13 ]. Individuals born with trisomy 21 exhibit a number of developmental anomalies including cardiac malformations, abnormal dermatoglyphics, skeletal changes, and muscular hypotonia, to name a few. As trisomy 21 infants mature, most exhibit degrees of mental retardation. By about age 50, these individuals invariably develop Alzheimer-like CNS degenerative changes that can be seen at autopsy [ 13 ]. Schizophrenia involves both developmental and degenerative features. From the time of Bleuler [ 14 ] and Kraepelin[ 15 ], "It is certain that many a schizophrenia can be traced back into the early years of the patient's lives..." [ 14 ] p. 252. The 'follow back' studies of schizophrenia support these views [ 16 ]. Likewise, prospective studies of children at high risk for schizophrenia report developmental anomalies in motor skills, cognition, and attention long before the onset of overt illness [ 17 - 19 ]. Overt psychotic symptoms for some individuals usually start in the late teenage years or early twenties, but the illness can start as early as middle childhood [ 20 ] and may, more rarely, start in old age [ 21 ] p 73]. The evidence suggesting early developmental perturbations in schizophrenia is compelling. At the same time, there certainly are examples of deterioration reminiscent of Kraepelin's suggestion for some people with schizophrenia. However, deterioration in clinical course may not indicate CNS deterioration. Instead, the decline could be a secondary consequence of an illness that disrupts education, economic achievement, and social functioning leading to a downward spiral in all aspects of adult life. Consistent with an early degenerative process, there are reports of declining cognitive function preceding onset of psychosis [ 22 ]. Proponents of neurodevelopmental models suggest that the premorbid cognitive abnormalities are developmental risk factors for future schizophrenia (c.f [ 23 ]) and argue that such abnormalities show little evidence of decline after onset [ 6 , 24 ]. Whether developmental or degenerative, the premorbid cognitive deficits seen in schizophrenia are also seen in other disorders [ 25 ] and lack specificity and sensitivity thus detracting from the concept that the cognitive abnormalities seen in schizophrenia are useful endophenotypes [ 26 ]. The strongest evidence for a neurodegenerative phenomenon comes from imaging studies showing progressive loss of brain volumes [ 27 - 29 ]. Neuropathological studies fail to find widespread classic signs of neurodegeneration such as gliosis though there are exceptions to this generalization [ 30 ]. Observations of abnormal dendritic arborization [ 31 , 32 ] are consistent with the neuroimaging evidence suggesting abnormal connectivity between brain regions [ 29 ]. As a cautionary note, most of the neuroimaging and neuropathology results are subject to confounds from the effects of medications and various other treatments, post-mortem intervals, possible effects of diet, smoking habits, as well as a myriad of other potential confounds associated with glucocorticoid mediated stress following chronic illness and associated life's limitations [ 33 , 34 ]. The symptoms of schizophrenia are highly variable. Within families (and thus presuming relative homogeneity of genetic and environmental factors) symptoms can vary widely over time, as illustrated by identical quadruplets concordant for schizophrenia [ 35 ]. Even within affected individuals, symptoms will wax and wane and may even remit [ 36 ] suggesting a life long process. The major behavioral symptoms of schizophrenia include alterations in cognition, memory, perception, thought (inferred from language), motor functions, and affect. People with schizophrenia may show abnormal dermatoglyphics and other minor physical anomalies [ 37 - 42 ]. Other oddities to be incorporated in a comprehensive explanation of schizophrenia include highly visible nail fold capillaries [ 43 , 44 ] and the rarity of rheumatoid arthritis among schizophrenic persons [ 45 ]. These physical characteristics suggest the need to look beyond the nervous system per se to have a comprehensive view of the illness. The fact that the schizophrenia syndrome, as currently defined, is relatively common provides important information about the frequency of causal factors. About 1% of the population will experience schizophrenia during the lifespan. Except for a few rare exceptions, this 1% risk is remarkably constant around the globe regardless of culture, geography, or ethnicity. Men and women are affected equally. These facts mean that the risk factors for schizophrenia must also be common and ubiquitous. Given that the concordance rate for schizophrenia in identical twins [ 46 ] is only about 50%, there must be at least two global risk-increasing categories for schizophrenia, i.e., something(s) genetic and something(s) environmental. Assuming these risk factors are independent of each other, the joint probability of acquiring both risk factors is the product of their population frequencies that, for schizophrenia, equals about .01. To make a simplifying assumption to allow easy calculations, let us say that the two risk factors are present with about equal frequency in the population. With this simplification, straightforward mathematics indicates that the individual frequencies of these factors are close to the square root of the population frequency of 1%. That would mean that about 10% of the population would encounter at least one risk factor. The math indicates that the greater the number of independent risk factors, the more common they are. [See [ 47 ] for further elaboration]. Our challenge is to develop a theory of schizophrenia that can plausibly explain an illness that affects all domains of behavior (thought, affect, motor performance, etc), that has elements of developmental perturbations early in life leaving clues such as minor physical abnormalities, and also has elements of degenerative changes. At the same time, the defect is so subtle that we can't find the cause(s) with our best modern technology. Furthermore, in spite of brain-wide dysfunctions, many individuals with schizophrenia remain sufficiently intact that, with good treatment and a bit of luck, can maintain jobs and function usefully in society. Thus, we need to find frequent and ubiquitous factors that can affect virtually all brain functions as well as creating somatic signs, but they operate in ways that leave these functions only slightly "off kilter" as compared to the complete disruption seen in strokes, or classical degenerative disorders such as Alzheimer, or as seen in Down syndrome where the behavioral pathology is apparent from earliest stages. As we try to explain schizophrenia, we must account for most all of the developmental and degenerative features of schizophrenia. To account for the panoply of signs and symptoms seen in schizophrenia, any complete theory of schizophrenia must include organism wide systems. In addition to the nervous system, the immune system and the vascular system are defensible candidates. Both are invoked in the following theory: Some schizophrenia psychoses are the result of damage to the micro-vascular system in the brain initiated by genetically influenced abnormal inflammatory processes acting in response to ubiquitous environmental factors that trigger inflammatory responses, including infection, trauma, or hypoxia. It is the relative infrequency of the vulnerable genotypes in the population [ 48 ] that results in only a small proportion developing overt psychosis. We wish to emphasize that our hypothesis specifically identifies the microvascular system as the critical site of inflammation. We postulate that the inflamed micro-vessels lose their coupling with astrocytes, leading to disrupted regulation of cerebral blood flow and damage to the blood brain barrier. These disruptions in homeostatic mechanisms then lead to abnormal signal processing. Our focus on inflammation of the vessels differentiates our hypothesis from models of widespread parenchymal inflammation such as seen in psychotic syndromes following, for example, encephalitis lethargica, or paraneoplastic syndromes. Many acute inflammatory disorders of the brain involve inflammation of both the parenchyma and the vasculature. By contrast, we are proposing a chronic, smoldering, inflammation of the vessels alone. And, finally, we distinguish our hypothesis from the theories of schizophrenia implicating direct parenchymal infection of the brain (c.f. [ 49 ]) and also differentiates our hypothesis from speculations about schizophrenia that invoke infectious agents altering DNA [ 50 ]. Many prior debates about inflammation in the brains of people with schizophrenia have focused on the presence of absence of gliosis (see [ 51 ] for review). The consensus opinion is that gliosis, though present in some cases, is not a consistent feature of the neuropathology of schizophrenia. However, as Harrison [ 51 ] points out, evaluating gliosis is fraught with a multitude of problems and is not a definitive indicator of degenerative/inflammatory changes in the brain. More recent efforts have demonstrated activation of microglia in the brains of some individuals with schizophrenia implying an ongoing immunopathological process in addition to what ever happened early in development [ 52 ]. Ongoing neurodegenerative processes are suggested by increased levels of S100B, a small calcium binding astrocytic protein that is involved in inducing apoptosis and modulating proinflammatory cytokines [ 53 - 55 ]. It is likely that the current clinical syndrome of schizophrenia is etiologically heterogeneous. We do not pretend to explain all (DSM or ICD) cases of syndromal schizophrenia. Instead, we put forward our hypothesis as an attempt to define a psychiatric syndrome in terms of a particular pathophysiology. Following this course may then help refine our nosology (see also section on 'specificity' below) and cause us to recalculate basics 'facts' such as prevalence rates. Discussion A primer on CNS blood supply Neurons derive their energy from oxygen and glucose delivered by the vascular system, plus lactate and glycogen derived from astroglia [ 56 ]. The combination of neurons, astroglia, and micro-vessels form a metabolic trio [ 56 ] whereby the glia extend processes interacting with neurons on the one hand and, on the other, form endplates interdigitated into capillary walls. Rather than being passive conduits, the CNS vascular system is the most precisely managed and the most complex fluid dynamic system known. Regulation of cerebral blood flow (CBF) is managed primarily by a coupling between astrocytic glial cells [ 56 - 59 ] and capillary endothelium [ 60 - 65 ]. Astrocytes sense local neuronal metabolic activity and adjust blood flow as needed. Cerebral vessels change caliber in response to vasoactive substances released by astrocytes activated by glutamate receptors [ 56 , 66 , 67 ]. Serotonin [ 68 ], acetylcholine [ 69 ] and dopamine [ 66 , 70 , 71 ] transmission between astrocytes and micro vessels also play roles. When the neuronal activation of discrete areas is sustained over longer periods, vasoactive substances stimulate angiogenesis resulting in increased capillary density [ 67 ] thus enhancing local neuronal circuitry. Conversely, decrease in capillary density is likely to reduce the functional capacity of brain areas so affected [ 67 ]. Consequently, capillary beds in the cortex are not distributed in uniform fashion [ 72 ]. There are close relationships among local neuronal activity, density of capillary bed, and the distribution of valve-like flow control structures [ 73 ]. Developmentally, the CNS vascular system originates from capillary endothelial cells that migrate into developing neuro-ectoderm under the influence of trophic factors such as vascular endothelial growth factor (VEGF) [ 74 ] and erythropoietin [ 75 ] both produced by astroglia. The developing micro-vasculature, although comprising only 0.1% of the entire brain, and operating under the influence of genetic directives, has a key role in the development, maintenance and repair of the brain [ 76 ]. In turn, VEGF has trophic effects on neurons and glial cells, and the activity of VEGF influenced angiogenesis is directly proportional to the high metabolic activity of neocortical development [ 77 ]. Thus, angiogenesis and neurogenesis occur simultaneously and synergistically [ 78 - 80 ]. In addition to formation of capillaries themselves, intricate anastomoses between micro-vessels further 'fine tune' the metabolic support of developing glia and neurons [ 81 ] The genetics of infectious & inflammatory diseases When infectious agents give rise to inflammatory vascular disease, the nature of the infectious agent may be less important that an individual's genetically influenced inflammatory response. The concept that infectious disease may have a genetic component is, of course, not new. Many agricultural geneticists make their livings by breeding disease resistance into both plants and animals [ 82 , 83 ]. One of the founders of behavioral genetics, Franz Kallmann [ 84 ], showed genetic factors influenced acquiring tuberculosis (DZ concordance = 26%, MZ concordance = 87%), an observation that was confirmed in modern times [ 85 , 86 ]. Many other infectious diseases appear to have genetic factors influencing susceptibility or resistance to the infection [ 87 - 97 ]. Mechanisms for genetically mediated responses to infection occur through genetic variations in immune mediators such as cytokines[ 96 ] and HLA factors [ 98 , 99 ]. Familial Mediterranean Fever (FMF) [ 100 , 101 ] provides a heuristic Mendelian example. The gene for FMF is located on the short arm of chromosome 16 and produces pyrin (marenostrin) that functions in a negative feed back loop to suppress inflammation. Absence of pyrin leads to exaggerated inflammatory responses. Vasculitis is one of the consequences [ 102 ]. Additionally, very high rates of rheumatic fever (RF) or rheumatic heart disease (RHD) are found in relatives of patients with FMF[ 103 ]. Having even one mutant gene appears to lead to immune hyperactivity to streptococcal antigens. We also know that antibody [ 104 ] production and cytokine activity [ 105 ] in RF patients is more marked than non-rheumatics. It is clear that genes influence the host's response to infection. A similar line of reasoning applies to other inducers of inflammation such as traumatic injury [ 106 ] or hypoxia [ 107 , 108 ]. Just as the CNS blood supply is highly regulated, the inflammatory systems in the brain require 'fine tuning.' Given the limited ability for adult brain to regenerate, and assuming there is little tissue to spare, it would make sense that the brain should be protected from overabundant inflammatory reactions [ 109 ]. Astrocytes play a key role in the expression of inflammatory cytokines, chemokines, and growth factors involving the modulation of gene expression for these factors [ 109 - 111 ]. Let us suppose that schizophrenia develops following an infection (or trauma or anoxia – the environmental contributors) but the host's response is determined by genetic factors regulating the nature and degree of inflammation. That infectious agents may be operative in schizophrenia is supported by several of lines of evidence. Summaries can be found in numerous sources [ 49 , 50 , 112 - 116 ]. The same concept applies to trauma [ 106 ] or anoxia [ 79 , 107 ] that may also stimulate inflammatory processes. Vascular disease and psychopathology The syndrome of schizophrenia is likely to be etiologically heterogeneous and a multitude of CNS disorders can give rise to schizophrenic-like psychoses [ 117 ]. The idea that CNS micro-vascular diseases, in particular, are factors in psychotic disorders is also an old idea [ 118 , 119 ] that deserves a second look in light of new perspectives offered by developments in the genetics of inflammatory diseases. There are many examples of psychoses resulting from micro-vascular CNS disease including lupus and Sjögren syndrome [ 120 ]. Neuroimaging and neurocognitive deficits in these disorders are similar to those seen in schizophrenia [ 121 ]. Psychoses associated with substance abuse are also associated with CNS vasculitis [ 122 ]. Furthermore, infectious agents such as syphilis [ 123 ] and rheumatic fever (RF – see below), lead to micro-vascular disorders of the CNS that are associated with psychiatric symptoms including psychoses. Thomas, et. al. [ 124 ] also demonstrated small vessel abnormalities in the depressed elderly. At the same time, there is growing interest in cytokines and other inflammatory agents in psychoses[ 125 ] as well as growing awareness that inflammatory reactions are modulated by neuropeptides [ 126 ]. Inflammatory processes often damage the precise regulation of cerebral blood flow. The wide spectrum of clinical conditions thought to be created, in part, by inflammatory CNS micro-vessel disease include Alzheimer disease where it is thought that inflammatory processed damage the micro-vascular endothelium causing insufficient blood flow leading to oxidative stress, a build up of amyloid, and eventual cell death [ 127 - 135 ]. Cerebral palsy is also conceptualized as an infectious-inflammatory-vascular disorder where the vascular lesion is complete thrombosis [ 136 ]. Neurotoxic effects of methamphetamine and cocaine appear to be due to induction of inflammatory genes in small vessel endothelial cells [ 122 , 137 ], thus explaining the vascular damage seen in amphetamine and cocaine abuse that was previously attributed to contaminants of injected drugs [ 122 , 138 - 140 ]. Returning to the early stages of life, we have seen that the development of the neurons and glia are intimately associated with, and dependent on, the parallel development of the CNS vasculature. If the stated theory is correct, and given the developmental perspective of schizophrenia ---early developmental perturbations of the CNS set the stage for later schizophrenia--- we would expect to find support for the idea that inflammatory events early in life affect CNS vascular function. Such is the case. Whether the early insults are traumatic, infectious, or hypoxic; inflammatory process are involved in the attempts to protect and repair by modulating angiogenesis [ 141 - 148 ]. Thus, the reports implicating pregnancy and birth complications (anoxia, trauma or maternal infections) in the development of some cases of schizophrenia [ 149 , 150 ] could all be mediated by the common pathway of inflammatory-vascular mechanisms. Individuals who's genes created perturbations in inflammatory-vascular regulation would continue to experience abnormalities of protection and repair in response to subsequent CNS insults. Over time, the accumulation of 'hits' could lead to brain dysfunction to the extent seen in psychoses. The greater the number and duration of 'hits,' the greater the risk for a deteriorating /degenerative course. That neuroleptics may alter the permeability of the blood brain barrier and modify immunoregulation in the CNS [ 151 ] strengthens the argument for early treatment as a strategy to prevent deterioration. Alterations of cerebral blood flow in schizophrenia Since the time of Seymour Kety's pioneering efforts [ 152 , 153 ], there has been interest in altered cerebral blood flow in people with schizophrenia. An in-depth review of this large literature is beyond the scope of this paper. The interested reader is referred to discussions of reduced anterior cerebral perfusion leading to the concept of 'hypofrontality' in schizophrenia [ 154 , 155 ] and to more recent reviews [ 156 - 158 ]. Bachneff's [ 159 ] review and theory about defects in regulation of CNS microvascular systems is particularly relevant. These reviews summarize a consistent body of evidence showing reduced cerebral blood flow in brains of people with schizophrenia especially to anterior regions. Flow deficits are seen in medication-naive new onset cases [ 160 , 161 ] and more established cases free of neuroleptics [ 162 ] suggesting that flow perturbations are neither the consequence of duration of illness nor treatment. Neuroleptics can alter cerebral blood flow [ 163 , 164 ] although the effects may be regionally and drug specific [ 165 , 166 ]. Decreased frontal flow is often associated with negative symptoms [ 167 , 168 ]. In addition to the frontal cortex, flow abnormalities in people with schizophrenia have been noted in the cingulate cortex [ 169 , 170 ], thalamus [ 171 ], basal ganglia [ 172 ], parietal cortex [ 167 , 170 ] and cerebellum [ 171 ]. Furthermore, in some instances, flow rates are increased [ 160 , 170 ]. Rather than a simple hypothesis of hypofrontality in schizophrenia, theorizing is evolving toward a concept of "dysfunctional circuits"[ 160 ] or "inefficient dynamic modulation" [ 173 ] of cerebral metabolism which is supported by other examples of abnormal modulation of cerebral blood flow in response to activation tasks [ 171 , 174 ]. Disturbances of blood flow in schizophrenia are well documented but are not limited to schizophrenia. Disturbed cerebral blood flow is also reported in obsessive compulsive disorder [ 175 ] and depression [ 176 , 177 ] as well as in Alzheimer disease (cited earlier). The usual interpretation is that alterations of blood flow arise as a consequence of abnormal neuronal metabolism. The theory proposed by this paper turns the causal arrow around to suggest that abnormalities of blood flow lead to altered neuronal-glial function that, in turn, leads to psychopathology. There has been scant direct visualization of the vascular system in schizophrenia, but at least one laboratory has found evidence of atypically simplified angioarchitecture and failure of normal arborization of small vessels [ 32 ]. Post- streptococcal behavioral syndromes as a model Post-streptococcal neuropsychiatric syndromes include Syndenham chorea, the PANDAS/obsessive compulsive syndrome, tics including Tourette syndrome, and possibly, ADHD [ 178 - 184 ]. Psychotic disorders are also implicated [ 183 , 185 ] and see citations below. Sydenham chorea is the best-known neuropsychiatric complication following streptococcal pharyngitis. The association of psychoses and Sydenham chorea as well as with RF even in the absence of chorea, was discussed in the 17 th and 18 th centuries starting with Sydenham himself (see [ 186 ]). The interest in psychoses associated with RF continued throughout the 1900's [ 187 - 197 ]. People with a history of Sydenham chorea and/or rheumatic fever are at high risk for developing psychopathology later in life [ 198 , 199 ] with a relative risk for schizophrenia as high as 8.9 in a 10 year follow-up of 29 Sydenham patients [ 200 ]. There is a suggestion that the family members of Sydenham patients are also at higher risk for psychosis [ 201 ]. During the 1940's-1960's when RF was still quite prevalent, people with psychoses appeared to have higher than expected rates of histories of RHD or RF)[ 195 , 202 , 203 ] or rheumatic chorea [ 204 ]. Psychotic patients with RHD more often had early (<age 19) onset, movement disorders, progressively insidious courses and poor long-term outcomes [ 203 ]. Preliminary data from a Minnesota study also finds increased rates of RHD in psychotic patients, a pattern of increased psychiatric hospitalization following an epidemic of RF, and a clinical course for "rheumatic psychoses" that disproportionately led to a severe and continuous decline in function [ 205 ]. Although schizophrenia-like psychoses were the most common psychopathology related to rheumatic syndromes, manic-depressive, involutional, and senile psychoses were also observed [ 183 , 197 ]. An inflammatory reaction of the CNS vascular endothelium (vasculitis) is a common denominator in the both acute and chronic cerebral consequences of rheumatic fever. [ 186 , 187 , 190 , 195 , 197 , 206 - 209 ]. The microvascular lesions suggest both an obliterating process likely due to micro-emboli from rheumatic cardiac valves and an inflammatory process involving irregular proliferative changes in the vascular endothelium, dilatation of the lymphatic spaces surrounding the capillaries suggesting increased permeability of the capillary endothelium, and inflammatory cell infiltrates. Disruption of the blood brain barrier suggested by the evidence of increased permeability of the small vessels could compromise the immunological protection of the brain leading to the formation of the anti-neuronal antibodies seen in post-streptococcal CNS syndromes. In parallel fashion, people with schizophrenia show evidence of altered blood brain barrier and consequent alterations in immunological markers [ 210 ] The post-strep psychopathologies provide a precedent for the hypothesis of this paper by demonstrating that an infectious process can trigger a series of inflammatory reactions that lead to a variety of somatic and psychiatric syndromes, including psychoses where vascular pathology is implicated. The pathogenicity of a strep infection is a function of the strain (genotype) of the bacterium and the genetically mediated inflammatory mechanisms of the host [ 211 ] and illustrates how a ubiquitous and often relatively benign environmental factor can create more serious sequelae in a limited number of genetically predisposed individuals-true genotype by environment interaction. Summary The ideas here are not completely new. Eugen Bleuler [ 14 ] remarked: "The fragility of the blood vessels which appears in many schizophrenics, both acute and chronic, seems to indicate a real vascular pathology (p.167)." We bring old ideas forward into the light of new understandings offered by molecular genetics and inflammatory diseases. Since the late 1800's there has been evidence of inflammatory neuro-vascular abnormalities in psychiatric illness that were initiated by infectious agents. CNS lues (syphilis) is the best-known example. This paper expands the concept to suggest that a variety of environmental insults (infection, trauma, anoxia) may follow a common final pathway to psychopathology by stimulating inflammatory processes that damage the capillary-glial-neuron triad as illustrated in Figure 1 . Abnormal behaviors develop as a result of disruptions in astroglial mediated coupling of cerebral blood flow to neuronal metabolic needs. These subtle disruptions are hard to find, as the microvasculature comprises only about 0.1% of the brain and are of a scale more appropriate for electron microscopy. None-the-less, the hemodynamic perturbations have sufficient impact to cause subtle but widespread disruption of the normally harmonious coordination of CNS function leading to a condition variously conceived as a "neurointegrative defect"[ 212 ], "synaptic slippage" [ 213 ], "abnormal signal transduction" [ 4 ], "inefficient dynamic modulation" [ 173 ] or "synaptic destabilization" [ 214 ]. The ultimate impact would lead to psychopathology including psychoses as the vascular-glial-neuron triad is progressively damaged over time after repeated inflammatory episodes. The resultant failure to regulate the delivery of oxygen and energy adequately would lead to oxidative stress [ 215 - 217 ]. Oxidative stress, in turn, can further damage the microvasculature and the blood brain barrier [ 218 - 220 ]. The astroglial-capillary partnership that protects the integrity of the blood brain barrier would be compromised, thus exposing neural tissue to damage from immunological attack [ 221 ]. Known precedents of such processes are found in the behavioral changes seen in CNS vascular inflammatory diseases such as lupus and the post-strep syndromes described above. This theory could explain how developmental events such as prenatal infections [ 150 , 222 ], and other birth and pregnancy complications [ 149 ] including anoxia [ 223 ] are linked to later schizophrenia – infection, trauma, or anoxia all stimulate inflammatory processes [ 224 ]. The data suggesting an (statistical) influence of season of birth [ 116 ] is also consistent with the hypothesis as infectious epidemics often follow seasonal patterns. Some of the minor physical anomalies such as unusual scalp hair patterns and dermatoglyphic changes are explained because the development of these phenomena are linked to each other [ 225 ], to the development of the central nervous system [ 226 ], and are developmentally modulated by the pleiotropic effects of the same substances that modulate brain vascular development (e.g., vascular endothelial growth factor/vascular permeability factor [ 227 ] and epidermal growth factor [ 228 ]). The waxing and waning of symptoms would correspond to waxing and waning of inflammations as individuals are exposed, recover, and then re-exposed in conjunction with other physiological and hormonal influences, as seen in lupus [ 229 ]. The nature and severity of symptoms would depend on where in the brain the inflammation takes place and this may be stochastic. As the micro- vascular system is everywhere in the brain, lesions could produce the variety of symptoms seen in schizophrenia including dysfunctions of thought, emotion, memory, motor skills and autonomic regulation. The developmental age of the individual will also make a difference. Inflammatory processes that alter angiogenesis during prenatal development will likely have more dire consequences than inflammatory reactions that start after CNS maturation although even the adult brain remains susceptible [ 230 ]. We have attempted to schematically illustrate this dynamic process in Figure 2 . This theory also captures many of the little oddities observed in schizophrenia. For example, the reported abnormalities of the nail fold capillary beds seen in some people with schizophrenia [ 44 ] are also seen in people with inflammatory disorders such as FMF [ 231 ] and rheumatoid arthritis [ 232 ]. Another oddity is the negative association between schizophrenia and rheumatoid arthritis [ 45 ]. There are parallels in the post-streptococcal syndromes where RF and acute post-streptococcal glomerulonephritis very rarely occur in the same patient [ 233 ]. Some strains of group-A-streptococci identified by their M-protein serotypes are rheumatogenic while others are nephritogenic [ 233 , 234 ]. Phage or phage-like elements inserted into the streptococcal DNA are a major source of variation between streptococcal strains and these elements determine pathogenicity [ 235 ]. Additionally, host variation in humoral and cellular immune response shape the outcome of infection[ 211 ] By analogy, individuals with vascular/CNS involvement following, for example, streptococcal infections may be systematically spared from joint involvement as a function of both the invading strain and the individuals susceptibilities. Alternatively, as postulated for Alzheimer disease (cited earlier) that is also less common in people treated for arthritis, the anti-inflammatory treatments for arthritis might reduce the risk of inflammatory brain disease. Another line of evidence compatible with this theory is the observation that genetic linkages for schizophrenia coincide with sites for glial growth factor cell regulators [ 214 ] and, as we have seen, the glia are key intermediaries of CNS inflammation and vascular regulation. More specifically, emerging data demonstrate associations between schizophrenia and genetic polymorphisms in regulators of inflammation such as tumor necrosis factor alpha genes [ 236 , 237 ] and interleukin-1 genes [ 238 ]. Another piece that fits into the puzzle is the fact that neuroleptics have inflammatory modulating properties [ 239 - 244 ] and neuroleptic treatment may be synergized by addition of anti-inflammatory drugs [ 245 ]. It may well be that the environmental components of psychiatric illness such as schizophrenia are relatively minor, ubiquitous, or chance events [ 246 , 247 ] that have the potential to stimulate the inflammatory systems. However, the nature of the insults may be less important than individuals' genetically influenced and idiosyncratic responses to the insults, similar to individuals with FMF who have an exaggerated inflammatory response. Thus, the genetic components of the inherited predisposition to mental illness may lie "upstream" in the immune system rather than in the CNS per se. The possibility that the environmental agents may be nearly universal (e.g. who has not had a strep throat or viral syndrome?), will mean that the prevalence of the etiological factor will be similar in control and experimental groups thus making it too easy to dismiss key environmental factors in null hypothesis designs [ 47 , 248 ]. Rather than focus on the environmental contributors that could be non-specific and ubiquitous, it will be more productive to look for genotypes that respond abnormally to triggers of inflammation and microvascular dysfunction (cf[ 48 ]). These individuals would be the ones who are at high risk for psychiatric illness. However, the inflammatory processes involve a cascade of steps involving many genes. But this, too, fits with the polygenic features of schizophrenia [ 249 ]. Identification of high-risk individuals, combined with such tools as immunizations or anti-inflammatory agents may promote prevention of much psychiatric morbidity. Already, the cytokine regulator and vascular growth factor erythropoietin is suggested as a possible neuroprotective factor in schizophrenia [ 250 ] Future directions The speculations about psychoses developing from vascular/inflammatory processes provide direction for future research across many domains. In addition to pursuing direct evidence of altered activities in inflammatory/immune systems in people with psychoses, the inflammatory/vascular theory has implications for epidemiology, genetics, neuroimaging and neuropathology. For the epidemiologist, the challenge will be to detect relatively small signals against a very noisy background. We hypothesize that the triggers for inflammation can be many and varied and are common factors in the environment. Imagine starting with the clinical syndrome of Sydenham chorea and comparing the rates of strep throat in those affected vs. comparison sample of people without Sydenham chorea. Null hypothesis testing with small sample sizes and nearly ubiquitous etiological agents are clearly not adequate. A second epidemiological challenge is to cast a broad enough net to capture the wide variety of possible contributing factors. Rather than taking a one by one approach to exploring the etiological contributions of, say, virus titers, anoxia, physical trauma, the epidemiologist should look for any and all. It would be predicted that individuals with multiple "hits" (e.g. in utero exposure to virus and low Apgar scores and childhood head trauma) would be at greater risk than those exposed to just one event. If in utero inflammatory processes are active in the genesis of schizophrenia we would also predict an increased rate of fetal deaths in families of schizophrenic probands. A third epidemiological opportunity lies in the search for non-psychiatric inflammatory-related disease or traits in people with psychosis. If something is askew in the inflammatory process in schizophrenia, the effects will show up in other parts of the body. Though requiring replication, the association of psychosis with hemolytic anemia in lupus [ 251 ] provides an illustrative example. In addition to rheumatoid arthritis, the associations of diabetes and cancer have been explored in schizophrenia; one of is exploring rheumatic heart disease [ 205 ]. Population-based health registries should be used in a search for co-morbid physical illness. For geneticists, the proposed theory obviously points to linkage/association studies using inflammation genes; a few examples were cited previously [ 236 - 238 ]. A simple step with extant data might start with a meta analysis defining chromosomal "hot spots" for linkage with schizophrenia and search the gnome maps for immune regulators at these sites as Moises, et al [ 214 ] have done for glial growth regulators. Family, twin, and adoption methodologies can all be applied to the issue of co-morbid or co-segregating physical conditions. The inflammatory/vascular theory has much to suggest to neuroimaging research especially in the realm of reinterpreting regional perturbations in metabolic activity as primary disturbances of flow regulation rather than intrinsic neuronal metabolic abnormalities. It would be interesting to assess the impact of vasoactive compounds and inflammatory modulators on neuroimaging studies of regional blood flow. Likewise, further pursuit of neuroimaging evidence of disrupted blood brain barrier, as initiated by Dysken, et al [ 252 ], and with manipulation of inflammatory systems as suggested by Mueller and Ackenheil [ 253 ] would test our hypothesis. The neuropathology of schizophrenia, focused mostly on the neurons, is notable for inconsistencies in findings (see [ 51 , 254 ] for reviews). Such inconsistency is exactly what would be predicted by an inflammatory/vascular theory where the lesions are truly functional in the sense that the function of the brain alters in relation to perturbations in blood flow regulation. Only the more prolonged and serious inflammation will leave visible traces of neuronal damage and such damage may be patchy and inconsistent from one patient to another. However, over the early years of CNS development, alterations in cellular organization or migration may result from disrupted angiogensis that must go hand in hand with neuronal and glial development. The location and extent of CNS change will be a function of severity of inflammation and timing during development. Such consequences will be hard to demonstrate in human post-mortem tissues and animal or in vitro models may be more fruitful areas for study the effects of inflammation on neurogenesis and blood flow regulation. To our knowledge, human post mortem studies have not utilized vascular cast methodology and this should be considered, perhaps casting one half of a specimen brain while subjecting the other half to cellular analysis. Specificity Because of our interests and expertise, we have focused our attention on schizophrenia as the behavioral phenotype resulting from inflammatory-vascular pathology but the theory presented here is likely to be more general. Indeed, our use of examples of psychoses associated with known inflammatory- vascular pathologies (e.g. autoimmune CNS vascular disease or infectious CNS vascular disease as seen in syphilis) makes it clear that a vascular-inflammatory theory may apply to a wide range of psychotic conditions that may also include psychoses associated with mood disorders. Whereas, the classical genetic studies support the separateness of schizophrenia and mood disorders [ 255 ], there are modern molecular signs that schizophrenia and mood disorders share genetic elements in common [ 256 , 257 ]. Furthermore, mood disorders, like schizophrenia, show evidence of frontal lobe pathology, enlarged ventricles, abnormal cerebral blood flow [ 33 , 258 ] and vascular abnormalities [ 124 ]. To what extent all of these changes are epiphenomena of being psychotic (treatment effects or stress, etc) remain debatable [ 259 ]. However, finding similar brain changes in a variety of psychotic conditions does not necessarily mean these changes are epiphenomena. Examples from neuropsychiatry teach us that the underlying pathology does not necessarily define the behavioral symptoms. Thus, psychoses with Huntington disease may be affective-like or schizophreniform. Similar pathophysiological mechanisms may underlie a variety of psychotic phenotypes. The evolution of behavioral symptoms for any given pathophysiology may depend on a variety of moderating variables such as an individual's developmental age when the disease process begins, gender, hormones, genetic 'landscape' upon which the disease process unfolds, along with the nature, frequency, and intensity of successive triggers of inflammatory response. Reprise A broad spectrum of observations leads to a working hypothesis that schizophrenia and, possibly, other psychiatric syndromes are the result of genetically mediated inflammatory reactions that damage the neuron-glial-capillary triad with resultant loss of ability to fine tune regional brain metabolism. This hypothesis incorporates genetic, epigenetic [ 260 ], and environmental factors. Furthermore, an inflammatory/vascular theory can explain the variety of behavioral symptoms seen in schizophrenia, the variable course of the illness, and the numerous other puzzling observations such as an excess of minor physical anomalies. Should this theory prove heuristic, it would point to the use of inflammatory modulators in treating the illness. Perhaps more importantly, identifying individuals who were at high risk for the disorder in high genetic risk families as well as the general population, because of abnormalities of their inflammatory systems, holds hope for prevention through early intervention using inflammatory modulators. List of abbreviations ADHD attention deficit hyperactivity disorder BDNF brain derived neurotropic factor CBF cerebral blood flow CNS central nervous system DZ dizygotic FMF familial Mediterranean fever MZ monozygotic NGF nerve growth factor NO nitric oxide PANDAS pediatric autoimmune neurological disorder associated with strep. RF rheumatic fever RHD rheumatic heart disease VEGF vascular endothelial growth factor Competing interests The author(s) declare that they have no competing interests. Authors' contributions This article was the joint effort of both authors with input as noted below. Pre-publication history The pre-publication history for this paper can be accessed here:

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Monomethyl Branched-Chain Fatty Acids Play an Essential Role in Caenorhabditis elegans Development

Monomethyl branched-chain fatty acids (mmBCFAs) are commonly found in many organisms from bacteria to mammals. In humans, they have been detected in skin, brain, blood, and cancer cells. Despite a broad distribution, mmBCFAs remain exotic in eukaryotes, where their origin and physiological roles are not understood. Here we report our study of the function and regulation of mmBCFAs in Caenorhabditis elegans, combining genetics, gas chromatography, and DNA microarray analysis. We show that C. elegans synthesizes mmBCFAs de novo and utilizes the long-chain fatty acid elongation enzymes ELO-5 and ELO-6 to produce two mmBCFAs, C15ISO and C17ISO. These mmBCFAs are essential for C. elegans growth and development, as suppression of their biosynthesis results in a growth arrest at the first larval stage. The arrest is reversible and can be overcome by feeding the arrested animals with mmBCFA supplements. We show not only that the levels of C15ISO and C17ISO affect the expression of several genes, but also that the activities of some of these genes affect biosynthesis of mmBCFAs, suggesting a potential feedback regulation. One of the genes, lpd-1, encodes a homolog of a mammalian sterol regulatory element-binding protein (SREBP 1c). We present results suggesting that elo-5 and elo-6 may be transcriptional targets of LPD-1. This study exposes unexpected and crucial physiological functions of C15ISO and C17ISO in C. elegans and suggests a potentially important role for mmBCFAs in other eukaryotes.

Introduction Fatty acids (FAs) belong to a physiologically important class of molecules involved in energy storage, membrane structure, and various signaling pathways. Different FAs have different physical properties that determine their unique functions. Among the most abundant in animal cells as well as the most studied are those of long-chain even-numbered saturated and unsaturated FAs. C15ISO and C17ISO are saturated tetradecanoic and hexadecanoic FAs with a single methyl group appended on the carbon next to the terminal carbon ( Figure 1 ). Monomethyl branched-chain FAs (mmBCFAs) in ISO configuration as well as in anteISO configuration (methyl group appended on the second to the terminal carbon) also seem to be ubiquitous in nature. They are present in particularly large quantities in various bacterial genera, including cold-tolerating and thermophilic species ( Merkel and Perry 1977 ; Annous et al. 1997 ; Ferreira et al. 1997 ; Batrakov et al. 2000 ; Jahnke et al. 2001 ; Groth et al. 2002 ; Nichols et al. 2002 ). There, mmBCFAs contribute to the membrane function, regulating fluidity ( Rilfors et al. 1978 ; Suutari and Laakso 1994 ; Cropp et al. 2000 ; Jones et al. 2002 ) and proton permeability ( van de Vossenberg et al. 1999 ). Figure 1 Structure of mmBCFAs of 15 and 17 Carbons C15ISO, 13-methyl myristic acid; C17ISO, 15-methyl hexadecanoic acid; C17anteISO, 14-methyl hexadecanoic acid. Other mmBCFAs mentioned in the text are the following: C13ISO, 11-methyl lauric acid; C15anteISO, 12-methyl tetradecanoic acid. C15ISO and C17ISO are readily detectable in C. elegans. Although comprehensive reports on mmBCFAs in eukaryotes are lacking, sporadic data indicate that they are present in the fungi, plant, and animal kingdoms ( Garton 1985 ; Seyama et al. 1996 ; Martinez et al. 1997 ; Cropp et al. 2000 ; Wolff et al. 2001 ; Destaillats et al. 2002 ). In mammals, mmBCFAs have been detected in several tissues, including skin ( Aungst 1989 ), Vernix caseosa ( Nicolaides and Apon 1976 ), harderian and sebaceous glands ( Nordstrom et al. 1986 ), hair ( Jones and Rivett 1997 ), brain ( Ramsey et al. 1977 ), blood ( Holman et al. 1995 ), and cancer cells ( Hradec and Dufek 1994 ). The fact that mmBCFAs are present in a wide variety of organisms implies a conservation of the related metabolic enzymes and consequently important and perhaps unique functions for these molecules ( Jones and Rivett 1997 ). Nevertheless, their physiological roles and metabolic regulations have not been systematically studied and thus remain fragmentary. It was found that C21anteISO is the major covalently bound FA in mammalian hair fibers. A removal of this FA from its protein counterparts results in a loss of hydrophobicity ( Jones and Rivett 1997 ). Other studies indicated that C17anteISO esterified to cholesterol binds to and activates enzymes of protein biosynthesis ( Tuhackova and Hradec 1985 ; Hradec and Dufek 1994 ). A potential significance of mmBCFAs for human health is associated with a long-observed correlation between amounts of these FAs and disease conditions such as brain deficiency ( Ramsey et al. 1977 ) and cancer ( Hradec and Dufek 1994 ). More recent studies have revealed a role of another mmBCFA, C15ISO, as a growth inhibitor of human cancer where it selectively induces apoptosis ( Yang et al. 2000 ). Given how important these FA molecules may be and how little is known about their biosynthesis and functions in eukaryotes, it is an opportune problem to study. De novo synthesis of long-chain mmBCFAs described for bacteria is quite different from the biosynthesis of straight-chain FAs ( Smith and Kaneda 1980 ; Oku and Kaneda 1988 ; Toal et al. 1995 ). While the latter uses acetyl-coenzyme A (acetyl-CoA) as a primer condensing with a malonyl-CoA extender, branched-chain FA synthesis starts with the branched-chain CoA primers derived from the branched-chain amino acids leucine, isoleucine, and valine. To synthesize branched-chain FAs, organisms must have a system for supplying branched-chain primers along with the enzymes utilizing them ( Smith and Kaneda 1980 ). No such enzymes have been previously characterized in vivo for any eukaryotic organisms. Here we describe our approach to characterize the biosynthesis and function of mmBCFAs using the free-living nematode Caenorhabditis elegans. Combining genetic, molecular, and biochemical analyses, we show that the worm is not only able to synthesize mmBCFAs de novo but is also absolutely dependent on these FA species for its growth and development. Results/Discussion C. elegans Synthesizes Branched-Chain FAs De Novo and Uses Two FA Elongation Enzymes to Produce C15ISO and C17ISO In characterizing FA elongation in C. elegans, we identified eight sequences homologous to the yeast long-chain FA elongation enzymes ( Kniazeva et al. 2003 ). To test for their possible functions in vivo, we applied RNAi to the corresponding genes, followed by an analysis of FA composition in whole animals using gas chromatography (GC). RNAi treatment of four genes— elo-3 (D2024.3), elo-4 (C40H1.4), elo-7 (F56H11.3), and elo-8 (Y47D3A.30)—did not produce any notable phenotypes, whereas suppression of elo-1 (F56H11.4) and elo-2 (F11E6.5) affected the elongation of straight long-chain saturated and polyunsaturated FAs ( Beaudoin et al. 2000 ; Kniazeva et al. 2003 ). Surprisingly, the RNAi treatment of the two remaining genes, elo-5 (F41H10.7) and elo-6 (F41H10.8), affected the levels of branched-chain FA. Transcriptional reporter constructs ( elo-5Prom :: GFP and elo-6Prom :: GFP ) indicated that both genes are expressed in the gut ( Figure 2 ). In addition, elo-5 was expressed in unidentified head cells and elo-6 was expressed in neurons, pharynx, and vulva muscles. Figure 2 The Expression of elo-5Prom :: GFP and elo-6Prom :: GFP Constructs in Wild-Type Worms (A, C, E, and G) DIC images; (B, D, F and H) fluorescence images. (A–D) Strong expression of the elo-5Prom :: GFP construct in the gut and in the head is shown. (E–H) The expression of the elo-6Prom :: GFP construct in the gut, vulvae (white arrows), and nerve ring is shown. Scale bars, 100 μm. The RNAi of elo-6 significantly reduced the amount of only C17ISO, while the RNAi of elo-5 dramatically reduced quantities of both C15ISO and C17ISO ( Figure 3 ). These results indicate that ELO-5 might be involved in the biosynthesis of C15ISO and possibly also C17ISO, whereas ELO-6 may function in elongating C15ISO to C17ISO ( Figure 3 C and 3 D). To our best knowledge, these are the first enzymes that have been shown to be involved in long-chain mmBCFA biosynthesis in a nonbacterial in vivo system and the first enzymes of the long-chain FA elongation family related to mmBCFA production. Figure 3 RNAi Treatment of elo-5 and elo-6 Significantly Alters the FA Composition (A and B) GC profiles showing the FA composition in the wild-type strain (Bristol N2) containing the RNAi feeding control vector and in the elo-5(RNAi) feeding strain. Arrowheads point to the peaks corresponding to C15ISO and C17ISO. (C) Comparison of FA composition in three strains: wild type, elo-5(RNAi), and elo-6(RNAi). C17ISO is decreased in both RNAi strains, while C15ISO is only decreased in elo-5(RNAi). (D) Suggested elongation reactions catalyzed by ELO-5 and ELO-6 in C15ISO and C17ISO biosynthesis. FAs are elongated by an addition of two carbon groups at a time. Combined data presented in this figure and in the text suggest that ELO-6 acts at the elongation step from C15 to C17, whereas ELO-5 may be involved in the production of both C15ISO and C17ISO. In bacteria, mmBCFA biosynthesis utilizes branched-chain α-keto-acids of leucine, isoleucine, and valine to produce mmBCFA acyl-CoA primers that substitute for acetyl-CoAs in conventional FA biosynthesis ( Oku and Kaneda 1988 ). Key enzymes engaged in synthesizing the mmBCFA acyl-CoA primers are branched-chain aminotransferase (BCAT) and the branched-chain α-keto-acid dehydrogenase (BCKAD) complex ( Figure 4 A). The elongation of the mmBCFA backbone is then carried out by fatty acid synthetase (FAS). Figure 4 The C. elegans BCKAD Homolog Is Involved in mmBCFA Biosynthesis (A) Early steps of mmBCFA biosynthesis in bacteria, based on Smith and Kaneda (1980) , Oku and Kaneda (1988) , and Toal et al. (1995) . IVD, isovaleryl-CoA dehydrogenase. Predicted corresponding C. elegans genes encoding predicted orthologs were identified (shown in italicized names of reading frames). (B) GC profiles reveal differences in the FA composition in the wild-type animals and animals treated with RNAi of E1 alpha subunit of BCKAD encoded by Y39E4A.3. Black arrowheads point to C15ISO and C17ISO. (C) A summary of several independent preparations shows a significant decrease in both mmBCFAs in the Y39E4A.3 dsRNA-treated animals ( p = 0.001 and 0.008 for C15ISO and C17ISO, respectively). The ability of C. elegans to grow on the chemically defined axenic medium CbMM ( Lu and Goetsch 1993 ), which lacks the potential mmBCFA precursors, has suggested that the animals can synthesize mmBCFA de novo. If so, a disruption of the BCKAD complex could affect mmBCFA levels. We identified a predicted C. elegans protein, Y39E4A.3, with significant sequence homology to the E1 alpha subunit of BCKAD (Y39E4A.3 scores expect value 8e-50 on 57% of the length with the Bacillus subtilis BCKAD and 1.4e-134 on 88.4% of the length with the Homo sapiens BCKADs). RNAi of Y39E4A.3 led to a significant decrease in C15ISO and C17ISO production ( Figure 4 B and 4 C). RNAi suppression of another predicted component of the BCKAD complex, pyruvate dehydrogenase (T05H10.6), resulted in a similar decrease in C15ISO and C17ISO (unpublished data), indicating a role for the C. elegans BCKAD protein in long-chain mmBCFA biosynthesis. Thus, C. elegans appears to use the same initial reactions to produce mmBCFAs as bacterial cells. In addition, the worm uses enzymes of the FA elongation family, ELO-5 and ELO-6, to complete the pathway. A connection between BCKAD functions and mmBCFA quantities has been previously reported in humans ( Jones et al. 1996 ). Normally hair fibers are densely covered with C21anteISO, which contributes about 38.2% to the total hair FAs ( Jones and Rivett 1997 ). It was observed that patients with maple syrup urine disease, which is caused by an inherited mutation in the BCKAD gene, had a drastically reduced level of mmBCFAs in their hair. Together, these data suggest that long-chain mmBCFA biosynthesis could be similar in bacteria, C. elegans, and human. Blocking ELO-5 Function Causes Growth and Developmental Defects While the suppression of elo-6 activity by feeding double-stranded RNA (dsRNA) to wild-type animals did not cause obvious morphological or growth defects, the suppression of elo-5 resulted in more pronounced phenotypes ( Figure 5 ). Worms originating from wild-type eggs laid on the elo-5(RNAi) plates displayed no obvious growth or morphological abnormality until the second day of adulthood, when they developed an egg-laying defect ( Figure 5 B). Eggs of the next generation hatched on time but the progeny arrested at the first of the four larval stages (L1). The small larvae maintained morphological integrity and could survive on a plate for up to 3–4 d. The arrest was only observed in progeny of parents exposed to elo-5 RNAi at the L1 stage. Figure 5 RNAi Treatment of elo-5 Causes L1 Arrest and Other Physiological Defects (A–C) Nomarski images of worms grown from eggs placed on RNAi plates. Scale bars, 100 μm. (A) Young adults had normal morphology and growth rates. (B) On the second day of adulthood, these animals displayed an egg-laying defect; eggs hatched inside the worms. Arrows point to the late embryos and hatched larvae inside a worm. (C) F1 generation arrested uniformly at the first larval stage (L1), and larvae arrested for 4–5 d died. (D–F) Images of worms derived from late larvae (L2–L4) placed on the RNAi plates. (D) The F1 progeny of worms developed from the treated larvae had smaller size and a scrawny morphology compared to the wild type shown in (A). Scale bar, 100 μm. (E) These animals produced very few oocytes, some of which gave rise to embryos and L1 worms. White arrows indicate embryos. Some oocytes remained unfertilized (black arrow). Scale bar, 10 μm. (F) The proximal part of the gonads undergoes deterioration resulting in sterility. The white arrow indicates spermatica, the black arrow shows an abnormally amorphous oocyte, and the two-way arrow points to the clumsy gonad arm that is finely ordered in wild-type animals. Scale bar, 10 μm. When parental animals were subjected to elo-5 RNAi at later larval stages (L2–L4), their progeny did not arrest in L1 but continued to develop into adulthood. These animals had no obvious defects in locomotion, pharyngeal pumping, intestinal contractions, chemotaxis response, touch sensitivity, or general anatomy (unpublished data). However, the growing worms became progressively sick ( Figure 5 D– 5 F). The gonads appeared normal at the L4 and early adult stages, but after fertilization of one to ten oocytes, oogenesis became impaired. Gonad degeneration began with a pronounced vacuolization in the midsection of the gonad followed by the appearance of disorganized clumps of nuclei in the proximal part. An egg-laying defect became apparent and only a few progeny arose from these worms, which then arrested at L1. The development of the elo-5 RNAi phenotypes is likely due to a gradual elimination of the ELO-5–associated functions. Our data suggest that these functions are crucial for larval growth and development. We also obtained a likely null mutant of the elo-5 gene, elo-5(gk208), which has a 245-bp deletion eliminating the predicted first exon (Genome Science Center, BC Cancer Research Center, Vancouver, British Columbia, Canada). This allele phenocopies the L1 arrest phenotype of the elo-5(RNAi) animals. A Deficiency of C15ISO and C17ISO FAs Is Solely Responsible for the Defects Caused by elo-5(RNAi) We reasoned that if the defects observed in the elo-5(RNAi) animals resulted directly from the deficiency of C15ISO and C17ISO, then feeding these worms with C15ISO and C17ISO should mask a shortage of endogenous C15ISO and C17ISO and permit the animals to grow normally. As predicted, the C17ISO and C17anteISO supplements rescued the elo-5 RNAi defects (in 52 of 60 and 58 of 60 plates, respectively). A partial rescue was observed on the plates supplemented with C15ISO and C15anteISO (23 of 38 and 20 of 28 plates, respectively). Corroborating results were obtained when homozygous elo-5(gk208) animals supplied with C17ISO grew normally. In sharp contrast, neither saturated or mono- or polyunsaturated FA molecules (C16:0, C16:1 n7, C17:0, C18:3 n6), mmBCFAs with shorter or longer backbones (C13ISO, C18ISO, C19ISO), nor polymethyl branched phytanic acid were able to rescue or reduce defects (0 of 30 plates in each experiment). Therefore, we have determined that only dietary 17-carbon mmBCFAs are competent to bypass the biochemical defect caused by loss of ELO-5 function. GC analysis of FA composition in elo-5(RNAi) worms grown on supplemented plates revealed that only C17ISO and C17anteISO are significantly incorporated into lipids ( Figure 6 A– 6 C). Because the addition of C15ISO did not result in elongation to C17ISO ( Figure 6 A), we wanted to determine whether ELO-6 was capable of extending an FA backbone in the absence of ELO-5, or whether the supplied free FA molecules could enter a different metabolic pathway, for instance, a degradation pathway. To distinguish between these two possibilities, we added mmBCFA-producing bacteria on top of the elo-5(RNAi) feeding Escherichia coli strain (HT115), which lacks mmBCFAs. This mmBCFA-producing strain was identified by chance; we noticed that in the presence of a certain bacterial contaminant the animals could overcome the elo-5(RNAi) effects. Using a rapid bacterial identification method ( Lane et al. 1985 ), we determined the contaminant to be Stenotrophomonas maltophilia. GC analysis revealed that this bacterial strain produced a high quantity of C15ISO and C15anteISO but not 17-carbon mmBCFAs ( Figure 6 D). GC analysis of elo-5(RNAi) animals fed with S. maltophilia indicated that they not only accumulated bacterial C15ISO and C15anteISO but also efficiently elongated these FA species to C17ISO and C17anteISO, which are absent in S. maltophilia ( Figure 6 D and 6 E). This suggested that elongation from C15ISO to C17ISO mmBCFA was not impaired in the elo-5(RNAi) animals. Therefore, ELO-6 function remains intact in elo-5(RNAi). Apparently, C15ISO added to the plates could not be utilized by ELO-6 whereas C15ISO-CoA and/or C15anteISO-CoA originating from the bacterial food could, suggesting that free and esterified mmBCFAs were likely to enter alternative pathways. Figure 6 The FA Composition in Worms Maintained on elo-5 RNAi Plates Supplemented with FA or with S. maltophilia Enriched with C15ISO and C15anteISO FA Black arrowheads indicate positions of mmBCFAs. (A) Animals grown with C15ISO supplements were partially rescued to the wild-type phenotype; however, no accumulation of C15ISO or its elongation to C17ISO was detectable. (B and C) Animals grown with the (B) C17ISO or (C) C17anteISO supplements were fully rescued. Peaks corresponding to C17ISO and C17anteISO are prominent. (D) The FA composition in S. maltophilia. Arrowheads point to the major FAs, C15ISO and C15anteISO. (E) The elo-5(RNAi) animals are able to elongate dietary C15ISO and C15anteISO into C17ISO and C17anteISO. Arrowheads indicate mmBCFAs. The horizontal arrow illustrates the elongation from C15 to C17 mmBCFA. The essential roles of C15ISO and C17ISO were also supported through an examination of the elo-5(gk208) deletion mutant. The homozygous mutants grew without any obvious morphological defects when maintained on the plates supplemented with C17ISO or seeded with S. maltophilia. However, removal of the FA supplements or S. maltophilia by bleaching resulted in the same L1 arrest phenotype seen for the elo-5(RNAi) worms. L1 Arrest of the elo-5(RNAi) Animals Is Reversible and Related to the Variations in Levels of C17ISO during Development We then asked if elo-5(RNAi) animals arrested at the L1 stage could be recovered by adding the 17-carbon mmBCFA supplements. Indeed, C17ISO and C17anteISO could effectively release L1 larvae from the developmental arrest; about 50% of 2-d-arrested and 1% of 4-d-old L1 were rescued to full growth and proliferation. Since C17anteISO could not be detected in the laboratory animals under normal conditions of culturing, C17ISO appeared to be the principal molecule conveying the ELO-5 function. Therefore, the L1 arrest of the C17ISO-depleted worms is both completely penetrant and reversible, indicating that C17ISO plays a critical role in growth and development at the L1 stage. The analysis of the FA levels of staged worms revealed that the C17ISO level increases gradually from a relatively low level at L1 to its peak in gravid adults containing eggs ( Figure 7 A). Based on the analysis of green fluorescent protein (GFP) reporter constructs (unpublished data) and in situ hybridization data (results from NextDB by Y. Kohara, Tokyo, Japan), neither elo-5 nor elo-6 is significantly expressed in eggs or L1. Therefore, C17ISO likely accumulates in embryos during oogenesis. It may be directly transported from gut to gonads, since both ELO-5 and ELO-6 were expressed mainly in the gut and since feeding C17ISO rescued the elo-5 mutant phenotypes. When RNAi-mediated disruption of elo-5 occurs at the L1 stage of a parent and consequently blocks C17ISO synthesis from that stage on, the eggs and L1 animals of the next generation are expected to contain a critically low concentration of C17ISO, halting further development. Because the arrested L1 can be rescued by a dietary supply of the mmBCFA, the deficiency is not likely to cause critical defects during the embryonic and early postembryonic periods. Figure 7 A Fluctuation of the C17ISO Amounts in Development (A) Relative amounts of C15ISO and C17ISO in the worm samples collected at different developmental stages. The amount of the mmBCFA molecule is presented as the percentage of total FA in each sample. Grey bars, C15ISO; black bars, C17ISO. (B) Proposed relationship between the levels of mmBCFA during development and the RNAi effects. Depending on the time of RNAi onset, the amount of C17ISO in F1 eggs varies. If elo-5 is suppressed in parental animals after they have begun to synthesize mmBCFA, then their eggs will have a reduced C17ISO level that is still above the critical low level, which permits these animals to grow but causes them to display gonadal defects. These worms produce a small number of progeny that is then arrested in L1. If parental animals are treated with elo-5(RNAi) right after hatching, they are unable to initiate mmBCFA biosynthesis and the levels of C15ISO and C17ISO in their eggs are reduced to below the critical low level, resulting in L1 arrest of their progeny. If elo-5 RNAi is applied to the parent worms at or after the L2 larval stage, when the amount of C17ISO has already been elevated and/or the RNAi effect is less penetrant, the progeny may receive sufficient C17ISO to pass the L1 arrest stage. The resulting animals, however, become visibly unhealthy at the L4 and adult stages as mentioned earlier, suggesting that C17ISO also plays a role in late developmental stages. Based on these results, we propose a relationship between the amounts of C17ISO and developmental stages ( Figure 7 B). In this model, the level of C17ISO is monitored at the first larval stage and the decision is made whether to proceed or pause in development. The analysis of GC data from staged animals has also indicated that the variation of the C17ISO level is correlated with only two other FA species, suggesting a potential compensatory and coregulatory mechanism. The C17ISO Level Correlates with the Levels of Two Other FAs during Development FA homeostasis implies that relative amounts of various FA species are coordinated and balanced for optimal performance. To obtain information that may help us understand why and how numerous FAs and their specific metabolic enzymes are maintained in nature, we carried out analysis to determine a possible correlation between changes in the levels of C17ISO and other FAs detected in worms. We have analyzed a large amount of GC data ( n = 50) obtained from mixed populations of wild-type animals where the fractions of eggs, larvae, and adults randomly varied. We also included GC data separately obtained from staged worms: eggs, L1, L2, L3, L4, and gravid adults. We found that the amounts of C17ISO significantly correlated with only two other FA molecules: linoleic acid (C18:2 n6) and vaccenic acid (C18:1 n7) ( Figure 8 ). A potential physiological significance of these correlations is intriguing. Figure 8 Correlation between the Level of C17ISO and the Levels of Linoleic and Vaccenic Acids during Development Graphical illustrations of the correlation between the levels of C17ISO and (A) vaccenic acid and (B) linoleic acid. Data were obtained by GC analysis of synchronized populations of worms. Combined with the GC measurements generated from 50 additional samples (see Materials and Methods), these data were used to calculate correlation coefficients: CORREL C17ISO/C18:2 n6 = 0.82772, T-TEST = 6.54814 × 10 −7 , and CORREL C17ISO/C18:1 n7 = −0.85162, T-TEST = 4.74094 × 10 −5 . Black bars, C17ISO; white bars, vaccenic acid; grey bars, linoleic acid. The observed negative correlation between the levels of C17ISO and C18:1 n7 throughout development may indicate a compensatory adjustment important for physiological functions, such as retention of the cell membrane physical properties. mmBCFAs and monounsaturated straight-chain FAs have been previously implicated in regulating membrane fluidity, which depends on the ratio of saturated FA to monounsaturated and branched-chain FA content in bacterial cells ( Rilfors et al. 1978 ; Suutari and Laakso 1994 ; Cropp et al. 2000 ). An elevation in monounsaturated FA amounts in response to the decrease of branched-chain FAs, but not vice versa, was observed in Streptomyces avermitilis ( Cropp et al. 2000 ), suggesting that monounsaturated FAs may sense a state of membrane fluidity. In the elo-5(RNAi) -treated worms, a substantial loss of C15ISO and C17ISO is also accompanied by a change in the FA composition, most noticeably by the elevation in C18:1 n7 (see Figure 3 C), a result consistent with the above observation. To estimate the effect of the C15ISO and C17ISO deficiency on the membrane saturation, the saturation index (SI = [saturated FA]/[mmBCFA + monounsaturated FA]) was calculated. No significant differences were detected in elo-5(RNAi) worm compared to wild type (SI = 0.325 ± 0.011 [ n = 6] and SI = 0.320 ± 0.032 [ n = 5], respectively). Therefore, elo-5(RNAi) may not cause massive cell membrane dysfunction. A positive correlation between the amounts of C17ISO and C18:2 n6 may suggest a potential common function during development. In addition to the importance of linoleic acid as a substrate for polyunsaturated FA biosynthesis, its hydroxylated fatty acid derivative (HODEs) is known as a signaling molecule affecting chemotaxis ( Kang and Vanderhoek 1998 ), cell proliferation ( Eling and Glasgow 1994 ), and modulation of several enzymatic pathways ( Hsi et al. 2002 ). A correlation between C17ISO and linoleic acid may also suggest a similar regulation of biosynthesis of the two molecules. The changes in the FA composition associated with a decrease in C15ISO and C17ISO indicate that the metabolism of straight-chain FA species is responsive to the mmBCFA levels and suggest a cross regulation. Interestingly, in the elo-5(RNAi) animals fed with C15ISO or C15anteISO containing bacterial supplement (S. maltophilia), the FA composition was significantly altered (see Figure 6 E). It appears that mmBCFAs become principal components in a range of 16–18-carbon FAs. This suggests that large quantities of mmBCFAs are not toxic. In contrast, because these worms grow and proliferate well, mmBCFAs seem to be efficient substitutes for saturated and monounsaturated straight-chain FAs. The Worm SREBP Homolog Controls Production of Branched-Chain FAs In mammals, straight-chain FA biosynthesis depends on the 1c isoform of sterol regulatory element binding protein (SREBP-1c), which promotes the expression of FA metabolic enzymes ( Edwards et al. 2000 ; Horton 2002 ; Matsuzaka et al. 2002 ). There is only one protein in C. elegans that is homologous to mammalian SREBPs, Y47D3B.7 (the gene has been named lpd-1, for “lipid depleted 1”) ( McKay et al. 2003 ). McKay and coauthors have shown that worms treated with lpd-1 RNAi display a lipid-depleted phenotype. They have also shown that lpd-1 regulates the expression of several lipogenic enzymes, acetyl-CoA carboxilase (ACC), FAS, and glycerol 3-phosphate acyltransferase (G3PA) ( McKay et al. 2003 ). Thus, similar to its mammalian homolog, lpd-1 is involved in straight-chain FA biosynthesis. We wanted to see if lpd-1 also plays a role in mmBCFA metabolism. We first applied RNAi to lpd-1 and determined the FA composition of the mutant worms. As expected, the FA content of treated animals was significantly changed, but surprisingly the most reduced were the levels of C15ISO and C17ISO ( Figure 9 ). Also significantly reduced was the amount of C18:2 n6. In contrast, the C16:0 level was elevated. These data indicate that, in addition to regulating the first steps of global FA biosynthesis through the activation of the ACC and FAS transcription, the worm SREBP homolog regulates mmBCFA elongation as well as desaturation of straight-chain FAs. Figure 9 RNAi of the C. elegans SREBP Homolog Alters the FA Composition (A and B) The GC profiles of (A) wild-type and (B) lpd-1(RNAi) -treated worms. (C) A summary of several independent GC runs. Bars represent the percentages of total FAs. The levels of C15ISO, C17ISO, and C16:0 are significantly altered by the RNAi treatment. Black arrowheads point to differences in the C15ISO and C17ISO amounts. Grey arrowheads indicate the changes in palmitic acid, C16:0. As reported previously, disruption of lpd-1 through a mutation or RNAi injection caused early larval arrest ( McKay et al. 2003 ). The effect of lpd-1 RNAi feeding in our experiments was apparently less severe. The RNAi-treated animals displayed slow growth, morphological abnormalities, and egg-laying defects but no larval arrest. Supplementing C17ISO to the plates did not significantly rescue these defects. LPD-1 and LPD-2 Diverge in Functions LPD-2 (C48E7.3) is another C. elegans homolog of a mammalian lipogenic transcription factor, CCAAT/enhancer-binding protein (C/EBP). McKay and coauthors have shown that the lpd-2(RNAi) and lpd-1(RNAi) phenotypes are quite similar; affected worms are defective in growth, pale and scrawny in appearance, and lacking in fat content ( McKay et al. 2003 ). They have also shown that LPD-1 and LPD-2 control the expression of the same lipogenic enzymes: ACC, FAS, ATP-citrate lyase, and G3PA. We tested to see if LPD-1 and LPD-2 function similarly in the regulation of mmBCFA biosynthesis. In contrast to the result from lpd-1 (RNAi), the FA composition in lpd-2(RNAi) worms was not significantly different from that of wild-type animals even though these animals had a noticeably sick appearance (unpublished data). This result suggested that, in addition to having some common targets, LPD-1 and LPD-2 have distinct functions. LPD-1 is important for production of mmBCFAs as well as other very-long-chain FAs, whereas LPD-2 has no specificity for any particular type of FA. elo-5 and elo-6 Are Likely Targets of LPD-1 The changes in FA composition observed in lpd-1(RNAi) would be consistent with downregulation of elo-5, elo-6 (decrease in mmBCFA), elo-2 (increase in C16:0) ( Kniazeva et al. 2003 ), and Δ 9- and/or Δ 12 -desaturase genes (decrease in C18:2 n6). The genes encoding mammalian orthologs of the C. elegans elo-2 and Δ 9 -desaturase genes are known targets of SREBP-1c ( Edwards et al. 2000 ; Horton 2002 ; Horton et al. 2002 ). To examine if elo-5 and elo-6 are targets of lpd-1, we analyzed the expression of elo-5, elo-6, and lpd-1. Evaluation of the expression from an lpd-1Prom :: GFP fusion construct (a gift of J. Graff) in transgenic animals revealed that, in addition to the previously reported expression in intestinal cells ( McKay et al. 2003 ), the construct is strongly expressed in a subset of head neurons ( Figure 10 A— Figure 10 D). Using a lipophilic dye, DiI, which highlights chemosensory ciliated neurons, we identified these neurons as amphids ( Murphy et al. 2003 ). In the strains carrying elo-5Prom :: GFP and elo-6Prom :: GFP reporter constructs, GFP fluorescence was also detected in the gut and several head neurons, including amphid neurons ( Figure 10 E– 10 H and Figure 2 ). Figure 10 The Expressions of elo-5 and lpd-1 Reporter Constructs Are Spatially Similar (A and B) Nomarski and GFP-filtered images of an adult animal containing the lpd-1Prom :: GFP construct, showing strong expression in two symmetrical head neurons, each of which has processes to the nose and around a nerve ring. Scale bars, 10 μm. (C) DiI staining of amphid neurons in lpd-1Prom :: GFP (dsRed filter). Arrows indicate neuronal nuclei shown in (D). Scale bar, 10 μm. (D) GFP expression in the animal shown in (C). Scale bar, 10 μm. (E and F) Nomarski and GFP-filtered images of an animal containing elo-5Prom :: GFP, revealing fluorescence in the similar amphid neuron. Scale bar, 7.5 μm. (G and H) The intestinal and intestinal-muscle GFP expression in (G) lpd-1Prom :: GFP and (H) elo-5Prom :: GFP constructs. Scale bar, 7.5 μm. If LPD-1 promotes elo-5 and elo-6 expression, then RNAi of lpd-1 should alter GFP intensity in elo-5Prom :: GFP and elo-6Prom :: GFP reporter strains. The level of GFP expression driven by elo-5 and elo-6 promoters is high in conventionally cultured animals. In the worms maintained on the lpd-1(RNAi) plates, the expression was noticeably weakened, suggesting a downregulation of the promoter activities ( Figure 11 A– 11 D). No significant changes in GFP expression were detected in a control strain containing a kqt-1Prom :: GFP construct that also expresses GFP in head neurons and the gut (unpublished data). Figure 11 The Expression of GFP Fusion Constructs Suggests the Involvement of lpd-1, acs-1, and pnk-1 in mmBCFA Biosynthesis (A–D) elo-5Prom :: GFP expression is downregulated in the lpd-1(RNAi) background. Scale bars, 100 μm. (A and C) GFP-filtered images of (A) elo-5Prom :: GFP and (C) elo-6Prom :: GFP in wild-type animals, showing the characteristic bright intestinal fluorescence. (B and D) GFP-filtered images of (B) elo-5Prom :: GFP and (D) elo-5Prom :: GFP in lpd-1(RNAi) animals, revealing diminished fluorescence in the gut. (E–H) lpd-1 expression is upregulated in neurons of the elo-5(RNAi) animals deficient for C15ISO and C17ISO. Scale bars, 15 μm. (E and F) Nomarski and GFP images of wild-type L1 larvae containing lpd-1Prom :: GFP. (G and H) Nomarski and GFP images of elo-5(RNAi) -treated animals (L1 arrested) containing lpd-1Prom :: GFP, showing a visibly brighter fluorescence than that seen in (E) and (F). Circles are centered on the pharyngeal back bulb. (I–K) acs-1Prom :: GFP expression is upregulated in the elo-5(RNAi) animals deficient for C15ISO and C17ISO. Panels show GFP images of acs-1Prom :: GFP animals grown on the (I) control, (J) elo-5(RNAi), and (K) lpd-1(RNAi) plates. The fluorescence from acs-1Prom :: GFP in (J) is significantly stronger than that in (I). Scale bars, 100 μm. (L–N) pnk-1Prom :: GFP expression is upregulated by elo-5(RNAi) but downregulated by lpd-1(RNAi). Panels show GFP images of pnk-1Prom :: GFP animals grown on the (L) control, (M) elo-5(RNAi), and (N) lpd-1(RNAi) plates. The fluorescence of the fusion construct is stronger in (M) but weaker in (N) than that in the control (L). Scale bars, 100 μm. To test if the disruption of FAS, a target of LPD-1 ( McKay et al. 2003 ), could contribute to the observed decrease of C15ISO and C17ISO in lpd-1(RNAi), we analyzed FA composition in FAS(RNAi) strains. There is one predicted FAS gene, F32H2.5, and its shorter homolog, F32H2.6, in the C. elegans genome. The latter can only encode the N-terminal portion of the protein. These genes share extended nucleotide identity, and RNAi of one could thus possibly affect the other. Consistent with a critical role for FAS in the first steps of FA biosynthesis, the RNAi-mediated disruption of F32H2.5 and F32H2.6 resulted in multiple defects and a lethal growth arrest (unpublished data). The FA composition (the content and relative amounts of various FA species) of the affected animals remained, however, unchanged. This suggests that disruption of FAS does not selectively alter FA biosynthesis and that neither FAS protein is specific for mmBCFA. Therefore, downregulation of FAS by loss of lpd-1 cannot account for the severe deficiency of mmBCFA in lpd-1(RNAi). Thus, we have shown that disruption of lpd-1 affects C15ISO and C17ISO biosynthesis. The fact that lpd-1, elo-5, and elo-6 are expressed in the same cells concurrently and that the GFP reporter analysis indicated that elo-5 and elo-6 transcription is downregulated in the absence of lpd-1 suggests that elo-5 and elo-6 are likely to be the targets of lpd-1. Since ACC and FAS catalyze the first steps in the biosynthesis of straight-chain FAs while ELO-5 and ELO-6 extend mmBCFA molecules, LPD-1 appears to integrate conventional and “unusual” FA biosyntheses. It seems reasonable to predict that in order to differentiate between these metabolic pathways and mediate compensatory or adaptive changes in FA composition, LPD-1 must interact with other factors such as nuclear receptors activated by specific FA ligands. It is thus important to screen for such interactions to better understand FA homeostasis in C. elegans. A Reciprocal Correlation between lpd-1 Expression and mmBCFA Levels Because mammalian SREBP-1c regulates polyunsaturated FA biosynthesis and is feedback inhibited by polyunsaturated FAs ( Jump 2002 ), we asked if lpd-1 could be regulated by mmBCFAs at the transcriptional level. Our microarray data (discussed below) indicated a 1.68-fold upregulation of lpd-1 in the elo-5(RNAi) animals, while no changes were detected in its levels between samples from wild-type animals at different developmental stages (see Materials and Methods ). To examine the influence of the mmBCFA deficiency on lpd-1 expression, we grew the lpd-1Prom :: GFP -containing strain on the elo-5(RNAi) and control plates to compare GFP fluorescence. No obvious difference in the GFP expression driven by the lpd-1 promoter in intestinal cells was detected on the elo-5(RNAi) plates versus the control plates. A modest change in the transcription level (1.68-fold) could be masked by a variability of the expression between individual animals and even between individual cells (unpublished data). In contrast to the observation for the intestinal cells, a strong induction of GFP was detected in amphid neurons of lpd-1Prom :: GFP ; elo-5(RNAi) animals ( Figure 11 E– 11 H). This suggests that a chronic deficiency of mmBCFA in elo-5(RNAi) animals may transcriptionally stimulate LPD-1 production, at least in neuronal cells. Collectively, our results suggest that the relationship between lpd-1 and C15ISO/C17ISO is reciprocal; while downregulation of lpd-1 transcription results in the C17ISO deficiency, the C15ISO and C17ISO deficiency upregulates lpd-1 transcription at least in a subset of cells. Therefore, the worm SREBP homolog, LPD-1, may play an important role in mmBCFA homeostasis. Screening for Additional Genes Involved in mmBCFA Homeostasis Because C15ISO and C17ISO play critical roles in animal development and growth, we suspected mechanisms might exist to respond to and regulate their levels. Regulation of mmBCFA homeostasis may involve transcription factors, metabolic enzymes, and transport and binding proteins. It is reasonable to suggest that a deficiency of mmBCFA triggers a compensatory alteration in the expression of these genes. It is also possible that a comparative analysis of global gene expressions between wild-type and mmBCFA-deficient animals may reveal these potential changes and the changes underlying developmental and growth functions of mmBCFA. We used DNA microarray analysis to compare the total gene expression in elo-5(RNAi) and wild-type animals. To select candidate genes, we applied restrictive criteria and excluded genes of which the expression was also changed in the spt-1(RNAi) strain ( Protocol S1 and Table S1 ). The spt-1 (C23H3.4) gene encodes a predicted C. elegans homolog of serine-palmitoyl transferase subunit 1. RNAi of spt-1 strongly affects the FA composition without reducing the C15ISO or C17ISO levels (unpublished data). The F1 generation of spt-1(RNAi) animals developed gonadal and egg-laying defects that were similar to the phenotype of F1 animals from parents treated with elo-5(RNAi) at a late larval stage (described earlier; see Figure 5 B and 5 F). We thought that by deselecting genes that have altered expressions in spt-1(RNAi), we would be able to eliminate variations in gene expressions unrelated to the mmBCFA deficiency. Such variations might emerge from altered straight-chain FA metabolism and from general sickness. Here, we discuss the analysis of the first set of candidate genes that are differentially expressed in elo-5(RNAi) and may relate to the C15ISO and C17ISO homeostasis. Twenty-five genes were selected in the screen ( Table 1 ) and each was functionally tested by RNAi and GC analysis for its role in C15ISO and C17ISO metabolism. RNAi of four of these genes ( pnk-1 [C10G11.5], nhr-49 [K10C3.6], acs-1 [F46E10.1], and C27H6.2) significantly affected the FA composition ( Figure 12 ). All four genes encoded products structurally homologous to the known proteins (PNK-1, human pantothenate kinase; NHR-49 , nuclear hormone receptor; ACS-1, very-long-chain FA CoA ligase; and C27H6.2, RuvB-like DNA binding protein). Figure 12 RNAi of Four Candidate Genes with Altered Expression in elo-5(RNAi) Worms Affects the FA Composition (A) GC profile of the wild type. (B–E) GC profiles of the RNAi-treated worms. (B–D) RNAi of the three genes resulted in a decrease of the C17ISO or both C15ISO and C17ISO levels, indicated by black arrowheads. In addition, a significant elevation in straight-chain saturated FA levels, indicated by grey arrowheads, is observed in K10C3.6(RNAi). (E) C27H6.2(RNAi) does not cause significant changes in mmBCFA but results in an elevation of straight-chain monounsaturated FA and C18:1 n7, indicated by white arrowheads. Statistical analysis of several GC runs on each of the samples was also carried out (unpublished data). Table 1 Candidate Genes and Their Encoded Proteins Selected from Microarray Data for Functional Tests (RNAi and GC Analysis) a Open reading frames (ORFs) predicted by the C. elegans Genome Project (WormBase.org) b Data from comparing arrays from the experimental sample with that from a baseline control sample (Sample I; see Protocol S1) Analysis of the Candidate Genes Circumstantial evidence suggests that these four candidate genes may be involved in feedback regulation of mmBCFA biosynthesis. First, the expression of these genes is not variable in nature, as judged by a comparison of the microarray data obtained from developmentally different populations of N2 ( Protocol S1 ) as well as for vulval development pathway mutants (data obtained for an unrelated project, J. Chen, personal communication). Second, the direction of the changes for three of the genes is in concordance with the proposed feedback regulation: pnk-1, nhr-49, and acs-1 were upregulated in C17ISO-deficient elo-5(RNAi) . Lastly, a functional analysis shows that these three candidate genes are required for the normal level of mmBCFA production (RNAi of the genes affects mmBCFA production). The fourth candidate gene, C27H6.2, affects the level of vaccenic acid (C18:1 n7), which is related to the levels of mmBCFA (see Figure 8 ), suggesting cross-talk. To detect potential feedback regulation involving acs-1 and pnk-1, we made reporter strains with GFP expression driven by acs-1 and pnk-1 promoters, acs-1Prom :: GFP and pnk-1Prom :: GFP, respectively. These two genes showed a higher degree of upregulation than the other candidates according to the microarray data. In addition, RNAi of these two genes resulted in a significant loss in the mmBCFA fraction. The GFP fluorescence from acs-1Prom :: GFP and pnk-1Prom :: GFP was readily detectable in the gut. Expression of acs-1Prom :: GFP was also detected in the canal-associated neurons in the head neurons and vulval cells. A comparison of synchronized animals grown on the control and elo-5(RNAi) plates indicated a significantly brighter fluorescence in the RNAi worms (see Figure 11 I, 11 J, 11 L, and 11 M), suggesting upregulation of acs-1 and pnk-1 under C15ISO or C17ISO deficiency. These results were in concordance with the microarray data. Moreover, pnk-1, but not acs-1, seemed to be regulated by LPD-1 because pnk-1Prom :: GFP expression was significantly reduced on lpd-1(RNAi) (see Figure 11 L and 11 N). It was interesting to note that the pnk-1 and acs-1 genes were previously selected in two different screens as potential targets of the daf-2/daf-16 (Y55D5A.5 and R13H8.1, respectively) pathway. pnk-1 had been identified in a screen for genes affecting the life span and metabolism of C. elegans through analysis of promoter regions, and it was confirmed as a direct target of DAF-16, a forkhead transcriptional factor ( Lee et al. 2003 ). acs-1 had been identified in a microarray screen for DAF-16 targets that influence life span ( Murphy et al. 2003 ). A third gene, nhr-49, had been previously selected in a screen for fat regulatory genes ( Ashrafi et al. 2003 ). It was shown that RNAi of this gene leads to an increase in fat accumulation in affected animals. Our analysis of nhr-49(RNAi) animals showed that reduction of nhr-49 activity results in upregulation of saturated FA biosynthesis that may contribute to fat accumulation. Although the regulatory path for this process remains unknown, the involvement of daf-2 has not been ruled out. A potential link between the candidate genes and DAF-2/insulin signaling is very intriguing. The C. elegans insulin-signaling pathway is involved in sensing nutritional state and metabolic conditions as well as controlling growth and diapause ( Kimura et al. 1997 ; Ailion et al. 1999 ). As we report in this paper, a mmBCFA deficiency causes transient L1 arrest. This phenotype strikingly resembles L1 arrest of worms hatched in the absence of food (a method commonly used to obtain synchronized animals). An investigation of possible roles for mmBCFA in food sensation and insulin signaling pathways is underway. Downregulation of the fourth candidate gene, C27H6.2, may result in a significant increase in monounsaturated FA levels ( Figure 12 ). This is consistent with the enlarged fraction of monounsaturated FAs observed in the elo-5(RNAi) animals (see Figure 3 C). Downregulation of C27H6.2 may have an adaptive effect to compensate for the loss of mmBCFAs in cell membranes. If so, C27H6.2 may be part of a mechanism that senses and tunes physical properties of membranes. C27H6.2 is homologous to RuvB/TIP49a/Pontin52, an evolutionarily conserved protein essential for growth and proliferation ( Kanemaki et al. 1997 ; Bauer et al. 1998 ; Qiu et al. 1998 ). Its mammalian ortholog acts as a transcriptional cofactor that binds to β-catenin, TATA-box binding protein, and likely to a number of other diverse transcription factors ( Bauer et al. 1998 ). Concluding Remarks Two mmBCFAs are normally detected in C. elegans: C15ISO and C17ISO. A deficiency of these FAs is lethal and cannot be compensated by any other FA present, indicating their crucial importance for growth and development. There are two sources of C15ISO and C17ISO available for worms. First, they possess a system for mmBCFA biosynthesis that includes two FA elongation enzymes, ELO-5 and ELO-6, which are regulated at least in part by the nematode homolog of SREBP-1c (lpd-1). Second, worms may obtain mmBCFAs from their diet (bacteria). Therefore, C. elegans is able to produce, activate, transport, and utilize mmBCFAs and is vitally dependent on this system. The level of C15ISO and C17ISO in eggs appears to be critical for growth and development, as animals depleted of C15ISO or C17ISO completely arrest at the L1 stage. The uniformity and reversibility of the arrest would be consistent with a regulatory role in growth and development for these mmBCFAs or for more complex lipid molecules containing them. However, it cannot be ruled out that the arrest is due to the failure of a metabolic or structural function that is essential for growth and development at the first larval stage. In addition, C15ISO and C17ISO may directly or indirectly regulate genes involved in FA homeostasis. Consistent with this, their deficiency triggers a large alteration in gene expression that may reflect a complex feedback mechanism. Among the potentially responsive genes are transcription factors and metabolic genes. Ubiquitous and unattended mmBCFAs come forth as physiologically important molecules that regulate essential functions in eukaryotes. Many interesting questions regarding mmBCFAs remain to be addressed. What are the other components of the mmBCFA biosynthetic machinery? What are the components of their transport system? Does an organism have a mechanism by which the mmBCFA level is measured? What are the signaling pathways involved in the mmBCFA responses? How do mmBCFAs exert their physiological function? Do mmBCFAs act alone or as parts of more complex lipids? How are mmBCFAs synthesized in mammals? Lastly, what are the specific physiological functions of mmBCFAs in mammals? Both genetic and biochemical approaches will be taken to address these questions. Materials and Methods RNA interference by feeding The RNAi feeding vectors were either made in our laboratory using Taq PCR and cloning genomic fragments into a double T7 vector, pPD129.36 (gift of A. Fire), or obtained from the C. elegans whole genome RNAi feeding library (J. Ahringer, MRC Geneservice, Cambridge, United Kingdom). The RNAi feeding strain was E. coli HT115 transformed with either empty pPD129.36 vector (controls) or with dsRNA-producing constructs. Plates were prepared as described in Kamath et al. (2001) . Unless stated differently, wild-type N2 Bristol animals were plated as synchronized adults. To obtain synchronized worms of various stages, a large quantity of N2 gravid adults were collected, bleached, and grown to the required stage on HT115 that had been transformed with pPD129.36 (control). GC analysis A mixed population of well-fed worms were washed off the plates with water, rinsed 3–4 times, and, after aspirating away water, frozen at −80 °C. FA methyl esters and lipid extraction were performed as described in Miquel and Browse (1992) . GC was performed on the HP6890N (Agilent, Palo Alto, California, United States) equipped with a DB-23 column (30 m × 250 μm × 0.25 μm) ( Kniazeva et al. 2003 ). Each experiment was repeated at least five times. Average values and standard deviations were then calculated for each of the compounds in the experiments. Staging worms to test for FA composition After bleaching gravid adults, an aliquot of the eggs was set apart, and the rest was incubated overnight in M9 at room temperature. On the next day, an aliquot of L1 was frozen for GC analysis. The rest of L1 was plated on agar plates. Subsequently, L2, L3, L4, young adults, and adults along with hatched L1 were collected as the separate samples. Mixed populations of worms starved for 24–100 h were included in the experiment to monitor a possible effect of the starvation. Phenotype rescue using FA supplements. Ninety microliters of the 4 mM solution of FA (Sigma, St. Louis, Missouri, United States) in 1% NP40 was dropped on the side of the bacterial lawn that contained either elo-5 dsRNA-producing plasmid or the control HT115 vector. Two synchronized young adults were plated and their progeny was scored 4 and 5 d later. Each experiment was performed in at least 30 replicates. For recovering elo-5(RNAi) worms from L1 arrest, wild-type adults were placed on the elo-5(RNAi) plates. Four days later, their progeny was removed and eggs of the next generation were left on the plates. Hatched L1 were kept for 2 or 4 d before transferring as agar chunks to new elo-5(RNAi) plates. FA supplements were added to spots next to the chunks. Ten plates were prepared for each FA supplement. Control plates contained no supplements. To verify that an addition of supplements did not affect RNA interference per se, we used let-418(RNAi) animals, which have a sterile phenotype, as a control. Neither C15 nor C17 mmBCFA added to let-418(RNAi) plates modified the expected phenotype. Designing of GFP reporter constructs. To prepare the GFP fusion constructs, genomic fragments were PCR amplified and cloned in frame into one of the GFP fusion vectors (gift of A. Fire). The locations of the genomic fragments and PCR primers used are listed below: (1) elo-5Prom :: GFP, starting at 3.894 kb genomic upstream of the first codon and ending 4 bp into the first exon; primers, F-BamHI-TTTAGGTCATTTTTTGAGTCGCCA and R-BamHI-TAGTCTGGAATTTTGAAATTGAACGG; vector, pPD95.69; (2) elo-6Prom :: GFP, a 4.764-kb fragment covering 3,104 bp upstream and 1,660 bp downstream of the predicted start codon and ending 14 bp into the third exon; primers, F-Sph1-GCCCTTGGAAACCATCTACGACGAATC and R-Sma1-TCCGAACAGAACGACATAAGAGATTTCC; vector, pPD95.77; (3) acs-1Prom :: GFP, a 3.142-kb genomic fragment containing 3,048 kb upstream of the first predicted ATG and ending 24 bp into the second predicted exon; primers, F-SphI-CATAATTACTATTGCGTCACATG and R-SphI-CTCTTCCAAACTGGCGATGTCGA; vector, pPD95.69; (4) pnk-1Prom :: GFP, a 1.14-kb fragment that includes 937 bp upstream of the first predicted codon of the C10G11.5 and 203 bp downstream, ending 24 bp into the second exon; primers, F-SphI-TCGTACGATCGGACCATAGGCTAA and R-SphI-CTGATCCTCTGTAGCAGCGGCCCT; vector, pPD95.69. These constructs were injected into C. elegans at 10–50 ng/μl to form extrachromosomal arrays. In the case of acs-1, the extrachromosomal array had been integrated into the C. elegans genome. Staining chemosensory ciliated neuron with DiI Worms were soaked in a 5-μg/ml solution of DiI (Molecular Probes, Eugene, Oregon, United States) in M9 buffer for 1 h. They were then rinsed three times with M9 and visualized by fluorescence using the Texas Red filter. Correlation analysis The FA quantities obtained by GC were expressed as a percentage of the total. t test (two-tailed distribution) and correlation analysis were performed using the Microsoft Excel program. Visualization and scoring of the GFP expression in promoter::GFP lines Synchronized adults were placed on control (HT115 bacterial strain transformed with empty vector, pPD129.36) and RNAi (HT115 bacterial strain transformed with dsRNA construct) plates. Several worms of the next generation were picked from the control and RNAi plates and mounted on the same microscopic slide. GFP images were obtained with the fixed settings and exposure. Microarray analysis One young adult of the N2 Bristol strain was plated on each control and RNAi feeding plate. Control plates contained the E. coli HT115 strain transformed with empty pPD129.36 vector. Experimental RNAi plates contained E. coli HT115 transformed with corresponding dsRNA constructs. The growth conditions, RNA preparations, and data analyses are described in detail in Protocol S1 . Expression data are presented in Dataset S1 . Supporting Information Dataset S1 Microarray Expression Data (1.8 MB TXT). Click here for additional data file. Figure S1 Expression of Collagen Genes as an Indicator of Developmental Differences in Mixed Populations of Worms Samples I, II, and III represent mixed populations of wild-type animals started simultaneously from one young adult. Each was harvested at three time points, when mostly adults represented the F1 generation and the embryos and larvae in different proportions represented the F2 generation (see Protocol S1 ). Sample III corresponds to the most diverse mixture of worms. Numbers of collagen genes that were differentially expressed between pairs of samples are shown above or bellow the arrow brackets. Sample I and Sample III, which originated from the most distal mixed populations, have the largest number of differentially expressed collagens. Sample I and an experimental sample corresponding to the elo-5(RNAi) phenotype have a lower number of the changed collagen genes, suggesting that populations on these experimental and control plates are similar. (24 KB PPT). Click here for additional data file. Protocol S1 Microarray Data Analysis (37 KB DOC). Click here for additional data file. Table S1 Filtering Candidate Genes by Comparing Different Mutant and Wild-Type Samples (28 KB DOC). Click here for additional data file.

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546041

Rice Genome Approaches Completion

In April 2002, Science published draft genome sequences for the two major subspecies of cultivated rice, Oryza sativa . The release of the rice genome—the first plant crop to be sequenced—was big news. Rice is a staple crop for more than half of the world's population, and it was hoped that the availability of the genome sequence might enable scientists to develop more productive rice strains or strains that are more environmentally friendly. Furthermore, the rice genome might provide the key to understanding the genetics of other major cereal crops, all of which have much larger genomes. But the sequences published in 2002 were only draft genomes, containing many gaps and errors—works-in-progress rather than finished products. Now, a large group of scientists led by the Beijing Institute of Genomics is publishing a much improved, near-complete genome analysis of the indica and japonica subspecies of O. sativa , which are eaten in India and China, and Japan, respectively. Their analysis team, led by Gane Ka-Shu Wong, provides important insights into the evolution of rice. First of all, the team improved their original whole-genome shotgun sequencing of indica by generating significantly more sequence data, and then they used better methods to assemble these data. In whole-genome shotgun sequencing, the entire genome is chopped into random fragments, each fragment is sequenced, and then powerful computer programs search for overlaps and put all these data in order. It's like putting a fiendishly difficult jigsaw puzzle together by looking for patches of matching color. The key to the improvement in the genome sequence analysis is that the researchers have used the combined DNA sequence data from the two subspecies to facilitate the sequence assembly. The result is a nearly 1,000-fold increase in contiguity for the two genome sequences. In other words, while the original draft was very fragmented, in the new version, 97.7% of the genes can be found, in either the indica or the japonica dataset, on one piece of DNA whose position along the chromosomes is well defined. The researchers have also used their improved genome sequence to investigate the evolutionary history of rice. Central to evolution is the development of new functions through mutation of existing genes. But when mutations occur in functional genes, the result is rarely beneficial, so it is thought that evolution is more likely to proceed first by duplicating existing genes and then experimenting on the “backup” copy of the gene. Wong and colleagues report that there is evidence in the rice DNA sequences for a whole-genome duplication event just before the grasses diverged from other flowering plants, about 55–70 million years ago. This genome duplication may have played a role in the origin of the grasses, which then spread rapidly across the world to provide important sources of food that, among other things, possibly influenced human evolution. A bowl of indica (white, long grains) and japonica (brown, short grains) rice Analysis of the rice genomes also indicates that a small chromosomal segment was duplicated about 21 million years ago and that there is massive ongoing duplication of individual genes. These individual gene duplications provide a continuous source of raw material for gene genesis and very likely contribute to the differences between members of the grass family. Now the challenge is to use the rice sequences as a basis for detailed genetic analyses of additional cereal crops and for the development of improved strains of not only rice, but wheat, maize, and other important food crops.

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529257

Adjuvant interferon gamma in patients with drug – resistant pulmonary tuberculosis: a pilot study

Background Tuberculosis (TB) is increasing in the world and drug-resistant (DR) disease beckons new treatments. Methods To evaluate the action of interferon (IFN) gamma as immunoadjuvant to chemotherapy on pulmonary DR-TB patients, a pilot, open label clinical trial was carried out in the Cuban reference ward for the management of this disease. The eight subjects existing in the country at the moment received, as in-patients, 1 × 10 6 IU of recombinant human IFN gamma intramuscularly, daily for one month and then three times per week up to 6 months as adjuvant to the indicated chemotherapy, according to their antibiograms and WHO guidelines. Sputum samples collection for direct smear observation and culture as well as routine clinical and thorax radiography assessments were done monthly. Results Sputum smears and cultures became negative for acid-fast-bacilli before three months of treatment in all patients. Lesion size was reduced at the end of 6 months treatment; the lesions disappeared in one case. Clinical improvement was also evident; body mass index increased in general. Interferon gamma was well tolerated. Few adverse events were registered, mostly mild; fever and arthralgias prevailed. Conclusions These data suggest that IFN gamma is useful and well tolerated as adjunctive therapy in patients with DR-TB. Further controlled clinical trials are encouraged.

Background Tuberculosis (TB) is not yet a defeated affection. Although controllable at a community level and curable in individuals, its eradication seems distant. At present, at least one third of the World's population, more than 1 500 million individuals are infected with the Mycobacterium tuberculosis. Every year, around 8 – 10 million new cases occur [ 1 ]. Two million people die annually due to non-AIDS related TB, which is the highest number of deaths attributable to a single infectious agent [ 2 ], and corresponds to the 7 th cause of death worldwide [ 3 ]. TB represents 26 % of avoidable deaths in developing countries [ 4 ]. The emergency of drug resistant (DR) or multidrug-resistant (MDR) strains has increased this global problem, leading to a high morbidity and mortality [ 5 ]. MDR-TB affected patients' mean survival ranges from 2 to 14 months [ 6 ]. According to the World Health Organization's (WHO) data, MDR patients global proportion is 2.2 % [ 7 ]. The infection is mainly transmitted by inhalation of the bacilli coming from respiratory airways infected secretions. Once inhaled, the bacilli are subjected to phagocytosis within the alveolar macrophages, where they can be destroyed. Nevertheless, Mycobacteria have developed mechanisms to adapt to the noxious intracellular environment. Thus, it can persist, replicate and disseminate, leading to new infectious foci. Resistance emergence depends on several factors such as initial bacillary load, inadequate or incomplete chemotherapy administration, and the patient's immune condition. Chemotherapy is successful in most cases, given that the treatment schedule is thoroughly followed. It is prolonged, costly, and needs to be directly observed. Otherwise it is inadequate to kill all the bacilli and drug resistance emerges. Toxicities are frequent as well [ 8 ]. The immunologic approach to TB treatment can be promising since only 10 – 20% of infected people develop the disease and many of them have spontaneous remission [ 9 ]. Interferon (IFN) gamma plays a main role in the immunity to TB. It is a glycoprotein, secreted by CD4+, CD8+ and NK cells. Nevertheless, CD4+ Th1 lymphocytes are the main producers in response to a stimulus [ 10 ]. Enough evidences exist related to IFN gamma action on the macrophages immunoregulatory activity [ 11 - 14 ]]. Lack of production of this cytokine [ 15 ] or expression of its receptor [ 16 , 17 ] is associated to the infection's most lethal forms. Interferon gamma has also a potent antifibrotic effect [ 18 - 20 ]. Therefore a pilot clinical study was done with the aim to evaluate IFN gamma effect on drug resistant pulmonary TB patients regarding their clinical, bacteriological and radiological evolutions. The results show that the use of this protein in eight DR-TB patients (four of them MDR) as adjuvant to antibiotics had short and middle-term beneficial effects. Methods An open-label, non-randomized, non-controlled, pilot trial was carried out at the "Benéfico -Jurídico" Hospital, Havana, which is the national reference unit for TB and other respiratory diseases. According to the national TB program, all patients with unfavorable response to treatment are remitted to this center. The study population was constituted by eight Cuban patients, both sexes, more than eighteen years old, with diagnosis of TB without a favorable response to the usual therapy, who gave their written, informed consent to participate. The diagnosis comprised clinical findings such as cough and expectoration, pulmonary lesions at thorax radiography, and positive DR-TB sputum-smear and culture. To confirm drug resistance the Canetti's multiple proportions method [ 21 ] was used as antibiogram. Exclusion criteria were another chronic disease, pregnancy or nursing, severe psychiatric dysfunction, multiple sclerosis or another autoimmune disorder, other pulmonary infections, HIV co-infection, and treatment with glucocorticoids or any other immunosuppressor medication. The previous drug therapy received by the patients included 4 drugs (isoniazid, rifampin, streptomycin, and pyrazinamide) daily during 2 months, then isoniazid and rifampin twice per week for 2 additional months. Since their sputum tests had not become negative at this point they were returned to the 4 drugs regime plus ethambutol daily for 3 months, and finally isoniazid, rifampin, and ethambutol three times per week for 5 months. The trial was done in compliance with the Helsinki Declaration. The protocol was approved by the hospital's Ethics Committee and by the Cuban Regulatory Authority. Data from nineteen historical control cases were obtained from the hospital's archives. Patients received 1 000 000 IU of human recombinant IFN gamma (Heberon Gamma R ® , Heber Biotec, Havana), intramuscularly, daily during 4 weeks and then 3 times per week for the next 20 weeks. Participants stayed as in-patients during the study period. They received anti-TB drugs (WHO schemes) [ 22 ], according to the resistance detected in each case by the antibiogram (Table 1 ). Drugs were given as follows: rifampin 10 mg/Kg (maximum 600 mg), ethambutol 20 mg/Kg (max. 1500 mg), ethionamide 10 mg/Kg (max. 750 mg), pyrazinamide 15–30 mg/Kg (max. 2000 mg), ciprofloxacin 15–20 mg/Kg (max. 1500 mg), amikacin 15 mg/Kg (max. 1000 mg), and kanamycin 15 mg/Kg (max. 1000 mg) daily. After the end of the 6-months IFN gamma treatment period, chemotherapy continued up to 9 months if the scheme included rifampin and 18 months otherwise. Evaluations were carried out at entry and monthly during IFN gamma treatment. A complete physical examination was done. Sputum samples were taken for acid-fast-bacilli smear and culture, as well as blood samples for hematological counts, globular sedimentation rate, alanine aminotransferase, and creatinin determinations. Thorax radiographies were also recorded. Afterwards, patients were followed up with half-yearly evaluations during one year. Treatment efficacy evaluation included clinical, bacteriological and radiological outcomes. Complete response was defined as total disappearance of all signs and symptoms, negative sputum acid-fast-bacilli smear and culture, and pulmonary lesions improvement at X-ray. Partial response included signs and symptoms decrease, negative sputum smear and culture and stable X-ray lesions. No response consisted in signs and symptoms persistence, positive bacteriological examinations, and lesions stabilization or progression. Safety and tolerability of the IFN gamma treatment were monitored by means of a rigorous control of the adverse events that could be presented. Results Eight patients were enrolled in the study. Those were all the pulmonary DR-TB cases in the country during the inclusion period, from December 1999 to February 2002. They had not responded to the usual Directly Observed Treatment Short-course (DOTS) chemotherapy regime. Strain resistance was acquired in all cases. This was well determined since in Cuba all detected cases are screened for resistance on their first isolate. The patients did not have any extra-pulmonary manifestation of the disease. Their demographic and baseline characteristics are shown in Table 1 . Five of them were men, six of them non-white. The age ranged between 23 and 54 years old, and body mass index (BMI) between 13.2 and 22.0 Kg/m 2 . Their main symptoms were cough, expectorations, dyspnea, stertors, distal cyanosis, and finger clubbing. Bacteriological tests codification was mostly high and all patients showed active lesions at thorax radiography. Most of them had accelerated globular sedimentation rates (GSR). Anemia or other hematological alterations were not recorded. A rapid favorable evolution was obtained after treatment with IFN gamma (Table 2 ). Clinical improvement was evident since the first month of treatment, when all signs and symptoms (except for finger clubbing) had disappeared in all patients and BMI increased in all but one of them. Sputum acid-fast-bacilli smears and cultures were negative since the 1 – 3 months of treatment. The eight patients had radiological improvement, with lesions size reduction (total disappearance in one case) (Figure 1 ). GSR decreased in five out of 6 patients who had abnormal values at inclusion. After six additional months follow-up, patients # 3 and 4 normalized it to 10 and 15 mm/h, respectively. At the end of the IFN gamma treatment all the patients were evaluated as complete responders. The treatment with IFN gamma was safe and well tolerated. Four patients presented at least one adverse event. These events were arthralgias, fever, headache and asthenia. All adverse events were mild, except for one moderate fever, which was efficiently controlled with acetaminophen. Significant differences were not detected in other clinical laboratory tests. After completion or the IFN gamma 6-months treatment the patients continued with the corresponding chemotherapy schedule. Seven of the eight patients remained bacteriologically, clinically and radiologically negative at least twelve months after the treatment with IFN gamma concluded. However, patient number five relapsed six months after the end of IFN therapy. He developed additional resistance to rifampin and ethionamide and a chronic obstructive respiratory disease that contributed negatively to his evolution. Table 3 shows the results obtained at the same hospital with the 19 DR-TB cases during the five years prior to the present study. These patients had also failed to the standard DOTS regime. They were all resistant to isoniazid and rifampin, 13 were resistant to streptomycin, 7 to kanamycin, 4 to ethambutol, and one to pyrazinamide. Resistance was primary in 2 cases and acquired in the rest. Their average age was 58 years. Management was essentially the same as for the patients included in the study, except for IFN gamma treatment. None of these DR-TB cases reached culture conversion at three months of treatment with chemotherapy and less than half had converted at six months. Their clinical outcome was also worse. Discussion In spite of the reduced size of the population studied, the results suggest the efficacy of IFN gamma on DR-TB, when used as adjuvant to chemotherapy. All eight patients were considered as complete responders at the end of IFN treatment with disappearance of the disease signs and symptoms, sputum tests conversion, and pulmonary lesions improvement. Bacteriological and radiological improvement correlated with the clinical evolution; BMI increased and manifestations as cough and expectoration did not recur after IFN gamma treatment. Clinical practice demonstrates that these results are very difficult to obtain in such a short period of time with the chemotherapy alone. Any conclusion from this study is also limited by the fact that it was not a controlled trial. This was not possible due to the low incidence of TB (7.6/100,000 inhabitants in 2002) [ 23 ] and DR-TB in Cuba [ 7 ]. Therefore a historical control with the results obtained at the same hospital by the same investigators in patients treated only with chemotherapy is used for comparison. Despite the fact that this kind of control is not fully comparable since they were not in the original design of the trial, a clear difference is shown regarding patients' performance. Literature reports on chemotherapy-treated DR-TB patients show similar unfavorable outcome [ 6 ]. The fact that four patients received rifampin, according to the strain sensitivity, which is one of the election drugs for the treatment of TB with excellent results in patients treated with DOTS regime, does not diminish the consideration regarding the possible benefit exerted by IFN gamma since these same patients had already not responded to this antibiotic as part of the DOTS. Moreover, one patient recurred after he had finished IFN gamma treatment for 6 months, despite still being under chemotherapy regime. This suggests that in his case a new IFN gamma cycle combined to the antibiotics would have been necessary, but this was not previewed in the protocol. However, only a controlled trial can definitely clarify the role of IFN gamma and second line antibiotics in this kind of patient's improvement. The radiological results demonstrated IFN gamma antifibrotic properties as well. All patients had a reduction in the pulmonary lesions size, while one showed a complete resolution. This effect cannot be attributable to the antibiotics, since it is well known that DR-TB patients only develop radiological improvement long time after sputum smears and culture become negative. In many cases extensive fibrotic lesions never improve, and stay stable for life. This antifibrotic action agrees with that obtained with IFN gamma in idiopathic lung fibrosis [ 24 ] and suggests that IFN gamma can have future indications in other pulmonary diseases where fibrosis is present. Therapy was well tolerated. It was not necessary to suspend the combined treatment due to adverse events; mostly mild. Adverse events such as arthralgias, fever and headache coincide with those reported for interferons [ 25 ]. Immunity to TB depends on the development of CD4+ cells- and macrophages-mediated Th1 response. The role of IFN gamma as the main macrophage-activator Th1 cytokine has been clearly established in animal models infected with M. tuberculosis [ 15 , 26 , 27 ]. IFN gamma action on the macrophages leads to kill intracellular Mycobacteria . It stimulates macrophages to produce tumor necrosis factor alpha (TNF α), oxygen free radicals and nitric oxide, increase surface display of MHC antigens and Fc receptors, decrease lysosomal pH, and increase the intracellular concentration of some antibiotics [ 11 - 14 , 28 ]. Regarding its antifibrotic effect, IFN gamma inhibits lung fibroblast proliferation and chemotaxis in a dose dependent manner, and reduces collagen synthesis [ 18 , 19 , 29 ]. Furthermore, this protein is a potent inhibitor of the transforming growth factor β (TGF-β)[ 20 ], involved in the pathogenesis of many fibrotic lung diseases [ 30 - 32 ]. On the other hand, mutations in the IFN gamma gene [ 15 ], or in the IFN gamma receptor alpha chain gene [ 16 , 17 ], increase susceptibility to develop the disease. Patients with disseminated BCG and other infections present defects in IFN gamma and other cytokines secretion and action [ 33 - 35 ]. IFN gamma therapy has been shown effective for cerebral tuberculosis caused by a multidrug-resistant strain [ 36 ]. The aerosol route of administration has been proposed as organ specific delivery method, obtaining a high release to infected alveoli [ 37 ]. Condos et al. reported clinical and bacteriological improvement and tolerability with aerosolized IFN gamma in five patients with MDR-TB [ 38 ]. In addition, other previous trials demonstrated promising results in patients infected with other Mycobacteria [ 10 , 39 - 42 ]. Conclusions These results can suggest a beneficial effect of IFN gamma when it is used as adjuvant in the treatment of tuberculosis patients that have resistance to standard chemotherapy, and encourage carrying out more extensive, controlled studies. Combination with second-line drugs can reduce the time of treatment, diminishing toxicities and possible relapses; in many cases could reduce the application of recessional surgery. Further controlled clinical trials are needed to confirm these results. Competing interests Authors IGG, CMVS, and PALS are employees of the Center for Biological Research, which is part of the Center for Genetic Engineering and Biotechnology, Havana network, where IFN gamma is produced. The rest of the authors have no competing interests at all. Authors' contributions RSM conceived the study and carried out the bacteriological determinations. IGG participated in the study design and coordinating, and wrote the manuscript draft. NFO, MVQ, MTM, DC, and DMM took care of patient recruitment, management, and follow-up. CMVS participated in the study design and result analysis. PALS took part in the design, results analysis and manuscript writing. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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Neural Activity When People Solve Verbal Problems with Insight

People sometimes solve problems with a unique process called insight, accompanied by an “Aha!” experience. It has long been unclear whether different cognitive and neural processes lead to insight versus noninsight solutions, or if solutions differ only in subsequent subjective feeling. Recent behavioral studies indicate distinct patterns of performance and suggest differential hemispheric involvement for insight and noninsight solutions. Subjects solved verbal problems, and after each correct solution indicated whether they solved with or without insight. We observed two objective neural correlates of insight. Functional magnetic resonance imaging ( Experiment 1 ) revealed increased activity in the right hemisphere anterior superior temporal gyrus for insight relative to noninsight solutions. The same region was active during initial solving efforts. Scalp electroencephalogram recordings ( Experiment 2 ) revealed a sudden burst of high-frequency (gamma-band) neural activity in the same area beginning 0.3 s prior to insight solutions. This right anterior temporal area is associated with making connections across distantly related information during comprehension. Although all problem solving relies on a largely shared cortical network, the sudden flash of insight occurs when solvers engage distinct neural and cognitive processes that allow them to see connections that previously eluded them.

Introduction According to legend, Archimedes shouted “Eureka!” (“I have found it!”) when he suddenly discovered that water displacement could be used to calculate density. Since then, “Eureka!,” or “Aha!,” has often been used to express the feeling one gets when solving a problem with insight . Insight is pervasive in human (and possibly animal [ Epstein et al. 1984 ]) cognition, occurring in perception, memory retrieval, language comprehension, problem solving, and various forms of practical, artistic, and scientific creativity ( Sternberg and Davidson 1995 ). The Archimedes legend has persisted over two millennia in part because it illustrates some of the key ways in which insight solutions differ from solutions achieved through more straightforward problem solving. We examine the neural bases of these different problem-solving methods. Although many processes are shared by most types of problem solving, insight solutions appear to differ from noninsight solutions in several important ways. The clearest defining characteristic of insight problem solving is the subjective “Aha!” or “Eureka!” experience that follows insight solutions ( Schooler et al. 1993 ). This subjective experience can lead to a strong emotional response—according to legend, Archimedes ran home from the baths shouting “Eureka!” without donning his clothes first. In addition, problem solving with insight is characterized by the following features. (1) Solvers first come to an impasse, no longer progressing toward a solution ( Duncker 1945 ). Archimedes, for example, was stymied by King Hiero's challenge to determine whether his new crown was pure gold without damaging the crown. (2) Solvers usually cannot report the processing that enables them to reinterpret the problem and overcome the impasse ( Maier 1931 ). Insight often occurs when people are not even aware they are thinking of the problem, as reportedly happened to Archimedes while in the baths. (3) Solvers experience their solutions as arising suddenly ( Metcalfe and Wiebe 1987 ; Smith and Kounios 1996 ) and immediately recognize the correctness of the solution (or solution path). (4) Performance on insight problems is associated with creative thinking and other cognitive abilities different from those associated with performance on noninsight problems ( Schooler and Melcher 1997 ). Some researchers have argued that all these characteristics of insight solutions are essentially epiphenomenal, that insight and noninsight solutions vary only in emotional intensity, and that they are attained with precisely the same cognitive (hence neural) mechanisms ( Weisberg and Alba 1981 ; Weisberg 1986 ; Perkins 2000 ). Persistent questions about insight concern whether unconscious processing precedes reinterpretation and solution, whether distinct cognitive and neural mechanisms beyond a common problem-solving network are involved in insight, and whether the apparent suddenness of insight solutions reflects truly sudden changes in cognitive processing and neural activity. Recent work suggests that people are thinking—at an unconscious level—about the solution prior to solving problems with insight. Specifically, while working on a verbal problem they have yet to solve, people presented with a potential solution word read the actual solution word faster than they read an unrelated word ( Bowden and Beeman 1998 ). This “solution priming” effect is greater—and in fact people make solution decisions about presented words more quickly—when words are presented to the left visual hemifield, which projects directly to the right hemisphere (RH), than when words are presented to the right visual hemifield, which projects to the left hemisphere (LH). This suggests that RH semantic processing is more likely than LH semantic processing to produce lexical or semantic information that leads to the solution. These RH advantages occur only when solvers experience insight—the “Aha!” or “Eureka!” feeling that comes with insight solutions ( Bowden and Jung-Beeman 2003a ). Moreover, when subjects try to solve classic insight problems, they benefit more from hints presented to the left visual field (i.e., the RH) than from hints presented to the right visual field (i.e., the LH) ( Fiore and Schooler 1998 ). Problem solving is a complex behavior that requires a network of cortical areas for all types of solving strategies and solutions, so solving problems with and without insight likely invokes many shared cognitive processes and neural mechanisms. One critical cognitive process distinguishing insight solutions from noninsight solutions is that solving with insight requires solvers to recognize distant or novel semantic (or associative) relations; hence, insight-specific neural activity should reflect that process. The most likely area to contribute to this component of insight problem solving is the anterior superior temporal gyrus (aSTG) of the RH. Language comprehension studies demonstrate that the RH is particularly important for recognizing distant semantic relations ( Chiarello et al. 1990 ; Beeman 1998 ), and bilateral aSTG is involved in semantic integration. For example, sentences and complex discourse increase neural activity in aSTG bilaterally ( Mazoyer et al. 1993 ; Stowe et al. 1999 ), and discourse that places particular demands on recognizing or computing distant semantic relations specifically increases neural activity in RH temporal areas ( St. George et al. 1999 ; Mason and Just 2004 ), especially aSTG ( Meyer et al. 2000 ; Kircher et al. 2001 ). If this prediction of RH aSTG involvement is confirmed, it will help constrain neurocognitive theories of insight. Other cortical areas, such as prefrontal cortex and the anterior cingulate (AC) may also be differentially involved in producing insight and noninsight solutions. We used functional magnetic resonance imaging (FMRI) in Experiment 1 and electroencephalogram (EEG) measurement in Experiment 2 to test the empirically and theoretically derived hypothesis that solving problems with insight requires engagement of (or increased emphasis on) distinct neural mechanisms, particularly in the RH anterior temporal lobe. Event-related experimental designs compared neural activity when people solved verbal problems with insight to neural activity when they solved problems (from the same problem set) without insight. As in earlier behavioral work, we used a set of compound remote associate problems ( Bowden and Jung-Beeman 2003b ) adapted from a test of creative cognition ( Mednick 1962 ). Figure 1 illustrates the sequence for each trial. Subjects saw three problem words (pine, crab, sauce) and attempted to produce a single solution word (apple) that can form a familiar compound word or phrase with each of the three problem words (pineapple, crab apple, applesauce) . We relied on solvers' reports to sort solutions into insight solutions and noninsight solutions, avoiding the complication that presumed insight problems can sometimes be solved without insight ( Davidson 1995 ) and circumventing the use of different types of problems requiring different cognitive operations. Thus, we made use of the most important defining characteristic of insight problems: the subjective conscious experience—the “Aha!” A similar technique revealed distinct behavioral characteristics when people recognized solutions with insight ( Bowden and Jung-Beeman 2003a ). Note that this is a very “tight” comparison. In both conditions problems are solved using a network of processes common to both insight and noninsight solutions. If insight ratings reflect some distinct cognitive processes, this contrast will reveal the distinct underlying brain activity. In other words, within the cortical network for problem solving, different components will be engaged or emphasized for insight versus noninsight solutions. FMRI ( Experiment 1 ) should reveal neuroanatomical locations of processes that are unique to insight solutions, and EEG ( Experiment 2 ) should reveal the time course (e.g., whether insight really is sudden) and frequency characteristics of neurophysiological differences. Figure 1 Sequence of Events for Each Trial (A) The “Compound” prompt was presented for 0.5 s, then persisted for a variable amount of additional time (0–2 s) until a cue from the scanner indicated the beginning of a new whole brain acquisition. (B) A three-word problem appeared in the center of the screen and persisted until subjects indicated with a bimanual button press that they had solved the problem, or until the 30-s time limit elapsed. Thus, event timing and condition were completely dependent on subjects' responses. (C) Following the button press or time limit, subjects were prompted to verbalize the solution (or press the buttons and say “Don't know” if the time limit expired prior to solution) then (D) prompted to indicate (with a bimanual button press) whether they felt insight, as described prior to the experiment. (E) Next, subjects performed 9 s of an unrelated filler task (three line-matching trials, 3 s each), allowing BOLD signal to return to baseline (in areas not involved in line matching). Results Experiment 1 Subjects solved 59% of the problems presented, and pressed buttons indicating “insight” for 56% (s.d. = 18.2) of their solutions, “no insight” for 41% (s.d. = 18.9) of their solutions, and “other” for 2% of their solutions. We marked a point about 2 s (rounded to the nearest whole second) prior to each solution button press as the solution event, and examined a time window 4–9 s after this event (i.e., 2–7 s after the button press) to isolate the corresponding hemodynamic response. Solving problems and responding to them required a strict sequence of events (reading of words, solving effort, solving, button press, verbalizing the solution, insight decision), but this sequence was identical whether subjects indicated solving with or without insight, so differences in FMRI signal resulted from the degree to which distinct cognitive processes and neural systems led to insight or noninsight solutions. Figure 2 illustrates the most robust insight effect: as predicted, insight solutions were associated with greater neural activity in the RH aSTG than noninsight solutions. The active area was slightly anterior to primary auditory cortex, posterior to temporal pole, and along the medial aspect of the aSTG, extending down the lateral edge of the descending ramus of the Sylvian fissure to midway through the middle temporal gyrus (MTG). (This site is also close to the superior temporal sulcus, which has been implicated in language). Across all 13 subjects, the peak signal difference at a single voxel within the RH aSTG was 0.25% across the 6-s window, and 0.30% at a single time to repetition (TR), i.e., the time needed to repeat the image of the whole brain. Overall signal in this region was robust, reaching 96.8% of the brainwide average (after removing voxels in other brain areas with signal below a standard criterion). Within the cluster of voxels identified across the group, 12 subjects showed from 0.03% to 0.35% greater signal for insight than for noninsight solutions; one subject showed 0.02% greater signal for the noninsight solutions. It is not likely that RH aSTG is involved only in output or in emotional response following insight solutions, because neural activity in this area also increased when subjects first encountered each problem ( Figure 3 ). Thus, RH aSTG is involved in processing the problem words both initially and at solution. (Of course, event-related FMRI signal occurred in many other cortical regions at problem onset, especially visual cortex). There was no insight effect in response windows immediately preceding or following the defined response window. All indications point to a striking transient event in the RH aSTG near the time when subjects solve problems with insight. Figure 2 FMRI Insight Effect in RH aSTG (A) Voxels showing greater FMRI signal for insight than noninsight solutions, overlaid on the averaged normalized structural image of all subjects. The active area has a volume of 531 mm 3 (peak t = 4.89 at 44, −9, −9 in Talairach space). (B) and (C) Group average signal change following the solution event, for insight (red line) and noninsight (blue line) solutions (yellow arrow indicates button press): (B) over entire LH aSTG region; (C) over entire RH aSTG region. (D) Insight solution signal change minus noninsight solution signal change, in RH aSTG (error bars show the standard error of the mean of the difference at each timepoint). Figure 3 FMRI Signal in RH aSTG during Initial Solving Efforts (A) Voxels in right temporal lobe showing baseline-to-peak event-related FMRI signal when subjects first encounter problems, overlaid on the averaged normalized structural image of all subjects. The cluster is in RH aSTG, with a volume of 469 mm 3 , with peak t value of 4.37 at 41, −6, −12 in Talairach space, clearly overlapping with the cluster showing an insight effect at solution. (B) Group average signal change following problem onset (time = 0), for the cluster defined by signal at the problem onset (green line) and the cluster (illustrated in Figure 2 A) showing the insight effect at solution (white line). Error bars show the standard error of the mean of the difference at each time point. The involvement of the RH rather than the LH for this verbal task is not due to greater difficulty in producing insight solutions: subjects produced insight solutions at least as quickly (mean solution time = 10.25 s, s.d. = 3.58 s) as they produced noninsight solutions (mean = 11.28 s, s.d. = 4.13 s) ( t < 1.0, p > 0.3). More importantly, the hemodynamic responses to both insight and noninsight solutions in the homologous area of the LH are about equivalent to the response to noninsight solutions in the RH aSTG—it is the strong response to insight solutions in the RH aSTG that stands out. There is no insight effect anywhere within temporal cortex of the LH. At statistical thresholds below significant levels ( p < 0.1 uncorrected), there are as many voxels in LH temporal cortex showing a noninsight effect as showing an insight effect. Several other cortical areas showing insight effects that did not meet significance criteria are listed in Table 1 (see also Figure S1 ). Some of these effects were in frontal cortex, which is notable because various frontal areas have been implicated in problem solving and reasoning. Patients with prefrontal damage have particular difficulty integrating relations in reasoning tasks ( Waltz et al. 1999 ), and when healthy subjects perform the same task, neural activity increases in rostrolateral prefrontal cortext ( Christoff et al. 2001 ). Some problem solving increases activity in dorsolateral prefrontal cortex ( Prabhakaran et al. 1997 ), perhaps because of working memory demands. Solving of poorly structured problems seems particularly impaired following damage to the prefrontal cortex of the RH ( Goel and Grafman 2000 ). Moreover, the inferior frontal gyrus (IFG) is highly active when people engage in directed semantic retrieval ( Wagner et al. 2001 ) or when they select particular semantic concepts over competing ones ( Thompson-Schill et al. 1997 ), e.g., to generate a response ( Frith et al. 1991 ). Usually in these circumstances the IFG activity is stronger in the LH, even when people are reasoning about spatial problems ( Goel et al. 1998 ), but the IFG responds particularly strongly in the RH when subjects select more distant semantic relations because of task demands ( Seger et al. 2000 ) or comprehension goals ( Robertson et al. 2000 ). Because of its putative importance for problem solving, semantic retrieval, and semantic selection, IFG was an a priori region of interest. One question we had hoped to answer was whether the semantic selection of insight solutions would preferentially evoke activity in RH or LH IFG, but the insight effects in both areas were too small (in area and in reliability) to test this question. When a more lenient statistical threshold was adopted, small clusters of signal were observed in both RH and LH IFG ( Table 1 ; Figure S1 A). Indeed, within the small region surpassing this weak statistical threshold, signal change in the RH IFG region was moderately strong (peak = 0.21% across the whole window). However, as is often the case, FMRI signal in this region was low (about 72% of the brainwide average) and variability was high, decreasing our confidence in the effect. Table 1 Full FMRI Results of Insight Effect All areas showing an “insight effect”—stronger signal for insight solutions than noninsight solutions (ordered by mean percent signal change). All cluster sizes represent active voxels at t (12) = 3.43, p < 0.005, except bilateral inferior frontal gyrus areas (*), shown at 2.83, p < 0.015, because it was an a priori region of interest. Location of cluster centers and peak t values are shown in Talairach coordinates After RH aSTG, the second largest area showing an insight effect in FMRI signal was the medial frontal gyrus in the LH ( Table 1 ; Figure S1 B). Although this area was 85% as large (453 mm 3 at p < 0.005 threshold) as RH aSTG, the event-related signal within it was weak and the insight–noninsight difference (peak difference = 0.15%) was relatively small. (The insight effect may be attributable as much to a negative response for noninsight solutions as to a positive response for insight solutions.) There also was an insight effect in small clusters in or near bilateral amygdala or parahippocampal gyrus. Again, regional signal was low (83% of the brainwide average), and the signal difference was small (peak = 0.16%). However, an amygdalar response may be expected, given the emotional sensation of the insight experience (Parsons and Osherson, 2001). Hippocampal or parahippocampal involvement is also plausible, if memory interacts with insight solutions differently from how it interacts with noninsight solutions. For instance, insight problems may encourage distinct memory encoding ( Wills et al. 2000 ) or may require distinct retrieval. Finally, a small cluster in the LH posterior cingulate (PC) also showed an insight effect. There was strong, sustained FMRI signal for both solution types in this region; on the fringe of this responding region, FMRI signal began earlier following insight than noninsight solutions. The lateness of the FMRI signal across LH PC suggests that this effect began later in the response sequence, rather than during solution generation. Finally, as in most FMRI studies, signal was relatively weak in temporal pole and orbitofrontal areas due to magnetic susceptibility artifact, so we cannot rule out undetected effects in those areas. Several cortical areas showed strong solution-related FMRI signal, but approximately equally for insight and noninsight solutions. Some of these areas (e.g., motor cortex) relate to the response sequence rather than solution processes; other areas probably reflect component processes of a problem-solving network common to both insight and noninsight solving, such as retrieving potential solutions. Two areas that may be of interest for future studies are AC and posterior middle/superior temporal gyrus. Both these areas, in the RH only, showed strong, negative solution-related signal, approximately equal in the two solution types. AC is an area that might be predicted to be involved in reorienting attention as solvers overcome impasses, given its role in performance monitoring and cognitive control ( MacDonald et al. 2000 ). RH posterior MTG is active when subjects “get” jokes ( Goel and Dolan 2001 ) and when they attempt to solve problems with deductive reasoning ( Parsons and Osherson 2001 ). However, in our experiment, only the RH aSTG showed a robust insight effect. Experiment 2 A separate group of subjects participated in fundamentally the same paradigm while we continuously recorded EEGs from the scalp. We then compared time-frequency analyses of the EEGs associated with insight solutions versus noninsight solutions. EEG provides temporal resolution greatly superior to that of FMRI and thus can better elucidate the time course and suddenness of the insight effect. Furthermore, complex EEG oscillations can be parsed into constituent frequency components, some of which have been linked to particular types of neural and cognitive processes ( Ward 2003 ). The high temporal resolution of EEG allows us to address one of the fundamental questions raised earlier: does insight really occur suddenly, as subjective experience suggests? For problems typically solved without insight, solvers report gradually increasing closeness to solution. In contrast, for problems typically solved with insight, solvers report little or no progress until shortly before they actually solve the problem ( Metcalfe 1986 ; Metcalfe and Wiebe 1987 ). Similarly, quantitative analyses of the distributions of response times and accuracies during anagram solving (a task frequently eliciting the experience of insight) reveal that a solution becomes available in a discrete transition from a state of little or no information about the correct response directly to the final state of high accuracy. This contrasts with various language and memory tasks not associated with insight, which yield partial outputs before processing has been completed ( Kounios and Smith 1995 ; Smith and Kounios 1996 ). We predicted that a sudden change in neural activity associated with insight solutions would produce an EEG correlate. Specifically, we predicted that high-frequency EEG oscillations in the gamma band (i.e., greater than 30 Hz) would reflect this sudden activity, because prior research has associated gamma-band activity with the activation of perceptual, lexical, and semantic representations ( Tallon-Baudry and Bertrand 1999 ; Pulvermüller 2001 ). Gamma-band electrical activity correlates with the blood oxygenation level–dependent (BOLD) response apparent in FMRI signal; lower-frequency EEG components do not seem to have direct correlates in FMRI signal ( Foucher et al. 2003 ; Laufs et al. 2003 ). Consequently, based on the language literature discussed earlier and on our FMRI results, we predicted a discrete insight-related increase in gamma-band activity at electrodes over the anterior temporal lobe of the RH. Participants solved 46% (s.d. = 8.2) of the problems correctly within the time limit. Of correctly solved problems, subjects reported more insight solutions (56%, s.d. = 8.4) than noninsight solutions (42%, s.d. = 9.0), ( t [18] = 3.47, p =0.003); there was no difference in mean response times (insight solutions = 9.94 s, s.d. = 2.60; noninsight solutions=9.25 s, s.d. = 3.06; t < 1.0). There was a burst of gamma-band activity associated with correct insight solutions (but not noninsight solutions) beginning approximately 0.3 s before the button-press solution response at anterior right temporal electrodes ( Figure 4 ), with no significant difference between insight and noninsight solutions over homologous LH sites. A repeated-measures analysis of variance (ANOVA) performed on log-transformed gamma-band (39 Hz) EEG power at left and right temporal electrode sites (T7 and T8, respectively) for insight and noninsight trials using two time windows (−1.52 to −0.36 s and −0.30 to −0.02 s, measured with respect to the solution response) yielded significant insight × time window ( F [1,18] = 6.68, p = 0.019) and insight × time window × Hemisphere ( F [1,18] = 8.11, p = 0.011) interactions. The overall interaction occurred because there was an insight × hemisphere interaction from −0.30 to −0.02 s ( F [1,18] = 4.61, p = 0.046) but no effect in the −1.52 to −0.36 s time window. Within the −0.30 to −0.02 s interval for these two electrodes, there was a significant insight effect at the right temporal (T8) site ( t [18] = 3.48, p = 0.003), but not at the homologous left temporal (T7) site or any other LH temporal electrode. Laplacian mapping of this effect ( Figure 4 B) is remarkably consistent with the FMRI signal in RH aSTG observed in Experiment 1 . (EEG does not have the spatial resolution of FMRI. However, we used the Laplacian transform [i.e., second spatial derivative] to localize observed activity. The Laplacian derivation acts as a high-pass spatial filter that reduces the contribution from activity in distant areas of the brain to the signal at a given electrode, and therefore reflects relatively focal and proximal brain activity. Given our FMRI results and the demonstrated correspondence between high-frequency EEG activity and FMRI signal [ Foucher et al. 2003 ; Laufs et al. 2003 ], we are confident in the localization of this effect.) Figure 4 Gamma-Band Power for Insight and Noninsight Solutions (A) Grand average time course of EEG power (in v 2 ) at 39 Hz estimated with the Morlet wavelet transform at right superior temporal electrode T8. The x -axis represents time (in seconds) with the yellow arrow and R marking the point in time of the solution button-press response (i.e., 0.0 s). The green horizontal bars above the x -axis represent the time intervals used in the statistical analyses and topographic maps. Note that gamma-band power for insight trials (red line) starts to increase above power on noninsight trials (blue line) by approximately 0.3 s before the button press. (B) Time-frequency plots of the insight minus noninsight difference shown in (A). The y -axis represents frequency (in Hz); the x -axis represents time (in seconds, with respect to the button press, exactly as shown in [A]). Red areas of the plot reflect times and frequencies at which insight EEG power is greater than noninsight EEG power; blue areas reflect times and frequencies at which noninsight EEG power is greater than insight EEG power. Note the sudden emergence of increased gamma power for insight solutions approximately 0.3 s before the button press. (C) Insight minus noninsight gamma-band differences plotted as topographic maps (LH and RH) of scalp current density (in v 2 /m 2 ) estimated by a spline-based Laplacian transform computed with a realistic FMRI-derived head model. The Laplacian transform acts as a high-pass spatial filter that minimizes the contribution of activity distant from each electrode, thereby manifesting discrete, relatively superficial sources. The maps are thresholded to show foci of current density at the upper and lower 20% of the scale. Note the prominent effect of insight (effect for insight greater than effect for noninsight, in red) at the right superior temporal electrode (T8) and surrounding electrodes present from −0.30 to −0.02 s (measured with respect to the solution response) that is not present in the earlier epoch (−1.52 to −0.36 s). The blue area over left inferior parietal cortex (electrode P7) indicates that noninsight gamma power is nonsignificantly greater than insight power ( F [1,19] < 1) over this region. The gamma burst in the right temporal area cannot be attributed to motor processes involved in making the response because (A) motor activity associated with the bimanual button press would have caused a bilateral gamma burst, not a unilateral one; (B) the location of the gamma burst as determined by Laplacian mapping ( Figure 4 B) is not consistent with hand-related motor cortex activity; and (C) both insight and noninsight solutions required button presses. Other planned statistical tests (ANOVAs) examined possible insight-related frontal theta (5–8 Hz), posterior alpha (8–13 Hz), and fronto-central beta (13–20 Hz) activity. There were no statistically significant theta or beta effects. (Visual inspection and post hoc statistical tests suggested insight-related frontal 4-Hz activity, but this effect cannot be reliably distinguished from possible artifacts due to small vertical eye movements.) There was a significant posterior alpha effect, which is discussed below. Discussion Complex problem solving requires a complex cortical network to encode the problem information, search memory for relevant information, evaluate this information, apply operators, and so forth. The FMRI and EEG results reported here conclusively demonstrate that solving verbal problems with insight requires at least one additional component to this cortical network, involving RH aSTG, that is less important to solving without insight. The insight effect in RH aSTG accords with the literature on integrating distant or novel semantic relations during language comprehension. When people comprehend (read or listen to) sentences or stories, neural activity increases in aSTG or temporal pole bilaterally more than when comprehending single words ( Mazoyer et al. 1993 ; Bottini et al. 1994 ; Stowe et al. 1999 ; Humphries et al. 2001 ; Meyer et al. 2000 ). Neural activity increases in predominantly RH aSTG during tasks that emphasize integration across sentences to extract themes ( St. George et al. 1999 ) or to form more coherent memories for stories ( Mason and Just 2004 ). RH aSTG is also selectively active when subjects must generate the best ending to a sentence ( Kircher et al. 2001 ) or mentally repair grammatically incorrect sentences ( Meyer et al. 2000 ), both of which likely require intense semantic integration. Like the results in language processing, the current results are predicted by the theory that the RH performs relatively coarse semantic coding ( Beeman 1998 ; similarly, Chiarello et al. 1990 ). This theory contends that when people encounter words, semantic processing in several LH areas engages in relatively fine semantic coding which produces small semantic fields—i.e., this processing strongly focuses on a few concepts closely related to the input word in the given context. This is very effective for most straightforward language processing. In contrast, the homologous RH areas engage in relatively coarse semantic coding, which produces large and weak semantic fields—i.e., this processing includes many concepts, even concepts distantly related to the input words and context. This process is ineffective for rapid interpretation or selection but increases semantic overlap among multiple semantic fields ( Beeman et al. 1994), which is useful when drawing together parts of a story or conversation that are only distantly related ( Beeman 1993 ; Beeman et al. 2000 ). In this view, the coarseness of semantic coding is largely influenced by slight asymmetries in neural microcircuitry that produce more discrete, less redundant input fields in pyramidal neurons of the LH language cortex, and more overlapping input fields in corresponding neurons in the RH (for reviews see Beeman 1998 ; Hutsler and Galuske 2003 ). We suggest that semantic integration, generally, is important for connecting various problem elements together and connecting the problem to the solution, and that coarsely coded semantic integration, computed in RH aSTG, is especially critical to insight solutions, at least for verbal problems (or problems that can be solved with verbal or semantic information). People come to an impasse on insight problems because their retrieval efforts are misdirected by ambiguous information in the problem or by their usual method for solving similar problems. Large semantic fields allowing for more overlap among distantly related concepts (or distantly associated lexical items) may help overcome this impasse. Because this semantic processing is weak, it may remain unconscious, perhaps overshadowed by stronger processing of the misdirected information ( Schooler et al. 1993 ; Smith 1995 ), and solvers remain stuck at impasse. Eventually, solution-related information bursts into awareness “in a sudden flash.” This can happen after misdirected processing decays or is suppressed, after solution-related processing grows, or after environmental cues occur—such as the water overflowing the bathtub when Archimedes got in. Archimedes had semantic and verbal knowledge about how to compute density from weight and volume, but struggled with measuring the volume of an irregularly shaped crown without harming the crown (e.g., melting it). His observation of water displacement allowed him to connect known concepts in new ways. This is the nature of many insights, the recognition of new connections across existing knowledge. A persistent question has been whether the cognitive and neural events that lead to insight are as sudden as the subjective experience. The timing and frequency characteristics of the EEG results shed light on this question. We propose that the gamma-band insight effect in Experiment 2 reflects the sudden transition of solution-related cognitive processing from an unconscious to a conscious state. Recent research associates gamma-band oscillations with the ignition of neural cell assemblies supporting the transient feature binding necessary to activate a representation ( Tallon-Baudry and Bertrand 1999 ; Pulvermüller 2001 )—in this case, a phonological, lexical, or semantic representation corresponding to the solution word and its associations to the problem words. According to this hypothesis, greater synchronous gamma-band activity for insight than for noninsight solutions could reflect a more integrated or unitized solution representation. Furthermore, synchronous gamma-band activity has been hypothesized to play a critical role in the accessibility to consciousness of such representations ( Engel and Singer 2001 ). The timing (with respect to the solution button press) of the insight gamma-band effect closely approximates estimates derived from cognitive behavioral studies of the amount of time required to access an available solution and generate a two-alternative, forced-choice button-press response (e.g., Kounios et al. 1987 ; Meyer et al. 1988 ; Smith and Kounios 1996 ). The present experiments had no response choice (i.e., always the same bimanual button press for solutions), so subjects could easily have responded 0.3 s after solving the problems. Thus, we infer that the observed gamma burst reflects the sudden conscious availability of a solution word resulting from an insight. Suddenly recognizing new connections between problem elements is a hallmark of insight, but it is only one component of a large cortical network necessary for solving problems with insight, and recognizing new connections likely contributes to other tasks, such as understanding metaphors ( Bottini et al. 1994 ) and deriving a story theme ( St. George et al. 1999 ). Similar tasks may depend on related cortical networks. For example, appreciating semantic jokes ( Goel and Dolan 2001 ) and engaging in deductive reasoning that sometimes involves insight ( Parsons and Osherson 2001 ) both increase activity in RH posterior MTG. It is striking that the insight effect observed in the RH in our experiments occurred when people solved verbal problems, which traditional views suggest should involve mostly LH processing with little or no contribution from the RH. It is possible that insight solutions to nonverbal problems would require different cortical networks. However, the observed effect cannot be due simply to verbal retrieval, which must occur for both insight and noninsight solutions; it could be due to a type of verbal retrieval specific to insight solutions, but not involved in noninsight solutions. We turn now to another result from the EEG time-frequency analysis, which was not predicted but nevertheless suggests a provocative interpretation. The gamma burst thought to reflect the transition of the insight solution from an unconscious to a conscious state was preceded by insight-specific activity in the alpha band (8–13 Hz). Specifically, there was a burst of alpha power (estimated at 9.8 Hz) associated with insight solutions detected over right posterior parietal cortex from approximately 1.4 s until approximately 0.4 s before the solution response, at which point insight alpha power decreased to the level of noninsight alpha power, or below ( Figure 5 ). An ANOVA was performed on log-transformed alpha-band (9.8 Hz) EEG power at left and right parietal-occipital electrode sites (PO7 and PO8, respectively) for insight and noninsight trials using three time windows: −2.06 to −1.56 s, −1.31 to −0.56 s, and −0.31 to 0.06 s (measured from the solution button press). This analysis yielded a significant insight × time window interaction ( F [2,36] = 4.13, p = 0.027, with the Huynh-Feldt correction). Follow-up t -tests in each time window yielded significant effects of insight in the first time window at both electrode sites (PO7: t [18] = 2.32, p = 0.033; PO8: t [18] = 2.42, p = 0.026) and in the second time window only at the RH site (PO8: t [18] = 2.17, p = 0.043), with a reversal of the direction of the effect. The third time window yielded no significant effects. Figure 5 Alpha-Band Power for Insight and Noninsight Solutions (Same conventions as in Figure 4 ). (A) Time course of EEG power at 9.8 Hz (in v 2 ) at right parietal-occipital electrode (PO8). The x -axis represents time (in seconds), with the green horizontal bars above the x -axis representing the time intervals used in the statistical analyses and topographic maps. The yellow arrow and R (at 0.0 s) signify the time of the button-press response. (B) Time-frequency plots of the insight minus noninsight difference shown in (A). (C) Insight minus noninsight alpha-band differences plotted as topographic maps of scalp current density (in v 2 /m 2 ). Note that alpha-band power is significantly greater for insight solutions than noninsight solutions during the −1.31 to −0.56 s interval, but not during the preceding (−2.06 to −1.56 s) or subsequent (−0.31 to +0.06 s) intervals. This alpha burst was embedded in a slow decrease in alpha (see [A]), probably reflecting a general increase in cortical activity as effort increases during the course of problem solving. Alpha rhythms are understood to reflect idling or inhibition of cortical areas ( Pfurtscheller et al. 1996 ). Increased alpha power measured over parietal-occipital cortex indicates idling or inhibition of visual cortex. This has been attributed to gating of visual information flowing into the perceptual system in order to protect fragile or resource-intensive processes from interference from bottom-up stimulation ( Ray and Cole 1985 ; Worden et al. 2001 ; Jensen et al. 2002 ; Cooper et al. 2003 ; Ward 2003 ). This interpretation assumes that brain areas are normally highly interactive, and that allowing one process to proceed relatively independently requires active attenuation of this interaction. For instance, when subjects attend to visual space in the hemifield projecting to one hemisphere, posterior alpha increases over the other hemisphere, which receives inputs from the unattended hemifield ( Worden et al. 2001 ). Analogously, the present results suggest selective gating of visual inputs to the RH during the interval preceding the insight-related right temporal gamma burst ( Figure 6 ). Hypothetically, this allows weaker processing about more distant associations between the problem words and potential solutions to gain strength, by attenuating bottom-up activation or other neural activity not related to solution that would decrease the signal-to-noise ratio for the actual solution. Figure 6 The Time Course of the Insight Effect Alpha power (9.8 Hz at right parietal-occipital electrode PO8) and gamma power (39 Hz at right temporal electrode T8) for the insight effect (i.e., correct insight solutions minus correct noninsight solutions, in v 2 ). The left y -axis shows the magnitude of the alpha insight effect (purple line); the right y -axis applies to the gamma insight effect (green line). The x -axis represents time (in seconds). The yellow arrow and R (at 0.0 s) signify the time of the button-press response. Note the transient enhancement of alpha on insight trials (relative to noninsight trials) prior to the gamma burst. This interpretation of the early insight-specific alpha effect is consistent with previous behavioral research suggesting that, prior to an insight, the solution to a verbal problem can be weakly activated ( Bowers et al. 1990 ), especially in the RH (Bowden and Beeman 1998 ; Bowden and Jung-Beeman 2003a ). Thus insight solutions may be associated with early unconscious solution-related processing, followed by a sudden transition to full awareness of the solution. We suggest that, in Experiment 2 , the early posterior alpha insight effect is an indirect correlate of the former, and the right temporal gamma effect is a direct correlate of the latter. In sum, when people solve problems with insight, leading to an “Aha!” experience, their solutions are accompanied by a striking increase in neural activity in RH aSTG. Thus, within the network of cortical areas required for problem solving, different components are engaged or emphasized when solving with versus without insight. We propose that the RH aSTG facilitates integration of information across distant lexical or semantic relations, allowing solvers to see connections that had previously eluded them. In the two millennia since Archimedes shouted “Eureka!,” it has seemed common knowledge that people sometimes solve problems—whether great scientific questions or trivial puzzles—by a seemingly distinct mechanism called insight. This mechanism involves suddenly seeing a problem in a new light, often without awareness of how that new light was switched on. We have demonstrated that insight solutions are indeed associated with a discrete, distinct pattern of neural activity, supporting unique cognitive processes. Materials and Methods Subjects Ten men and eight women were paid to participate in Experiment 1 ; 19 new subjects (nine men, ten women) were paid to participate in Experiment 2 . All were young (18–29) neurologically intact, right-handed, native English speakers; Experiment 1 participants met safety criteria for FMRI scanning. After hearing about all methods and risks and performing practice trials, they consented to participate. In Experiment 1 , data from four men and one woman were excluded due to poor FMRI signal or because subjects provided fewer than ten insight or noninsight responses. This research was approved by the University of Pennsylvania Institutional Review Board. Behavioral paradigm Following practice, subjects attempted 124 compound remote associate problems during FMRI scanning. These problems ( Bowden and Jung-Beeman 2003 b) can be solved quickly and evoke an “Aha!” experience, producing a distinct behavioral signature ( Bowden and Jung-Beeman 2003 a), roughly half the time they are solved. Figure 1 illustrates the sequence of events for each trial. Each trial began with the task label “Compound” presented on liquid crystal diode goggles for 0.5 to 2.5 s. A gating signal from the scanner triggered the central presentation of three problem words, which persisted until subjects solved the problem or 30 s elapsed. If subjects solved the problem, they made a bimanual button press, after which the word “Solution?” prompted them to verbalize their solution. After 2 s the word “Insight?” prompted subjects to press buttons indicating whether they solved the problem with insight. Prior to the experiment subjects were told the following: “A feeling of insight is a kind of ‘Aha!' characterized by suddenness and obviousness. You may not be sure how you came up with the answer, but are relatively confident that it is correct without having to mentally check it. It is as though the answer came into mind all at once—when you first thought of the word, you simply knew it was the answer. This feeling does not have to be overwhelming, but should resemble what was just described.” The experimenter interacted with subjects until this description was clear. This subjective rating could be used differently across subjects (or even across trials), blurring condition boundaries; yet the distinct neural correlates of insight observed across the group demonstrate that there was some consistency. If subjects failed to solve problems within 30 s, the “Solution?” prompt appeared, and subjects pressed the “no” buttons and verbalized “Don't Know.” Then the “Insight?” prompt appeared, and subjects pressed the “no” buttons again. After the insight rating, subjects performed three line-matching trials (3 s each) to distract them from thinking about the problems, allowing the critical BOLD signal to return to baseline ( Binder et al. 1999 ). The total time from the end of one problem to the onset of the next was 14.5–16.5 s. The condition (e.g., insight or noninsight solution) and time of events was determined by subjects' responses. Image acquisition Imaging was performed at the Hospital of the University of Pennsylvania, on a 1.5 Tesla GE SIGNA scanner with a fast gradient system for echo-planar imaging and a standard head coil. Head motion was restricted with plastic braces and foam padding. Anatomical high-resolution T1-weighted axial and sagittal images were acquired while subjects performed practice trials. Functional images (21 slices, 5 mm thick; 3.75-mm × 3.75-mm in-plane resolution; TR = 2000 ms for 21 slices; time to echo = 40 ms) were acquired in the same axial plane as the anatomical images using gradient-echo echo-planar sequences sensitive to BOLD signal ( Kwong et al. 1992 ; Ogawa et al. 1992 ). Each functional run was preceded by a 20-s saturation period. Subjects participated in four 15-min runs and a fifth run of varying length, depending on the number of remaining problems. Image analysis Images were coregistered through time with a three-dimensional registration algorithm ( Cox 1996 ). Echo planar imaging volumes were spatially smoothed using a 7.5-mm full-width half-maximum Gaussian kernel. Within each run, voxels were eliminated if the signal magnitude changed more than 10% across successive TRs, or if the mean signal level was below a noise threshold. Functional data were transformed ( Collins et al. 1994 ) to a standard stereotaxic atlas ( Talairach and Tournoux 1988 ) with a voxel size of 2.5 mm 3 . Data were analyzed using general linear model analysis that extracted average responses to each trial type, correcting for linear drift and removing signal changes correlated with head motion. Each TR was divided into two 1-s images to improve time locking of the solving event and the functional image data (time-course data were temporally smoothed in Figures 2 and 3 ). Solution-related responses were calculated using the average signal change within the window 4–9 s (to account for hemodynamic delay) after the solving event (beginning about 2 s prior to the button press). Differences between insight and noninsight solution events were estimated for each participant, then combined in a second-stage random effects analysis to identify differences consistent across all subjects. A cluster threshold was set at regions at least 500 mm 3 in volume (32 normalized voxels, or 7.1 original-sized voxels) in which each voxel was reliably different across subjects, ( t [12] > 3.43, p < 0.005 uncorrected). Monte Carlo simulations with similar datasets reveal low false positive rates with these criteria. RH aSTG was the only cluster to exceed these criteria, and converging evidence and the a priori prediction about RH aSTG strengthen confidence in this result. Experiment 2 Behavioral procedures were similar to those of Experiment 1 , except that (A) problem words were presented at smaller visual angles to discourage eye movements, (B) there were 2-s delays between each event in the response sequence, and (C) subjects triggered a new problem directly after responding to the previous problem (i.e., no line task occurred between problems). EEG methods Continuous high-density EEGs were recorded at 250 Hz (bandpass: 0.2–100 Hz) from 128 tin electrodes embedded in an elastic cap (linked mastoid reference with forehead ground) placed according to the extended International 10–20 System. Prior to data analysis, EEG channels with excessive noise were replaced with interpolated data from neighboring channels. Eyeblink artifacts were removed from the EEG with an adaptive filter separately constructed for each subject using EMSE 5.0 (Source Signal Imaging Inc., San Diego, California, United States). Induced oscillations were analyzed by segmenting each subject's continuous EEG into 4-s segments beginning 3 s before each solution response. (An analysis epoch beginning at an earlier point in time would have resulted in the loss of trials associated with response times of less than 3 s.) Time-frequency transforms (performed with EMSE 5.0) were obtained by the application of complex-valued Grossmann-Morlet wavelets, which are Gaussian in both time and frequency. Following Torrence and Campo (1998 ), the mother wavelet, ω 0 , in the time domain has the form where ω 0 is a nondimensional frequency. In this case, ω 0 is chosen to be 5.336, so that ∫ϕ 0 ( t ) ≅ 0. The constant π−¼ is a normalization factor such that ∫(ϕ 0 ( t )) 2 = 1. For the discrete time case, a family of wavelets may be obtained as where δ t is the sample period (in seconds), s is the scale (in seconds), and n is an integer that counts the number of samples from the starting time. The Fourier wavelength λ is given by In the frequency domain, the (continuous) Fourier transform of Equation 2 is where One reasonable way to measure the “resolution” of the wavelet transform is to consider the dispersion of the wavelets in both time and frequency. Since the wavelets are Gaussian in both domains, the e -folding time and frequency may serve as quantitative measures of dispersion. Note that these dispersions are a function of the scale, s . For a selected frequency, 𝒻 c = 1/λ, or from Equation 3 Then substituting into Equation 2 , we find that the e -folding time is for frequency 𝒻 c . From Equation 2 , the e -folding frequency is . To make this concrete, we find that for a 10-Hz (alpha-band) center frequency, the e -folding time is 0.12 s and the e -folding frequency is 2.6 Hz. For a 40-Hz ( gamma-band) center frequency, the e -folding time is 0.03 s and the e -folding frequency is 10.5 Hz. Note that these e -folding parameters imply that wavelet scaling preserves the joint time-frequency resolution (equal areas in time-frequency space), with higher temporal resolution but broader frequency resolution as the wavelet scale decreases. Segments corresponding to trials for which individual subjects produced the correct response were isolated and averaged separately according to whether or not the subject reported the experience of insight. Planned statistical tests (repeated-measure ANOVAs) were performed in order to detect insight-related effects on frontal midline theta (5–8 Hz), posterior alpha (8–13 Hz), fronto-central beta (13–20 Hz), and left and right temporal gamma (20–50 Hz). Response-locked event-related potentials (ERPs) were also computed using the same analysis epoch. Standard ERP analyses yielded no evidence of statistically significant effects, likely because ERPs reflect phase-locked activity rather than the induced (i.e., nonphase-locked) activity examined in the wavelet analyses; due to the long response times evident in this experiment, phase locking resulting from problem presentation would not be expected. EEG effects were topographically mapped by employing spline-based Laplacian mapping with an FMRI-derived realistic head model and digitized electrode positions. Localization of EEG/ERP signals is a form of probabilistic modelling rather than direct neuroimaging. In contrast to other techniques, source estimation by Laplacian mapping indicates the presence of superficial foci of neuroelectric activity with minimal assumptions. Supporting Information Figure S1 Cortical Regions Showing “Insight Effects” Below Cluster Size Threshold The far left lane shows for each region a single slice best depicting the cluster activated above threshold; middle lane shows time course of signal following insight (red line) and noninsight (blue line) solutions, across the entire active cluster; right panel shows the “insight effect” (insight signal minus noninsight signal, error bars show the standard error of the mean of the difference at each timepoint). (A) depicts bilateral IFG with lowered threshold ( t [12] = 2.83, p < 0.015); (B–D) depict clusters of FMRI signal at the same t -threshold used in the main paper ( t [12] = 3.43, p < 0.005), but the clusters are too small to surpass cluster criterion. (B) LH medial frontal gyrus; (C) LH PC gyrus; (D) LH amygdala (there was also a small cluster near RH amygdala). Spatial coordinates and other are details listed in Table 1 . (914 KB PDF). Click here for additional data file.

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Use of email for patient communication in student health care: a cross-sectional study

Background Citizens increasingly use email in personal communication. It is not however clear to what extent physicians utilize it for patient communication. Our study was designed to examine physicians' activity in using email and to estimate the proportion of email messages missing from documentation in electronic patient records (EPR). Methods All physicians (n = 76; 48 general practitioners and 28 specialists) at the Finnish Student Health Service received a questionnaire by email, and were asked to print it and keep a daily tally of visits, phone calls and email messages over the study period of one working week (5.5. – 9.5.2003). The response rate was 70%. The data originating from the questionnaire were compared with statistical data from the EPR during the study period. Results The majority (79%, 41/52) of doctors reported using email with patients, averaging 8.6 (range: 0–96) email contacts and a percentage rate of "email / visit" 20% (range: 0–185%) in one working week. Doctors in the capital city region and those doctors who had a positive attitude toward email for patient communication were most active in email use. Up to 73% of email contacts were not documented in the EPR. Conclusion The activity in using email with patients verified among Finnish physicians is compatible with recent study results elsewhere. The notable proportion of un-recorded email messages establishes the need for an electric communication system built into the EPR to improve the quality of patient care and to limit medico-legal risks.

Background Citizens increasingly use email in personal communication [ 1 ]. It is not however clear to what extent physicians utilize it for patient communication. In reports from the 1990s 1–14% of doctors in the USA and Norway used email in patient work [ 2 , 3 ]. In recent studies up to 73% of physicians had used email for patient communication [ 4 - 6 ]. International and national recommendations and guidelines have been published on email use between doctor and patient; the contents of the Finnish guidelines are well in line with European guidelines [ 7 , 8 ]. These guidelines emphasize the suitability of email for only certain limited purposes and stress the risks to information security. Use of email between doctor and patient has been studied in one controlled and randomized and three cross-sectional studies [ 4 , 5 , 9 , 10 ]. In a study by Katz and colleagues the number of email contacts of physicians in their study group (46 email messages/100 scheduled visits) was greater than that in the control group (9/100) [ 5 ]. Houston and associates found that the majority of doctors received daily 1–5 email messages from their patients [ 9 ]. According to Sittig, physicians received daily in average 2.6 messages, and monthly an average of 40 per 140 visits [ 10 ]. Gaster and colleagues noted that physicians on average received 7.7 email messages in a month from their patients. Physicians in university clinics were most active in email use, while those in municipal primary health care were least active. Of physicians 58% reported in the questionnaire that the email contacts with patients were for the most part not registered in patient records [ 4 ]. Among Finnish citizens of working age young adults are the most active users of email and Internet [ 1 , 11 ]. University students use these electronic net services even more actively than the young adult population as a whole. In a study from 2002 99% of students reported using email and Internet at least weekly [ 12 ]. All students have an email address at the university and their health providers at the FSHS can be reached by email. The student health care system can be seen as an appropriate setting to use email for patient communication [ 13 ]. The students represent a young, well educated, relatively healthy part of population which has been identified to be the most active to use email in patient-doctor communication [ 2 , 5 , 14 ]. The Finnish Student Health Service The Finnish Student Health Service (FSHS) provides primary health care services to approximately 140.000 university students in Finland. The FSHS has health stations in 16 university cities. Services include health promotion, consultations with general practitioners and with other clinical specialists, mental health care, and dental care. Since 1993 FSHS has provided health counseling in Internet. Since 1999 all physicians have had an email account at their disposal in health stations and an email address of type: firstname.surname@yths.fi . Principles of communication by email with FSHS' employees and of other forms of electronic services (email service for cancellation of appointments, health counseling service on the Internet, and email service for feedback) are available at the FSHS' website. The Social Insurance Institution, the university cities, the State of Finland, and the students themselves finance FSHS services. Students pay an annual obligatory health care fee as a part of the Student Union's membership fee. There is no other fee for preventive services, visits to general practitioner or public health nurse, and laboratory or X-ray examinations prescribed during these visits. Use of Internet services is also free of charge. The FSHS employs 560 persons and 63% of the physicians are general practitioners. In this paper general practitioners also include specialists of general practice/family medicine, whereas "specialists" refers to clinical specialists other than psychiatrists or oral surgeons. Aims of the study The aim of the study was to seek answers to the following questions: 1. How actively did physicians at student health care use email in communication with their patients? 2. How much did they use email compared to phone calls and patient visits? 3. Who were the active doctors using email with patients? 4. What proportion of visits and phone calls could be candidates for substitution by email communication? 5. Did the volume of visits, phone calls and email messages documented in the EPR of the FSHS during the study period differ from that of visits, phone calls and email messages registered in the study? Methods All physicians (n = 82) in the FSHS' functionary register in April 2003 received a questionnaire by email. We excluded six physicians, who were not any more working for the FSHS and took exception to the two authors. The actual number of survey population was 74. The questionnaire (see Additional file 1 ) included background factors and a registration (in form of daily tally) of numbers of patient contacts, phone calls and email messages over one working week. Respondents were also asked to assess the number of visits and calls replaceable by email, and the number of email messages including a request, which could not be fulfilled without face-to-face contact. Also doctors' attitudes toward email use for patient communication were asked. The first mailing of questionnaire took place 28.4.2003 and a reminder was sent 5.5.2003. Recipients were asked to print the survey form, fill it in by hand, and return it by internal mail. Overall 52 out of 74 (70%) physicians returned a completed survey. Respondents were grouped according to age, location, speciality licence, and type of employment (Table 1 ). Facts on years of birth were collected from the register book "Finland's Doctors 2002" and other background factors from the FSHS' functionary register. Table 1 Background variables of all physicians at the FSHS and of the respondents. All physicians Respondents (n = 76) (n = 52) % % Gender Male 34 29 Female 66 71 Age (years) 45 or under 33 31 46–55 39 42 56 or over 28 27 Location Capital city region 38 42 Turku and Tampere 28 27 Other 1) 34 31 Speciality licence General practitioner 63 69 Specialist 37 31 Type of employment Permanent 68 85 Non-permanent 2) 32 15 Distribution (%) of background variables of all physicians at the Finnish Student Health Service and of the respondents of the survey. 1) Small university towns 2) Vicars and fee-based working doctors All physicians at the FSHS utilize EPR (Medicus ® ). We collected the numbers of patient contacts documented at Medicus ® for the study period using the statistical software Cognos ® . We entered data using Microsoft Excel ® software and performed statistical analysis using StatsDirect Statistical ® (3,2,7 -version) software. Statistical analyses were conducted using the proportion test for two independent groups, the χ 2 test, Fisher's exact test, the unpaired t-test, the Mann-Whitney test and the Kruskal-Wallis test. All tests were made two-sided and p-values below .05 were regarded as statistically significant. Results Respondents Of all respondents 29% were men and 71% women (Table 1 ). The respondents and all doctors at the FSHS were compared according to the background variables. The doctors who answered represented well the overall body of physicians working in the FSHS. The mean age of male respondents was 52.8 (range 34–65) and of female 48.5 years (range 29–65). This was in the same range as the mean age of all doctors at the FSHS (men 51.3 and women 48.3 years). Activity of using email In one working week 79% of doctors had used email and 98% the phone for patient communication. Respondents reported 2296 patient visits, 948 phone calls and 449 email contacts. They had on average 8.6 email contacts and 18.2 phone calls per week with their patients (Table 2 ). They reported a mean percentage for "email per visit" of 20%, and phone calls in proportion to visits ("phone call per visit") averaging 40%. Eleven doctors (21%) reported more email contacts than phone calls. Table 2 Characteristics of the use of email. Characteristic Mean Median Minimum Maximum n 1) n 1) n 1) n 1) Type of patient contact Visit 44.2 46.5 7 78 Phone call 18.2 18.0 0 59 Email 8.6 3.5 0 96 % % % % Proportion of the respective contact types Phone call / visit 40 36 0 100 Email / visit 20 12 0 185 Email / phone call 55 22 0 600 Characteristics of the use of email in one week (5.5. – 9.5.2003) among physicians (n = 52) at the Finnish Student Health Service. 1) Number of contacts We tested the variables showing email usage and phone calls with respect to background factors. There were no statistically significant differences by gender, age group, speciality licence or type of employment. Of physicians in the capital city area 41% reported more email contacts than phone calls. Among doctors working in Turku and Tampere the proportion was 7.1%, and among those working in small towns 6.3%. The difference between capital city region and other locations was statistically significant (p = .015). Respondents' general attitude toward email for patient communication was evaluated by the statement "Email contacts with patients facilitate my work." The group of "positives" was formed of the 56% of respondents who on a five step Likert scale replied: "same opinion" or "nearly same opinion." The "positives" reported more email contacts than others (email per visit: median18% versus median 3%, p < .001). All eleven who had reported more email contacts than phone calls belonged to the "positive" group. Patient visits and phone calls replaceable by email Doctors estimated that 2% (57/2296) of patient visits could have been replaced by email. Of phone calls 21% (196/948) could have been substituted with email. Respondents estimated that 10% (45/449) of patients' email messages required a personal consultation. Documentation in the EPR The number of visits registered in the survey did not differ from the presumed number documented in the EPR (Table 3 .). The difference noted between registered and presumed email contacts shows that 73% of email contacts were not entered in the EPR. Table 3 Patient contacts documented in the EPR and registered in the survey. Type of patient contact Number of patient contacts Statistical significance of the difference between expected and registered contacts 2 ) All physicians Respondents Documented data in electronic patient record (Medicus R ) Expected 1) Registered n n n p Visit 3098 2107 2296 0.237 Phone call 1115 758 948 < 0.001 Email 178 121 449 < 0.001 Patient contacts documented by all physicians at the Finnish Student Health Service (n = 76) in the electronic patient record (Medicus R ) and registered by respondents (n = 52) in a survey over one working week (5.5. – 9.5.2003), as well as the statistical significance of the difference in contact numbers. 1) Based on the proportion of respondents among all physicians 2) Proportion test for two independent groups Discussion Even if university students do not represent the whole population, they can act as "pilot population" representing adults of working age of a future information society. Our study population – doctors taking care of students – was small with only 52 respondents. Thus the results of the study cannot be indiscriminately generalized. Because of the small study population comparison of the subgroups may not be reliable. Although the study group was small, it well represented all the doctors at the FSHS, and the response rate was high. A further strength was that we compared the number of patient contacts documented in the questionnaires during the study week to the statistical data of contact numbers from the EPR at the same time. In other studies no corresponding comparison has been made. The doctors were asked to keep a daily tally of visits, phone calls and email messages, and to evaluate how many visits or phone calls could have been replaced by email. Many doctors undoubtedly did this simultaneously with patient work. Some doctors might have been in a hurry, they probably supplemented the questionnaire at the end of the day. To achieve a more accurate evaluation of visits and phone calls replaceable by email, a continuous assessment (visit by visit, phone call by phone call) could have been stressed even more in the instructions. The doctors at the FSHS do not have a specific electronic communication system focused on patient communication. They use their general, unprotected email system also to communicate with patients. A specific communication system used only for patient communication would enable an automatic collection of the patient communication data and create a more accurate database than our data collection method. Katz and colleagues have made the only controlled and randomized study concerning physicians' use of email [ 5 ]. Our own results on the average number of doctors' email contacts and email usage are of the same magnitude as those referred to above and in other recent studies in the USA [ 4 , 9 , 10 ]. In the present study 79% of respondents used email for patient communication. The proportion of those who had used email is clearly larger than in older studies, and at the same level as reported in recent international studies [ 2 - 6 ]. Our study revealed individual differences in the use of email in patient work. Differences in physicians' activity in using email have previously been reported in only one study [ 4 ]. Deriving of our small study group only especially glaring association between subgroups of respondents could be verified. Our findings still support the results published by Gaster and associates. Physicians working in the capital area were more active email users than their colleagues elsewhere in Finland. Physicians reckoned that email could replace only 2% of visits. This confirms Sittig's evaluation in 2003 that email could possibly cover a small percentage of visits [ 10 ]. Increasing the use of email can thus not considerably reduce the number of patient visits. On the other hand it could make physicians' crowded telephone hours easier [ 15 ]. When we compared contacts in the EPR with contacts registered daily on the questionnaires we found that the majority of email contacts were not registered in the EPR. This finding is supported by Gaster and colleagues who asked physicians themselves to describe how often they usually registered email contacts in patient records [ 4 ]. FSHS provides specific electronic services for focused issues (email service for cancellation of appointments, health counseling service on the Internet, and email service for feedback). Principles of recommended issues to use email between health providers and patients are available for students at FSHS' websites. We have had a presumption that email messages between FSHS' physicians and their patients mainly handle patient care. Nyström's congress report from 2004 supports our presumption. He explored 139 email messages from 103 individual patients at his GP practice at the FSHS and noticed that 77 % of email messages handled medical tests, and 16 % handled follow-ups of symptoms or illnesses [ 16 ]. Thus the information in email communication should be entered in patient records. All university students in Finland have access to Internet and email at their universities. Use of email as communication method in health care does not in their case cause inequalities in health. A general tendency in the societies to provide also health services widely in electronic form (in Internet or by email) can contribute to inequalities for those who are not able to use modern technologies [ 13 , 17 ]. Conclusions Doctors at the FSHS had an average of 8.6 email contacts with their patients during one week. The proportion of email contacts per visit was 20%. Physicians estimated that email contacts could substitute 2% of patients' visits and 21% of phone calls. Of email contacts 73% were not documented in the EPR. Our study indicates that email communication really constitutes a part of patient work. This should be taken into account in planning working time and daily timetables. Use of software not integrated with the EPR increases the physician's registering load and currently involves extra work. It is not possible to confirm the patient's identity reliably using two separate systems. A system allowing retrieval of patient's identity safely and with no need to register separately the email communication in the EPR would promote the patient's adequate treatment and reduce the physician's medico-legal risks. There is a need for a larger study on email utilization between patient and physician which better covers the medical profession. The consumer point of view should also be better taken into account. A content analysis of email messages for patient communication combined with assessments of email documentation in the EPR could have strengthened present study regarding the importance of email registration in patient records. In Finland the Ministry of Social Affairs and Health has instigated a major project to safeguard health care [ 18 ]. One part of this programme demands that the whole public health care field should be using EPR by 2007 [ 19 ]. More research data are needed on electronic communication with patients and on users' experiences. The future EPRs should include a purposeful, safe and secure means of patient communication. Competing interests The author(s) declare that they have no competing interests. Authors' contributions JC participated in the design of the study, coordinated it, keyed data into computer, performed the statistical analysis and drafted the manuscript with co-authors. MN conceived of the study, participated in the design of the study and wrote parts of manuscript in English. IV participated in the design of study and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Questionnaire Click here for file

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Factors associated with psychotropic drug use among community-dwelling older persons: A review of empirical studies

Background In the many descriptive studies on prescribed psychotropic drug use by community-dwelling older persons, several sociodemographic and other factors associated with drug use receive inconsistent support. Method Empirical reports with data on at least benzodiazepine or antidepressant drug use in samples of older persons published between 1990 and 2001 (n = 32) were identified from major databases and analyzed to determine which factors are most frequently associated with psychotropic drug use in multivariate analyses. Methodological aspects were also examined. Results Most reports used probability samples of users and non-users and employed cross-sectional designs. Among variables considered in 5 or more reports, race, proximity to health centers, medical consultations, sleep complaints, and health perception were virtually always associated to drug use. Gender, mental health, and physical health status were associated in about two-thirds of reports. Associations with age, marital status, medication coverage, socioeconomic status, and social support were usually not observed. Conclusions The large variety of methods to operationalize drug use, mental health status, and social support probably affected the magnitude of observed relationships. Employing longitudinal designs and distinguishing short-term from long-term use, focusing on samples of drug users exclusively, defining drug use and drug classes more uniformly, and utilizing measures of psychological well-being rather than only of distress, might clarify the nature of observed associations and the direction of causality. Few studies tested specific hypotheses. Most studies focused on individual characteristics of respondents, neglecting the potential contribution of health care professionals to the phenomenon of psychotropic drug use among seniors.

Background The use of prescribed psychotropic drugs by older persons has been a subject of interest for several decades [ 1 , 2 ]. Psychotropic drugs are defined as substances that act directly on the central nervous system, affecting mood, cognition, and behavior, and usually include anxiolytics, sedatives and hypnotics, antidepressants, neuroleptics, anticonvulsants, and stimulants. The topic continues to draw researchers' attention for several reasons, including (1) the high prevalence of older users (especially of benzodiazepines) and their typically long-term consumption, (2) their special vulnerability to drug induced iatrogenesis, (3) the discrepancy between rates of mental disorder and rates of drug use among older people, and (4) inappropriate prescribing. The prevalence of psychotropic drug use among community-dwelling older persons (usually defined as those 65 years and older) varies from about 20% to 48% [ 3 - 5 ]. More than half of them take psychotropics for six months or longer, and are therefore considered long-term users [ 4 , 6 , 7 ]. For example, 69% of Canadian elders using benzodiazepines have done so for at least a year [ 3 ]. Long-term use is contraindicated because benzodiazepines lack effectiveness beyond a few weeks or months of sustained use for their principal indications, the relief of insomnia and anxiety [ 8 - 17 ]. Since aging increases the likelihood of drug accumulation and intoxication, older persons are particularly vulnerable to adverse effects from psychotropic drugs [ 18 ]. Notable harmful consequences of psychotropic drug use include memory impairment, psychomotor slowing, delirium, falls with a risk of hip fracture, automobile accidents, and psychiatric hospitalizations [ 19 - 21 ]. Although more psychotropic drug users are found among older persons than any other age group, the prevalence of mental disorders appears to be lower among older than younger adults. This has been shown for major depression, anxiety disorders and sleep disorders [ 6 , 22 - 30 ]. Other observations, such as the small rate of hospital admissions for psychiatric reasons among older persons (1.1%) [ 4 ] compared to their high rate of psychotropic drug use relative to younger adults' rate, confirm these findings. The inappropriate prescription of psychotropic drugs may account partially for the discrepancy between low levels of distress and high levels of drug use among older persons. Inappropriate prescriptions include questionable drug combinations (such as two benzodiazepines), excessive treatment duration, and drugs contraindicated for use by older people (such as long half-life benzodiazepines). Authors estimate that one-fifth to one-half of psychotropic drug prescriptions to the elderly are "inappropriate" [ 4 , 16 , 31 , 32 ]. These findings suggest that older persons' psychological well-being is not the principal determinant of their psychotropic drug use. In the present article, we review recent empirical studies in order to identify other factors that may account for psychotropic drug use among older persons. A large body of work bears on this age group and their use of psychotropic and other drugs [ 33 , 34 ]. Earlier reviews examined prevalence rates [ 35 - 39 ], the issues of long-term use [ 40 ] and dependence [ 41 , 42 ], as well as various models of drug use [ 43 , 44 ]. However, to our knowledge none focuses on community-dwelling psychotropic drug users. Consequently, the present review critically examines various factors associated with such drug use among this population. Each factor is discussed in terms of the empirical support it has received, of hypotheses put forth to explain its association with drug use, and of suggestions for future research. Methods Reports were selected on April 20, 2001, from the following bibliographic databases: MedLine, Cumulative Index to Nursing & Allied Health Literature , Psychlit , Eric and Sociological Abstracts for the years 1990 to 2001. Various keywords were used. Selected reports had to: (1)constitute either a peer-reviewed journal article or a government publication published in English or French; (2) present specific retrievable empirical results concerning older persons, regardless of other age groups studied (some reports [ 16 , 39 , 45 ] were rejected because of this criterion); (3) report on non-institutionalized, community-dwelling participants at the time of the study; and (4) provide specific results on at least benzodiazepine or antidepressant drug use, regardless of other drug classes studied (some reports [ 29 , 46 , 47 ] were rejected because of this criterion). A total of 61citations met these criteria in the databases. Eliminating overlaps left only 32 separate reports, all of which were retrieved and are included in this review. Their reference lists were also consulted, but no other study was identified by this mean. The 32 publications report on 30 different studies conducted in ten different countries. Unless indicated otherwise, each report is treated as a separate study. Most reports originated from the United States (31%) and Canada (28%), followed by France (12.5%), Sweden (9.5%), and 3% each (one report) from Australia, Austria, Ireland, the Netherlands, Spain, and the United Kingdom (Table 1 [see additional file 1 ]). Of 26 reports that disclosed a source of funding, 16 (61.5%) identified government agencies, 6 (23%) government-industry or government-foundation partnerships, and 4 (15.5%) industry. Most studies (62%) collected their data during the 1990s, but several did so during the 1980s (34%) and 1970s (3%). As far as could be determined, the median time from end of data collection to publication was 6 years (range: 1 – 12 years). Most reports (24, 75%) presented multivariate data analyses, usually multivariate logistic regression, where the independent contribution of various variables to the variance in psychotropic drug use could be ascertained while controlling for the contribution of other variables. Although variables with statistically significant associations (p < .05) to drug use that remained in the regression equation were often considered "predictors," strictly speaking only an association is demonstrated in this fashion, and the direction of causality can rarely be established. This is especially so when a theoretical model is not specified prior to the data analysis, as was the case in the vast majority of reports. The eight remaining studies conducted bivariate analyses solely, and most examined three or less variables. In the following review, associations of sociodemographic factors and life conditions with psychotropic drug use are examined first. A factor was deemed well supported by the review studies when 70% of the results from all studies that considered this factor were statistically significant and pointed in the same direction. Second, methodological and conceptual issues relevant to the study of psychotropic drug use among the elderly, and salient in the reviewed studies, are discussed. Results Psychotropic drug use and sociodemographic characteristics Prevalence of psychotropic drug use Weighted average prevalence rates of psychotropic drug use were estimated from all reports having collected data from probability samples or entire populations (n = 22). In longitudinal studies, data from the last year of the study were used. Average prevalence of any psychotropic drug use from 9 studies was 29.0% (range 11.8% – 42.5%). For drugs identified in the reports as benzodiazepines, minor tranquilizers, anxiolytics, or sedative-hypnotics, the average prevalence in 13 studies was 21.5% (range 6% – 43.8%). In 11 studies with data on antidepressants, the average prevalence was 6.9% (range 2.3% – 14%). Finally, for neuroleptics, it was 3.1% (6 studies, range 1% to 6%). Age Older people are more likely than any other age group to use any type of medication [ 29 ]. However, it has been observed that medication use decreases in those over 75 years [ 4 ]. Some suggest that this occurs because: doctors exercise more caution when prescribing to the oldest old [ 48 , 49 ], survivors into advanced age are healthier and thus use fewer medications [ 4 , 50 ], the elderly face fewer stressful events [ 50 ]. The observation of a decline in psychotropic drug use with very advancing age is not universal [ 51 ]: Blazer et al. [ 6 ] and Mamdani et al.[ 49 ] found that it reaches its peak prevalence among those older than 85 years. As Table 2 [see additional file 2 ] shows, 22 studies carried out 23 tests of the association between age and drug use, but only 8 (35%) of the results showed an age-specific trend (5 found an increase and 3 a decrease). This low percentage prevents firm conclusions about any age trend in regard to psychotropic drug use among older people. Gender Over the past three decades, numerous hypotheses have been proposed to explain the higher prevalence of psychotropic drug use among women than men: women are more inclined to reveal their emotional problems to their doctor[ 35 , 36 , 52 - 55 ], to request prescriptions explicitly [ 36 , 54 , 55 ], to hold more positive views of psychotropic drugs [ 50 ]. Some authors have suggested that men prefer to use alcohol rather than prescribed drugs to deal with emotional problems [ 56 , 57 ]. Graham and colleagues [ 42 ] failed to support this last hypothesis in a sample of 826 older persons. Other authors suggest that since women live longer than men, they experience more effects of aging, losses and health problems, all of which increase their likelihood of using a psychotropic [ 37 , 49 , 55 , 58 ]. Physicians might be more willing to prescribe a psychotropic drug to a woman than to a man [ 49 , 54 , 55 , 59 ]. Also, women visit physicians more often, increasing their chance of receiving a prescription [ 36 , 55 , 57 ]. However, in studies using population-wide databases, Brown et al.[ 60 ], Jorm et al.[ 55 ], and Weyerer and Dilling [ 45 ] controlled both the number of health problems and physician visits and showed that women still used more psychotropic drugs than men. Kirby et al. [ 53 ] found the same result when controlling for mental health status. Contradictory results are reported by Mayer-Oakes et al., [ 61 ] and Swartz et al., [ 62 ] who showed the use of benzodiazepines to be associated not to gender but to physical health status, poorer in women than in men. In this review, 73% of the relevant studies support the established finding that women are more likely to use psychotropic drugs than men. In two studies, the relationship with gender holds only among those aged between 65 and 74 years, and in a third, the results are opposite. However, the studies shed little light on reasons for this disparity. Only two studies identified as a secondary objective to look at the gender issue in relation to psychotropic drug use [ 42 , 49 ]. A few studies tested hypotheses to examine the role of physical health status and mental health status relative to gender among seniors, but none received consistent support [ 42 , 45 , 53 , 55 , 60 - 62 ]. No other hypothesis was directly tested in this body of studies. Some authors borrowed hypotheses from studies with middle-aged adults to discuss their results [ 49 , 53 , 55 , 63 ]. Authors in other studies which found a statistically significant relationship between gender and psychotropic drug use did not discuss or interpret the association [ 6 , 48 , 55 , 60 , 64 - 74 ]. In sum, many studies confirm that more women than men use psychotropic drugs, but no single compelling explanation for this difference among the aged emerges from this body of studies. Race The role of culture, ethnicity, and race has received little attention so far in the literature on older persons' psychotropic drug use. Explaining cultural, ethnic, or racial disparities in use of health services is a complex endeavor, requiring analysis of predisposing, structural, access, and other variables in interaction [ 75 - 78 ]. In this review, two studies from Canada compared likelihood of use among French and English speakers, one finding an association with French speakers, albeit in a non-probability sample [ 79 ]. In addition, six studies from the United States compared the prevalence rate of psychotropic drug use between Whites and African-Americans. All studies but one controlled for income or education, and all found that Whites are significantly more likely to use psychotropic drugs. These results concur with many findings showing differential use of health services along racial and ethnic lines in the United States [ 75 ]. Interpretations of the race-specific findings from these six studies focused mostly on professional-and individual-level determinants. Brown et al. [ 60 ] suggested that doctors prescribe fewer psychotropic drugs to older African-Americans by prudence, since the former are more sensitive to some drug effects. Other authors proposed that different prescription patterns result from differences in the expression of psychological distress [ 62 , 80 ] or from the lower rate of depression among African-Americans [ 6 ]. The smaller proportion of African-Americans and other minorities in the United States with private health insurance also may play a role, especially with regard to newer, more expensive drugs [ 81 ]. In that country, more studies are needed to test these hypotheses and others involving structural and access variables, also with other significant minority groups such as Latinos. Beyond racial or ethnic status, studies are needed to understand whether distinct cultural attitudes independently predict psychotropic drug use among older people after various sociodemographic and structural variables are controlled. Marital status It has been suggested that older widows would be more likely to use psychotropic drugs because of the distress of bereavement [ 82 ]. Eleven of 32 studies examined psychotropic drug use in relation to marital status, but only 4 (36%) confirmed an association between drug use and not being married (single, widowed, divorced, or separated). A spouse's death, especially if preceded by long illness, is likely to represent a significant stressful event accompanied by insomnia, anxiety or depression for any person and this can lead to psychotropic drug use. However, strictly speaking, stressful events or psychological distress rather than marital status as such would be implicated, and perhaps only on a relatively short-term basis. In summary, marital status so far is not revealed as a significant factor accounting for psychotropic drug use in older people. Socioeconomic status Living in deprived environments and having an unskilled occupation, low income and little formal education are variables associated with psychological distress among adults, and it might be assumed that the same would hold among older people. However, the studies reviewed fail to support this association. Socioeconomic status (SES) is typically operationalized by education, income, and occupation. As Table 2 [see additional file 1 ] shows, 13 studies examined one of more of these variables. Lower education was significantly associated with drug use in only 3 of 11 results (27%). Lower income was significantly associated with drug use in 3 of 7 results (43%). Two studies examining occupational status before retirement found an association: one found that the self-employed were less likely to use psychotropics [ 83 ], the other, using a non-probability sample, found more blue-collar workers among users [ 79 ]. In sum, most studies did not support the impact of lower educational level or lower income on psychotropic drug use among older persons. The sparse findings regarding occupation are difficult to interpret. While the importance of SES as a predisposing or mediating influence on drug use might be more established in general population studies, it may be less relevant in the older age group because of a lower rate of psychological distress and greater access to income security plans and retirement pensions that prevent extreme poverty. Insurance status In the United States and until recently in Canada, older persons benefited from Medicare or provincial health insurance plans without medication coverage. Insurance coverage has been shown to influence psychotropic drug use by the elderly [ 84 ]. Five studies in this review (four from the USA and one from Canada) examined this issue in six different tests but only two (33%) supported a relationship between having insurance coverage for medication and being more likely to use psychotropic drugs. Besides the fact that the most frequent psychotropic drugs used by older persons during the time period covered by the 32 studies in this review (for example, benzodiazepines like lorazepam or tricyclic antidepressants) were available in inexpensive generic versions, no compelling hypothesis exists to account for these results. However, newer psychotropics, such as selective serotonin reuptake inhibitors which reached peak usage during the late 1990s, have been marketed at much higher prices, and the impact of insurance coverage on their use might be shown to differ in future studies. Proximity to health centers No study examined meso-level variables related to SES, such as census-tract or neighborhood poverty, except proximity to health centers. The use of health services, including medications, may be increased by proximity to such services [ 85 ]. Close or easy availability of health services augments medical consultations (and consequently the request of medication or its prescription). This hypothesis is supported in six of seven studies (85%) in the present review. Life conditions Stressful events Stressful events may increase people's likelihood of using psychotropic drugs. Only three studies examined this relationship, each reporting a different result: a positive association [ 65 ], a negative association [ 50 ], and no association [ 79 ]. The precise role of stressful events within a model of psychotropic drug use by older persons clearly needs to be elucidated. To explain the mixed results, future research might consider the transformation of stressors over time: stressful events may impact significantly on the initiation of psychotropic drug use, but may recede to a negligible level in long-term consumption. A longitudinal design is needed to examine this relationship more adequately, but unfortunately none of the 12 longitudinal studies in this review explored stressful events in relation to psychotropic drug use. Illnesses and other medications A recent qualitative synthesis of the literature concludes that the pathway to psychotropic drug use among older people typically includes the presence of organic disease [ 86 ] (and loneliness, see ahead). Since elderly people suffer from more diseases than younger people, they might use more psychotropic and of course non-psychotropic medications. In another vein, some anxious elderly users of psychotropic drugs might worry excessively about their health and be more likely to consult physicians and receive other medications [ 85 ]. In this review, nine of 16 studies (56%) found a significant association between the presence or the number of physical illnesses and psychotropic drug use. In their conceptual model, Gustaffson et al. [ 69 ] suggest that affliction by a new disease constitutes a stressful event that worsens an older person's mental health status, leading to the need (personally or professionally perceived) for a psychotropic drug. Nonetheless, the results overall are equivocal, perhaps because researchers neglected to consider both the type and the duration of health problems. For example, living with high blood pressure and being struck with congestive heart failure are dissimilar experiences. Typically, researchers counted only the presence or number of illnesses and did not distinguish between individuals who have been living with a disease for a long period of time and those recently experiencing it. As actually measured, the illness factor did not clearly discriminate between older users and non-users of psychotropic drugs. Taking into account both the nature and the duration of illnesses in future studies might better elucidate the relationship between health status and psychotropic drug use. Four of six studies (60%) examining the relationship between psychotropic drug use and use of other medications found a significantly positive association, but the direction of causality remains unclear. Health perception Health perception–the self-evaluation of one own's health–has been shown to be more strongly associated with psychotropic drug use than actual diagnosis of disease [ 33 ]. Some researchers see here an indirect relationship, where poor health perception negatively influences the mental health status of a person, which in turn leads to the request for psychotropic drugs [ 65 , 69 ]. Previous studies support the correlation between mental health status and health perception [ 87 - 90 ]. In this review, seven of eight studies (87.5%) examining the association between health perception and drug use found a positive relationship. However, two thirds of the reviewed studies were cross-sectional, preventing conclusions about cause-effect relationship, and typically more than 80% of the psychotropic drugs used by their subjects were benzodiazepines. This raises the possibility that some subjects had poor health perception as a result of benzodiazepine consumption. Iatrogenic effects of long term benzodiazepine use may worsen health and functional capacity, hence health perception [ 73 , 91 ]. The present findings confirm that health perception has a place within a model of psychotropic drug use among the elderly, yet leave its precise role unclear. A longitudinal study would help to elucidate this role. Social support It has previously been proposed that low social support is associated with psychotropic drug use among older people [ 92 , 93 ]. In 10 studies here reviewed, various scales operationalized constructs identified as social support, social network, social relationships, family relationships, and social isolation. A variable of living alone or with others was included in two of these studies, and subjective reports of loneliness in two studies. However, in 13 tests of the association between some of these variables and drug use, 9 results (69%) failed to observe any significant relationship. All five tests of association with "social support" and all three tests of association with "social relationships" found no relationship with drug use. The single association of "social relationships" was positive, as was one of two of "living alone." Overall, results are counter-intuitive since one would expect that without significant social support to aid in time of difficulties, a person might be more likely to seek medical help, increasing the chances of receiving a drug prescription. Either social support is a minor strand in the tapestry of psychotropic drug use, or the concept of social support may be inadequately operationalized by standard scales. These may need to measure, beyond frequency of social contacts and residential status, subjective judgments of loneliness and of opportunities for social intimacy. Whereas a summed scale score indicating "social isolation" was not associated with benzodiazepine use in one study [ 61 ], in both studies where the older person's feeling of "loneliness" as such was elicited, its association with psychotropic drug use was found to be significant after controlling for other variables, including "social support" scores [ 69 , 70 ]. Rather than the presence or extent of social support, a subjective feeling of loneliness may influence, directly or indirectly, psychotropic drug use [ 86 ]. Mental health status The association between a diagnosis or rating of mental disorder or psychological distress and the use of psychotropic drugs is evidently anticipated. In 18 studies, 22 tests of this association were carried out, and it was significant in 17 (77%). An intriguing observation is that two of the four studies that failed to support the association included only the antidepressant drug class [ 67 , 71 ]. While the association between mental health and psychotropic drug consumption is well supported, it is worth noting that the magnitude of the association varies considerably across studies. The following two pairs of longitudinal and cross-sectional large-sample studies using multivariate logistic regression analysis illustrate this variability: benzodiazepine users were 2.13 times more likely than non-users to report depressive symptoms at 10-year follow-up [ 6 ], and 6.7 more likely to report emotional or nervous problems [ 63 ]. Psychotropic drug users were 2.78 times more likely to report depressive symptoms at 6-year follow-up [ 68 ], and 4 times more likely to report anxious or depressive symptoms [ 94 ] (Table 3 ). We discuss ahead how this variability may stem from methodological differences, especially in the measurement of the key variables in the associations. Table 3 The association between mental health variables and psychotropic drug use Study Results 65 Allard et al. (1995), n = 500 In bivariate analysis, drug use negatively correlated with morale (r = -0.32, p < 0.001) 6 Blazer et al. (2000), n = 4,162 In multivariate logistic regression, benzodiazepine use significantly associated with depressive symptoms at 10-year follow-up (OR: 2.13, p < 0.05) 68 Dealberto et al. (1997), n = 2,812 In multivariate logistic regression, drug use significantly associated with depressive symptoms at 6-year follow-up (OR: 2.78, p < 0.001) 69 Gustafsson et al. (1996), n = 421 In LISREL model, drug use positively correlated (0.63) with poor mental health (chi 2 = 3.38, p = 0.33) 94 Paterniti et al. (1998), n = 1,389 In multivariate logistic regression, drug users 4 times more likely to report anxious and depressive symptoms than non-users (OR: 4.0, CI: 2.5–6.5) 63 Gleason et al. (1998), n = 5,181 In multivariate logistic regression, benzodiazepine users 7 times more likely to report emotional or nervous problems than non-users (OR: 6.66, CI 5.1, 8.7) 74 Taylor et al. (1998), n = 5,222 Depressive symptoms multiply by 3 the likelihood of using hypnotics and by 5.1 the likelihood of using anxiolytics. Anxious symptoms multiply by 4.2 the likelihood of using hypnotics and by 3 of using anxiolytics. Sleep In the general population, Ohayon and Caulet [ 28 ] have clearly shown that a sleep disorder, such as primary insomnia diagnosed according to DSM-III-R criteria [ 95 ], is associated with the use of psychotropic drugs. These authors also noted that complaints of poor or inadequate sleep were associated with drug use. In the present review, all five studies (100%) that evaluated this latter association among older persons supported it. Although as mentioned earlier they do not report more sleep problems than younger persons, the presence of sleep complaints appears to play an important role in their psychotropic drug use. Medical consultations The number of annual visits to a doctor's office has an obvious influence on the number of prescriptions since it has been shown that up to 75% of visits by older people end up with a prescription [ 96 , 97 ]. All seven studies (100%) having examined this factor in this review supported its association with psychotropic drug use. Because no drug can be prescribed without the active participation of a physician, the physician's role is undoubtedly critical. However, it might differ over time. It has been previously suggested [ 98 ] that the first prescription of a benzodiazepine drug and the renewal prescription are two separate phenomena: the doctor might be more directive during the visit leading to the first prescription, whereas the older patient might be more directive to ensure its renewal. Thus, the physician's role in psychotropic drug use among community-dwelling older persons might best be modelled according to the duration of use. In previous research, several characteristics of physicians, notably a high number of consultations, have been significantly associated with the likelihood of prescribing psychotropics to older patients [ 99 ]. Summary of findings on sociodemographic factors and life conditions Among the 16 factors identified in this review of 32 empirical reports, only the variables of language and stressful events were examined in less than five different studies. Gender, age, and mental health status were most frequently examined. The variables of race, medical consultations, proximity to health centers, sleep complaints and health perception are significantly associated with drug use in all or almost all studies incorporating them. Gender and mental health status are associated with drug use in over 70% of studies, while physical illness and number of medications are associated in slightly over half of relevant studies. However, significant associations with age, marital status, socioeconomic status, and social support are observed in only 27% to 36% of studies in which these factors are examined. Methodological issues in the study of psychotropic drug use by the elderly Conflicting results could be explained partially by methodological and conceptual characteristics of many of the studies reviewed. The following discussion focuses on study design, sample, concept definitions and measurements (particularly of mental health status and psychotropic drug use), as well as distinction between short-and long-term psychotropic drug use. Table 1 [see additional file 1 ] lists these and other methodological characteristics of the studies reviewed, as well as summaries of findings on prevalence of psychotropic drug use. Design Descriptive methods (100%) using cross-sectional data (62%) predominate in this body of recent reports of psychotropic drug use in the elderly. Cross-sectional data are vulnerable to sample bias, especially when respondents are not randomly selected, as was the case in 9 of 30 (30%) studies. Given the greater costs involved, the proportion of longitudinal research (38%) seems respectable. Possibly because of cost factors, these studies examined fewer variables in relation to psychotropic drug use. However, besides highlighting the phenomenon of long-term use of psychotropic drugs by the elderly [ 55 ], they provided clear demonstrations of the deleterious effect of psychotropic drug consumption on cognitive capacities [ 19 ] and of how nursing home admission increases psychotropic drug prescriptions [ 5 ], for example. Despite their greater cost and demanding logistics, longitudinal designs are essential to understand more accurately how and why an older person begins, ceases, continues, and modulates psychotropic drug use. Finally, the absence of qualitative studies, which usually allow for a better grasp of elderly users' own perspectives on their use, is noteworthy; most studies so far have reflected only the researchers' points of view. Samples Virtually all samples (29 in the 30 distinct studies) were composed of older consumers and non-consumers of psychotropic drugs. Comparisons between these two groups allow researchers to identify what differentiates users from non-users of psychotropic drugs. However, one notices from this review that researchers do not always succeed. One possible reason may be that short-and long-term users are combined in one group. Indeed, in almost every study it appears that investigators are comparing long-term users with non users. About 20% to 30% of community-dwelling older persons use psychotropic drugs [ 3 , 84 ], with over two-thirds having been users for more than a year, and over one half for more than one year. Among the very old, up to 93% have consumed for more than a year [ 100 ]. Thus, the characteristics of users with less than six months of use are not well known. In one longitudinal study, the one factor that most accurately distinguished long-term users of psychotropic drugs from non-users was having been a user of the drug at the first measurement three years earlier [ 68 ]. The researchers found that older users were 15 times more likely to be users 3 years later – 71 times more likely in the case of antidepressant drugs – whereas the fact of being depressed increased the likelihood of drug use at follow-up by 4.7 times. Thus, if most psychotropic drug use among the elderly transforms into long-term use, the process of transformation itself requires more observation and explanation, again by means of longitudinal research. Immediate problems leading to the first prescription of a psychotropic drug, such as psychological distress or insomnia, might have little relevance among long-term users. Possibly, drug dependence has developed among some long-term users and sustains long-term consumption [ 41 ]. The six-month cut-off period habitually used to distinguish short-from long-term consumption in studies may be too long if dependence is taken into account, as patterns of physiological and psychological dependence may already have become established within such a time frame. However, current studies have not taken into account the dependence issue. For instance, one can wonder how withdrawal symptoms are influencing beginning and long-term users of psychotropic drugs. Fear of withdrawal symptoms might explain long-term use of a certain percentage of elderly users. In one previous study, 71% of middle-aged psychotropic drug users wanted to stop consuming these medications, but half of them feared stopping because of withdrawal symptoms [ 47 ]. It seems desirable to increase knowledge on this aspect of the phenomenon among older people, given the well documented potential of benzodiazepines to provoke dependence. It also appears that over the past decade, the use of mixed samples of users and non-users of psychotropic drugs has provided little information about older consumers themselves. Although comparison is essential for understanding, perhaps more studies should target older drug users exclusively. For instance, we know of no study examining mental health outcomes of older psychotropic drugs users over time. One might deem it illogical if no studies had been conducted on the long-term effects of antihypertensive medication on blood pressure. Grad [ 101 ] made the same observation when commenting on the few empirical studies of the impact of long-term use of benzodiazepines on the quality of sleep of community-dwelling older persons. Mental health status, distress, and psychological well-being The concepts of mental health, psychological distress, depression, and social support are often studied in relation to psychotropic drug use, but operationalized differently across studies. For instance, in the body of studies here reviewed, mental or emotional state is generally measured by standardized, self-rated scales that focus on the manifestation or experience of various symptoms of psychological or bodily distress, or by structured diagnostic interviews aiming to identify mental disorders according to official diagnostic criteria. In all, at least 12 different instruments or scales, 4 different systems of diagnostic criteria (ICD-9 and ICD-10, DSM-III-R and DSM-IV), and 3 unstructured open questions were used in 18 studies to measure concepts identified as depression (12 studies), anxiety (6 studies), psychological distress, mental disorders, life events (3 studies each), psychiatric syndromes (2 studies each), and morale, nervous or emotional disorders, melancholy, nerves, emotional condition, dementia, and sleep problems (1 study each). This variability in concepts and instruments surely impacts the variability of observed relationships between drug use and mental health status (Table 3 ). In addition, it is relevant to ask whether the sole use of scales measuring psychiatric symptoms is appropriate to understand psychotropic drugs use among older people. As discussed, the rate of mental disorders, including sleep problems, is lower among older than middle-aged adults, and largely outpaces their use of psychotropic medications. This suggests that many older people are prescribed psychotropic drugs for mild to moderate psychological difficulties that would not qualify as DSM mental disorders, and that might not produce significant impairment in daily functioning. For example, in two studies [ 61 , 79 ], while drug users displayed more depressive symptoms than non-users according to the Center for Epidemiologic Studies Depression scale (CES-D), their scores did not reach the standard threshold of 16 points necessary for a diagnosis of depression. Results such as these suggest that scales less oriented to symptoms, distress, or disorder, such as measures of psychological well being that focus on so-called "positive" psychological dimensions such as self-acceptance, autonomy, relationships, and purpose in life [ 102 - 104 ], should supplement traditional-type scales. Psychological well-being is not merely the reverse of psychological distress, and the relationship of psychological well-being to symptoms of distress is complex [ 105 ]. No scale of psychological well-being was used in any of the reviewed reports. However, in one recent study of older persons, Guerette [ 106 ] found that among several measures of mental status, measures of psychological well-being produced the strongest associations with benzodiazepine use. Measures of psychotropic drug use Some conflicting or ambiguous results across studies can be attributed to how psychotropic drug use should be measured. Two issues are discussed here: what drug classes to include, and what time period of use to consider. Depending on the study, one or more different classes of drugs are included among the substances measured as psychotropic medications, although, for example, indications for the prescription of benzodiazepines differ from those for the prescription of neuroleptics. Using a composite variable of any psychotropic drug use when examining the association with relevant sociodemographic factors or life conditions probably affects the observed association and consequently our understanding of the phenomenon. It would seem better to determine separately what variables are associated with benzodiazepine, antidepressant, and other drug use. Benzodiazepines warrant careful attention, as these drugs may be classified as anxiolytics, sedatives, hypnotics, and anticonvulsants–and we would not expect logically the correlates of anticonvulsant use in the elderly to resemble those of hypnotic use. In addition, researchers rarely specify which drugs they classify as "minor tranquilizers," or how they distinguish between "anxiolytics" and "sedatives." These definitional issues have long existed in pharmacoepidemiology and we should not expect them to be solved in these studies, but without assurances that drug categories do not overlap, measures of the dependent variable occasionally remain imprecise. The average prevalence of neuroleptic drug use among community-dwelling older persons, estimated at 3.1% from six studies with probability samples, deserves comment in this respect. It has been common wisdom that such drug use has been rare except in institutionalized samples. The studies also confirm that the main psychotropic drugs prescribed to seniors are benzodiazepines and antidepressants. However, most of these studies, conducted in the early 1990s, probably missed the increased popularity of atypical neuroleptics. These drugs have so far enjoyed a reputation as less toxic drugs than conventional neuroleptics, encouraging their prescription among the older age group [ 98 ]. Several problems associated with benzodiazepine use among seniors are not associated with neuroleptics. However, all neuroleptics carry their own substantial risks of adverse effects, such as tardive dyskinesia and cognitive dysfunction (conventionals) and weight gain and diabetes (atypicals) [ 107 , 108 ]. In future studies, investigators will probably need to examine the prevalence of these drugs' use among the elderly. The category of reversible cholinesterase inhibitors, also known as anti-dementia, anti-Alzheimer's, or "cognition enhancers," is also unmentioned in any of the reviewed studies. Introduced in the mid-1990s, drugs such as donazepil and rivastigmine are increasingly prescribed to community-dwelling elderly showing subtle signs of dementia and simple forgetfulness (often termed "mild cognitive decline") according to a preventive ethos [ 109 ]. The second measurement issue to consider relates to the period of time for which data are collected, a source of confound noted by other reviewers [ 41 , 42 , 110 , 111 ]. In the 30 studies, temporal windows varied widely, from "current use" (4 studies), "current and past use" (1 study), "regular use" (2 studies), two days (2 studies), one week (1 study) two weeks (3 studies), one month (3 studies), three months (5 studies), and one year (7 studies). In two studies this period is undefined. When drug utilization is measured based on more than a week, one risks inflating the prevalence rate by including consumers who no longer use the drugs. Conversely, one risks omitting occasional users when referring to the last two days only [ 45 ]. Graham and Vidal-Zeballos [ 42 ] suggested asking respondents about short-term consumption (past 2 days and "recent use") and about long-term use (past year). This suggestion does not however solve problems of inaccurate self-report. Studies have found that older persons frequently hide or deny their use of psychotropic drugs [ 112 - 114 ]. To avoid these pitfalls, researchers in six studies determined psychotropic drug use according to municipal, provincial, state or national prescription databases held by government agencies or health maintenance organizations. The number of prescriptions, however, can greatly differ from actual drug consumption [ 16 ]. For example, about 50% of the elderly do not comply with their prescription regimen [ 115 , 116 ]. and an unknown proportion share their pills with relatives or friends [ 85 ]. Finally, researchers in at least 10 studies measured drug use by inspecting medication containers at the home of the respondent [ 83 ]. This method confirms that drugs have been obtained but leaves the compliance problem unresolved. A good rationale exists nonetheless to encourage this method, probably the most reliable way to get as close as possible to actual consumption [ 55 , 63 , 83 , 111 ]. Given that one third of studies in this review used this method suggests that its cost may be relatively acceptable. Interestingly, only two studies used telephone surveys. Owing to their relatively lesser cost than personal interviews, and since the development of reliable technologies, telephone surveys using random-digit dialing and random-select dialing are increasingly used to contact respondents in health related surveys. Conclusions Without being exhaustive, the present literature review is comprehensive and the range of reports, methods, samples, and geographical locations provides a reasonably solid base to support most recommendations. This review was limited to empirical studies. To better understand the phenomenon of psychotropic drug use among community-dwelling elderly, it is desirable that historical, psychological, sociological, political and regulatory, as well as medical aspects of the phenomenon be considered. While some factors are clearly associated with psychotropic drug use in the reviewed studies (such as race, proximity to health centers, sleep complaints, and health perception), few investigators test specific hypotheses to account for the associations. For example, sparse work focuses on cultural factors that might explain drug use disparities between Whites and African-Americans. Similarly, with most drug users being long-term and most drug treatments for insomnia losing their effectiveness with long-term use, the strong association between sleep complaints and drug use needs thorough examination. One of the least studied aspects of the phenomena in the reviewed studies concerns the role of health care professionals. Physicians, nurses, and pharmacists all interact significantly with community-dwelling older persons, especially with those who take psychotropic medications. Only through a health care professional such as a physician and a pharmacist would the vast majority of older people obtain a psychotropic drug to relieve a sleep problem. Recently, the Internet and mail-order pharmacies without face-to-face interaction have facilitated individuals' access to prescription drugs. Each professional's role, as mentioned, is also likely to alter as the individual's phase of consumption transforms from short-to long-term. Researchers must face the challenge to incorporate variables related to health care professionals' attitudes and behaviors, as well as to new modes of distribution of psychotropic drugs to consumers, in their study of the phenomenon. Approximately one third of community-dwelling older persons use psychotropic medications. If the rate of psychiatric disorders among a population serves as a guideline, then obviously older persons' use of psychiatric drugs far outpaces these drugs' standard indications and has extended into areas where drugs have little documented effectiveness. Viewed in this light, the ubiquitous phenomenon of long-term psychotropic drug use should evoke concern and caution. The discipline of nursing can definitely contribute to the rational use of these agents among older people. As suggested in this review, researchers do not still fully grasp the dynamics of psychotropic drug use among this population and creative and rigorous research from several disciplines, and from interdisciplinary perspectives, is needed. However, nurses concerned by the problem of the overuse of medication and its adverse consequences can already implement and evaluate programs to educate older people and allied health care and social service professionals about the risks of psychotropic drugs and alternatives to drugs for the management of everyday anxiety, loneliness, depression, and especially insomnia. Nurses can also actively implement and evaluate drug withdrawal programs aimed at long-term users who have had difficulty in withdrawing, and especially at short-term users who might soon be trapped into dependency and thus long-term use. Conversely, diverse patterns of psychotropic drug use undoubtedly exist among older persons, and positive patterns of use, emanating from users' own experiences and discoveries, need to be documented and disseminated. Competing interests None declared. Authors' contributions PV and DC conducted the literature review and drafted the manuscript. SL and JC revised the literature review and subsequent drafts. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Characteristics of 32 empirical reports on psychotropic drug use among community-dwelling older persons, 1990–2001. Reports on psychotropic drug use among community-dwelling older persons. Click here for file Additional File 2 Factors associated with psychotropic drug use among community-dwelling older persons in 32 empirical reports, 1990–2001. Factors associated with psychotropic drug use among community-dwelling older persons. Click here for file

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514698

Linear fuzzy gene network models obtained from microarray data by exhaustive search

Background Recent technological advances in high-throughput data collection allow for experimental study of increasingly complex systems on the scale of the whole cellular genome and proteome. Gene network models are needed to interpret the resulting large and complex data sets. Rationally designed perturbations (e.g., gene knock-outs) can be used to iteratively refine hypothetical models, suggesting an approach for high-throughput biological system analysis. We introduce an approach to gene network modeling based on a scalable linear variant of fuzzy logic: a framework with greater resolution than Boolean logic models, but which, while still semi-quantitative, does not require the precise parameter measurement needed for chemical kinetics-based modeling. Results We demonstrated our approach with exhaustive search for fuzzy gene interaction models that best fit transcription measurements by microarray of twelve selected genes regulating the yeast cell cycle. Applying an efficient, universally applicable data normalization and fuzzification scheme, the search converged to a small number of models that individually predict experimental data within an error tolerance. Because only gene transcription levels are used to develop the models, they include both direct and indirect regulation of genes. Conclusion Biological relationships in the best-fitting fuzzy gene network models successfully recover direct and indirect interactions predicted from previous knowledge to result in transcriptional correlation. Fuzzy models fit on one yeast cell cycle data set robustly predict another experimental data set for the same system. Linear fuzzy gene networks and exhaustive rule search are the first steps towards a framework for an integrated modeling and experiment approach to high-throughput "reverse engineering" of complex biological systems.

Background While similarity (homology) of DNA sequence between organisms can be used to propose potential gene functions, transcriptional regulation, and protein pathways (e.g., [ 1 ]), there are often major differences in the protein products, functions, and pathway involvement of genes with nearly identical sequences [ 2 ]. Consequently, sequence homology may be viewed as a means of generating an initial "draft" hypothesis for the gene network of a newly sequenced organism that can be built upon using high throughput experimental techniques such as DNA chips and microarrays for mRNA transcript profiling [ 3 ], protein abundance profiling with mass spectroscopy and 2-D gel electrophoresis [ 4 ], and protein-protein and protein-DNA binding assayed using SELDI mass spectrometry [ 5 ] and protein chips [ 6 ]. In addition, new genetic technologies, in particular small interfering RNA (siRNA) for selective gene suppression facilitate high-throughput massively parallel perturbation of the gene and protein networks of biological systems [ 7 ]. Given the potential scale and complexity of experiments and resulting data sets, biologists need a modeling and simulation framework to optimally design experiments and interpret results. The problem is not simply one of "reverse engineering" to find the optimal "best fit" gene, protein, and/or metabolite interaction model to explain a set of experimental results; rather, modeling should suggest the range of hypotheses that can potentially explain the results of one experiment and select the optimal next experiment to reduce the number of possible alternative hypotheses, with the goal of converging to a biological system model that can be used to predict the effect of molecular perturbations. A major challenge of modeling biological systems is that conventional methods based on physical and chemical principles require data that is difficult to accurately and consistently obtain using either conventional biochemical or high throughput technologies, which typically yield noisy, semi-quantitative data (often in terms of a ratio rather than a physical quantity) [ 3 ]. In particular, microarray gene expression ratios are ultimately obtained from pixel counts of relatively messy images. Boolean networks (e.g. [ 8 ]) are computationally simple and do not depend on precise experimental data, and thus they are suitable for handling both the complexity of biological networks and the challenge of generating and comparing multiple hypothetical networks as described in the above scheme. However, Boolean models have inadequate dynamic resolution to accurately describe the behavior of a biological network [ 9 ]. In contrast, differential equation models (e.g., [ 10 ]) can be computationally expensive and sensitive to imprecisely measured parameters. Even the lower throughput RT-PCR method for gene expression measurement (as described in [ 10 ]) cannot produce quantitatively precise data that can be accurately mapped to actual mRNA concentrations in the sample. Because of computational limitations, continuous modeling approaches (e.g., [ 10 , 11 ]) are limited to finding the single model that best fits experimental data given some set of constraints, such as a maximally sparse gene interaction network [ 11 ]. Fuzzy logic [ 12 ] provides a mathematical framework that is compatible with poorly quantitative yet qualitatively significant data. Fuzzy logic is a natural language for linguistic modeling, thus it is consistent with the qualitative linguistic-graphical methods conventionally used to describe biological systems. Fuzzy models are rule-based; accordingly, there is a potential scalability problem as the number of antecedents ("inputs" to the rule) and variable states ("resolution" of inputs and rule outputs) increase, causing combinatorial explosion. Non-scalable conventional fuzzy logic has previously been used to analyze microarray data [ 13 ]. However, because of the nonlinear scalability of the modeling method and resulting computational expense of generating rules for multiple inputs, this method allows for only one possible positive and one possible negative regulator for each gene, thus yielding few biologically meaningful insights and experimentally testable hypotheses. The problem of rule set combinatorial explosion is addressed by the union rule configuration (URC) developed by Combs and Andrew [ 14 ], which allows for linear growth in rule set complexity with both resolution (number of states) and number of inputs (rule antecedents) at the cost of having to represent nonlinear relationships between inputs as hidden layers [ 15 ]. In the linear (URC) fuzzy logic scheme, there are distinct fuzzy rules for each individual input to a given output. For example, given input variables A and B to an output C, there would be a set of rules relating A to C (e.g., "If A is LOW then C is LOW", "If A is HIGH then C is HIGH") and another set of rules relating B to C (e.g. "If B is LOW then C is HIGH", "If B is HIGH then C is LOW"). After each rule is applied individually, the intermediate evaluations of the fuzzy state of the output variable ("node") are aggregated by a fuzzy union (logical OR) operation (e.g. by summing or taking the resulting memberships in the fuzzy sets defining the state of the output). This contrasts with conventional fuzzy logic (or the "intersection rule configuration"), which has rules relating all combinations of inputs evaluated by a fuzzy intersection (logical AND). For the example with inputs A and B to output C, rules would read as, e.g., "If A is LOW and B is LOW then C is LOW", "If A is LOW and B is HIGH then C is HIGH", etc., leading to a combinatorial explosion avoided by the URC. The utility of linear (URC) fuzzy logic has been demonstrated in its ability to qualitatively model the lac operon of E. coli [ 16 , 17 ]. In our previous work, a URC fuzzy logic model was constructed from existing qualitative biological knowledge about the interaction of genes and limited quantitative data on protein and metabolite concentration and enzyme kinetics, showing the power of linear fuzzy logic to describe complex multi-component regulation. Here, the linear fuzzy logic method is extended to tackle the inverse problem of gene network reconstruction from real quantitative microarray data where there are many inputs. This involves both methods for mapping the experimental data to the fuzzy logic membership functions and a useful implementation of the URC fuzzy logic to represent the gene networks. In addition, a robust algorithm for performing searches through the exponentially large space of possible gene networks is presented. To address the problem of generating all plausible hypothetical network models that explain an experimental data set, we are initially proceeding with an exhaustive search of possible gene interactions to find those that fit the data within some error threshold. Thus, the problem we are tackling is of exponential complexity with O( m N ) growth in the number of possible rules for the behavior of a given "output" gene of a gene interaction node, where N is the number of (input) genes that can possibly control it and m is the number of possible rules describing the effect of each single input gene on the output gene. On the other hand, if a linear fuzzy logic scheme is not used, the problem would grow at an unacceptably high O( m N^N ) rate. The number of possible rules for each gene-gene interaction ( m ) is given by n n , where n is number of fuzzy sets that describe the state of a variable. Hence, we will constrain the size of the problem by (i) setting the minimum number of fuzzy sets to three, the minimum for meaningful resolution, (ii) limiting the number of possible input genes that are allowed to control the output of the output gene at each node of the fuzzy network model, and (iii) not allowing nonlinear gene interactions which would require hidden layers. The last condition is not particularly severe, as a typical nonlinear interaction (e.g., "xor") interaction between two regulatory proteins at a gene is mediated by an intermediate complex between the proteins that can be represented as an independent node in a network model. Therefore, "hidden layers" may generally be avoided by including more biological detail as explicit nodes in the model: for example, explicitly including the temporary interaction between proteins within a scaffolded cellular signal transduction complex, or by incorporating as model nodes the various topological states of a region of DNA influencing transcription factor binding, or in general, adding sufficient biological detail such that interactions between inputs can be linearly modeled. We apply partially scalable, linear fuzzy network modeling to a data set commonly used for demonstrating computational methods in systems biology, microarray experiments of yeast cell cycle gene expression [ 18 ]. These data were obtained in 1998, prior to subsequent technical and statistical advances to improve data quality. However, to keep our case study as general as possible and demonstrate the ability of the fuzzy logic approach to handle other similarly noisy data sets, we do not do any data processing other than the fuzzy modeling process (described in the Methods). Exhaustive search is used as a brute force "reverse engineering" method to find all possible gene network models that fit the data for a set of twelve genes known to participate in the yeast cell cycle. We show that the search converges to a small number of models describing the expression of each gene within a fit tolerance. Models found from the data for one particular yeast cell cycle time series are also capable of qualitatively predicting data from another time series experiment (i.e., one using a different cell synchronization method). In addition, given the constraints of the search algorithm (described in more detail in the Methods) and our limitation to pure transcriptional data, we find that the best fitting fuzzy network models collectively recover some direct and indirect functional relationships between genes predicted by interactions found by previous biochemical experiments as well as quantitative and statistical methods based on transcriptional correlations. Results Yeast cell cycle data set As a proof of concept, we have used exhaustive search to generate fuzzy gene networks based on yeast ( Saccharomyces cerevisiae ) cell cycle microarray time series data sets presented in [ 18 ] (which included data from [ 19 ]). Researchers frequently use these data sets to demonstrate and validate statistical and clustering analysis (e.g., [ 20 , 21 ]), mathematical modeling [ 22 , 23 ], and reverse engineering methods [ 21 , 24 ]. Biological details of the yeast cell cycle transcriptional network and some computational methods for its analysis are reviewed in [ 25 ]. S. cerevisiae cell cycle regulatory protein-DNA interactions were also the subject of a recent extensive experimental study [ 26 ] and there is a large amount of previously obtained biological knowledge on the interaction of yeast cell cycle proteins, i.e., information contained in the Yeast Proteome Database [ 27 ] and the KEGG pathway database [ 28 ]. Consequently, predicted transcriptional network models we derive for the Spellman et al . [ 18 ] data set can be tested against numerous independent data sets and compared with models obtained using other methods. We focus on the 12 key yeast cell cycle genes listed in Table 1 with descriptions taken from the Yeast Proteome Database. The protein products of these genes have been extensively studied using conventional biological techniques and are known to regulate each other and play key roles in controlling cell cycle. Consequently, observed correlations between the genes of Table 1 in cell cycle microarray data are most likely the result of real biological activity rather than noise. In addition, cell cycle gene subsets similar to this one have been the subject of other recent gene network modeling and reverse engineering publications (e.g., [ 21 , 24 ]). Figure 1 shows the current understanding of the interactions of the cell cycle protein subset. There are three sets of gene expression time series in [ 18 ] measured for cells synchronized by different methods, called the cdc15 , alpha , and cdc28 sets. We fit models on the basis of the cdc15 data set since it contains the least number of missing data. Time points in the cdc15 set for which there is missing data for one or more of the 12 genes are excluded from the rule search. We perform an exhaustive search with a maximum of 4 inputs per node, as detailed in the Methods. A Microsoft Excel workbook with the complete fitting data set is provided in Additional File 1 , including all the fuzzy rule models for each gene obtained from exhaustive search with an E MIN threshold of approximately 0.6. Results of fitting to data Figure 2 shows the number of rule models found in the exhaustive search that fit the expression time series of the CLN1 gene (using the cdc15 data set) at different error tolerance levels ( E MIN , as defined by Equation 4 in the Methods). It shows typical behavior for the exhaustive rule search. The number of fuzzy models that fit a gene expression time series decreases exponentially as the fit tolerance ( E MIN ) increases, up to a maximum tolerance above which no models fit the data. A successful search generally converges to a small number of distinct models at the maximum fit tolerance, representing "plausible" hypothetical transcriptional networks that can explain the available data. In some cases, though it did not occur for any of the genes analyzed here, the search fails and there are a large number of models with similar poor fit scores and no suitable subset of "plausible" models. The plausible model subset generally contains common rule patterns. For example, Table 2 lists the models for CLB5 expression with the highest fit scores found in the exhaustive search. The rules are in the format used for the example described in the Methods section. Table 3 shows three models for each gene in the network: the best fitting rule (highest score) and the two highest scoring rules with different combinations of input genes. The scores for each of the three models are provided in corresponding rows at the bottom of the table. Figure 3 shows the best fitting interaction network diagrams for each node gene from Table 3 . To test whether the linear fuzzy gene network models found for one set of experimental data (i.e., cdc15 synchronization time series) can accurately predict another set of results for the same system, we analyzed the microarray time series for alpha cell synchronization presented in [ 18 ]. There are some missing values for some genes at some time points in the alpha data set, which are set to zero and could potentially lead to discrepancies between the modeling and experimental data only at those points. Figure 4 shows the predicted time series for the expression ratio of four genes ( CLN1 , CDC28 , SWI6 , and CLB5 ) given the highest (except for CLN1 , second-highest) scoring models in Table 3 . (The second-best fitting model is used for CLN1 because it consists of four inputs, including all three inputs in the highest-scoring model along with another gene. Thus, it represents a more general "consensus" model for CLN1 .) These models fit the original cdc15 training data with very different calculated tolerances (as measured by the fit error E ) ranging from 0.510 ( CDC28 , Figure 4B ) to 0.930 ( CLN1 , Figure 4A ). Discussion Using exhaustive search, we found linear fuzzy networks that predict cdc15 cell cycle microarray data for the expression of most of the twelve yeast genes we analyzed. The rule search typically converged to a small set of "plausible" models at a given fit error ( E ) tolerance for each gene (with exponential convergence as shown in Figure 2 ). Even for genes for which no highly fitting model could be found, such as SWI4 , the best model (fitting at E = 0.620) predicts the qualitative behavior of independently measured alpha time series data (Figure 4C ). Moreover, models that are more predictive ( E > 0.8) of the cdc15 training data provide quantitatively accurate predictions of the alpha data (Figures 4A and 4D ). Notably, these consistently good fits for the alpha data set were achieved using exactly the same arctangent data normalization and fuzzification scheme applied to the cdc15 data set. This suggests that the fuzzy processing methods described here can be generally applied for data sets obtained from different microarray experiments, provided a roughly symmetric distribution of Log2 ratios about 0, such that sets 1 and 3 both remain meaningful – though the ratios could be re-centered if necessary. In general, our results demonstrate the ability of qualitative fuzzy rule models to interpret the results of quantitative data and make predictions that can be statistically analyzed. Consequently, these models can be used to pose experimentally testable hypotheses. Measurements of mRNA expression from microarray experiments complement information from additional gene knockout, DNA-protein and protein-protein experiments. A model based on pure transcriptional data will thus necessarily contain indirect relationships between proteins and miss other direct purely protein-protein interactions. However, gene network models can suggest functional roles and relationships for genes and proteins, and these models are necessary in complex system analysis to design and interpret further experiments that will specifically determine protein function and identify actual chemical interactions. To see what biological insights can be derived from fuzzy gene network models, we can examine areas of agreement and discrepancies between the best-fitting models found in our exhaustive search (shown in Table 3 and Figure 3 ) and the current understanding of the yeast cell cycle network (summarized in Figure 1 ). Focusing on CLN1 , we found positive regulation by CLN2 and negative regulation by CDC20 (Figure 3 ), which are correlations expected from biological knowledge (as shown in Figure 1 ) and found by Soinov, et al . using a supervised learning method [ 21 ]. In addition, the model for CLN1 includes a direct connection with CDC28 and an indirect connection with MBP1 (through regulation of the SBF complex) that are consistent with their relative positions in the cell cycle (Figure 1 ). The best-fit model for CLN1 depended solely on a positive interaction with CLN2 , revealing the strong co-transcriptional connection between CLN1 and CLN2 . The connection between CLB5 and CLB6 was also found in the model for CLB5 . Other successfully found interactions include the negative regulation of CDC6 by CLB6 and the positive regulation of CLB6 by MBP1 . Some biologically accurate relationships were found that were absent from the supervised learning analysis of [ 21 ]. Notably, the model successfully recovers the apparent inhibition of CLB5 by CDC20 , which is not shown in Figure 1 (based on the KEGG pathway) but arises from cdc20 protein presenting clb5 protein to proteases for degradation (as included in the model of [ 22 ], references within). There are several biological relationships that are not found in the best-fitting networks of Figure 3 , such as an interaction between SWI6 and SWI4 (which form a multiprotein complex). The best-fitting models for SWI4 include a repressing action by MBP1 , which is inconsistent with biological knowledge (Figure 1 ) suggesting that MBP1 and SWI4 activity should correlate (since they act at the same stage in the cell cycle). However, closer examination of the Spellman data set reveals that the amplitude of MBP1 transcription varied within a small range, and the measurement could have been very noisy, resulting in a potential error by the algorithm. (It should be noted that no correlation is identified between MBP1 and SWI4 by the supervised learning algorithm in [ 21 ].) In general, determining which relationships found in the fuzzy gene network represent biologically accurate interactions is a question that must be resolved by analyzing other data sets or from new experiments. The multiple plausible hypothetical input gene combinations can be used to optimally design experiments to add most information for least effort (time and cost) to revise fit errors and produce a new, more realistic set of hypothetical networks. Conclusions In this work, we describe partially scalable, linear fuzzy logic models for biological network modeling. We demonstrate our approach by developing network models that accurately predict transcriptional data from typically noisy and semi-quantitative microarray experiments. Looking at the transcription network alone provides us with a view of the system at the "gene interactions" level. As measurement technology rapidly advances, the methods we describe can be extended to comprehensive heterogeneous data sets. To address the problem of analyzing the complex results of an exhaustive fuzzy model search and designing optimal experiments, we are currently developing pattern recognition methods to better visualize and interpret potentially large sets of models. In addition, we are considering stochastic methods to accurately sample and characterize the "space" of all possible fuzzy models to (a) more efficiently identify the subset of plausible models and (b) identify common patterns among all the models to gain a better understanding of the system and its evolution. While it is tempting to develop methods to obtain a single "optimal solution" as in a classic inverse problem, this is not appropriate for complex biological systems. Scarcity of both data and biological understanding mean that at best experiments will merely limit the space of potential solutions. Biological system analysis is a dynamic reverse engineering problem, requiring continuous acquisition of new experimental data – data that should be acquired from experiments designed and informed by continuous modeling. Linear fuzzy rule network models are a promising methodology for an integrated modeling and experimental approach. Since fuzzy rule models are enumerable, methods developed for combinatorial optimization can be extended to them. Moreover, linear fuzzy network models can simultaneously contain both quantitative and qualitative information, providing a common framework for a broad range of biological data, including mass spectrometry analysis, RT-PCR, single cell imaging, metabolite profiling, and other technologies yet to be developed. Methods Converting between numerical data and fuzzy sets We use three fuzzy sets, Low (or 1 ), Medium ( 2 ), and High ( 3 ) to represent the magnitude of gene expression, as defined in Figure 5 . Fuzzification (conversion to fuzzy representation) of a numerical datum x is performed by finding the corresponding fuzzy set memberships y 1 , y 2 , and y 3 (with values ranging from 0 to 1.0) given the linear functions shown in Figure 5 , where Defuzzification (conversion back to numerical representation) is performed using the "simplified centroid method" [ 29 ], with point set definitions shown in Figure 5 . Following a fuzzy rule evaluation that returns fuzzy set memberships y = [ y 1 y 2 y 3 ] in sets 1 (Low), 2 (Med), and 3 (High) respectively, the estimated numerical result of the evaluation, , is given by the centroid for the points located at -1, 0, and +1 for each set respectively, or The fuzzy set definition and centroid defuzzification of Equations 1 and 2 were selected to maximize computational efficiency during exhaustive search: all 27 rules can be represented by easily implemented algebraic functions and it is possible to design the implementation to avoid as many costly if/then comparisons as possible. In addition, the scheme perfectly reproduces monotonic linear positive and negative interactions (i.e., the functions f ( x ) = x and f ( x ) = - x are quantitatively equal to monotonic fuzzy rules, which can be written using notation from the following section as [1 2 3] and [3 2 1] respectively) so it generally will not introduce systematic error in the model. To apply this scheme for defuzzification and fuzzification scheme, experimental data must be projected on to the interval -1.0 through +1.0. Thus, log base 2 expression ratios are normalized by taking the arctangent of each ratio and dividing by π/2, yielding a symmetric transformation covering the desired interval. Previous work normalized expression ratios by the maximum value found in the experiment [ 17 ] or used different fuzzy set definitions for each variable [ 16 ], but those approaches suffer from a lack of universality across data sets and makes it difficult to compare and integrate data from different experiments. On the other hand, the arctangent method is defined across infinity, so no data will be "out of range". It also takes into account the fact that gene expression ratios often "saturate", and the difference between different degrees of high and low ratios are not necessarily biologically significant (this is because of the optical methods for measuring microarrays and the exponential error introduced using RT-PCR). When used in conjunction with the overlapping fuzzy set mappings shown in Figure 5 , these "middle" values will tend to land in the Medium set ( 2 ). Comparing fuzzy predictions to numerical data The fuzzy rule relating the input of a single gene to an ouptut node gene can be expressed as a rule vector r . For example, the rule r = [3 2 1] corresponds to the linguistic rules: If Input is Low ( 1 ) then Output is High ( 3 ) If Input is Med ( 2 ) then Output is Med ( 2 ) If Input is High ( 3 ) then Output is Low ( 1 ) Given the fuzzified expression of an input gene y = [ y 1 y 2 y 3 ] obtained using Equation 1 and the general fuzzy rule r = [ r 1 r 2 r 3 ], the resulting fuzzified expression of the output gene z will be: In general, node behavior is the result of N input genes acting on the output gene simultaneously. In the linear fuzzy logic scheme, the rule for each input gene is evaluated separately, leading to intermediate outputs z i : These intermediate fuzzy values are summed algebraically to obtain the final resulting fuzzy value for node gene expression: This result is defuzzified using Equation 2 to evaluate the output of the node. For three fuzzy sets, there are 3 3 or 27 possible rules describing the effect of a single gene on another gene. Thus, if there are N input genes for a node, there are 27 N total possible rule combinations describing the behavior of the node gene. In general, no rule combination will be an exact fit to real experimental data. Given some tolerance to fitting error, there will be multiple possible rule combinations, representing plausible hypothetical gene network models. In our present work, we define the error of the fit for the M data of the output gene x = { x 1 , x 2 ,..., x M } as where is the set of defuzzified numerical predictions (typically log expression ratios) and is the mean of the experimental data set x . A perfect fit results in a maximum E of 1.0. This error score was chosen because while it is quantitative, it emphasizes the correlation in qualitative behavior between the fit and prediction instead of the absolute numerical fit, which can be difficult to model with the limited resolution of three fuzzy sets. We can use the fitting error to rank these models, and use rule patterns consistent throughout all plausible models as a basis for constructing the template of a final network model that can be tested experimentally. Example of fuzzy rule evaluation As an example to illustrate fuzzy gene networks using a simple rule combination, we consider three genes ( G1 , G2 , G3 ) with log base 2 expression ratios measured at three different times: G 1 = {-3.0 0 +3.0} G 2 = {0.3 0 -0.3} G 3 = {+1 0 -1.0} Using the arctangent normalization to project the ratios on [-1,1], we obtain G 1 = {-0.795 0 +0.795} G 2 = {+0.186 0 -0.186} G 3 = {+0.500 0 -0.500} which can be fuzzified using Equation 1 to yield: G 1 = {[0.795 0.205 0] [0 1.0 0] [0 0.205 0.795]} G 2 = {[0 0.814 0.186] [0 1.0 0] [0.186 0.814 0]} G 3 = {[0 0.5 0.5] [0 1.0 0] [0.5 0.5 0]} with vectors for each time point containing set membership in Low ( 1 ), Medium ( 2 ), and High ( 3 ). Consider the following rules for G1 and G2 as input genes to G3 : G 1: G 3 = [3 2 1] G 2: G 3 = [1 2 3] where the rules can be written in English as If G1 is Low ( 1 ) then G3 is High ( 3 ) If G1 is Med ( 2 ) then G3 is Med ( 2 ) If G1 is High ( 3 ) then G3 is Low ( 1 ) If G2 is Low ( 1 ) then G3 is Low ( 1 ) If G2 is Med ( 2 ) then G3 is Med ( 2 ) If G2 is High ( 3 ) then G3 is High ( 3 ) Now, the evaluations of the rules taken individually are G 1: G 3 = {[0 0.205 0.795] [0 1.0 0] [0.795 0.205 0]} G 2: G 3 = {[0 0.814 0.186] [0 1.0 0] [0.186 0.814 0]} The sum of two intermediate outputs (Equation 3) is the predicted fuzzy behavior of G3 for the three time points, which can be defuzzified using the point-centroid method (Equation 2) and transformed back to real numbers on [-1,1]: G 3 = {[0 1.019 0.981] [0 2.0 0] [0.981 1.019 0]} = {0.491 0 -0.491} These numbers can be transformed back to a Log2 expression ratio by inverting the normalization (multiplying by π/2 and taking the tangent): G 3 = {0.97 0 -0.97} Finally, we use Equation 4 and the experimental data for G3 to calculate the fit error for this rule combination: which compares to a maximum E = 1.0 for a perfect fit. Exhaustive network search In general, a possible model for a node can include any combination of the genes available to act as inputs. In the work described here, we consider potential interactions of 12 genes. Thus, a rule for any one gene can include as inputs any combination of any number of up to all 11 other genes. Since each input gene can influence the node by any one of the 27 possible fuzzy rules, there are approximately 10 16 possible rule combinations for each of the 12 genes, making the exhaustive search method practically impossible. Thus, the number of possible inputs to a node must have a maximum constraint to make exhaustive search tractable. Studies of network topology through the experimentally observed association of proteins suggest that in many cases only few regulatory proteins are observed to directly influence the expression of a gene [ 26 , 30 - 32 ]. For our transcriptional network searches, we use the constraint of up to 4 input genes to any node. Thus, for each node gene, each of the other 11 genes occurs as an input alone and also in combination with any of up to 3 of the other genes as multiple inputs. Our use of this input constraint does not necessarily restrict the full range of interactions that can be found for the genes in our network, since all possible combinations of 1 through 4 of the genes are searched sequentially. For example, in our fitting of rules to CLN3 , we considered the following potential input combinations: SIC1 alone, SIC1 and CLN1 together, SIC1 - CLN1-CLN2 , SIC1-CLN1-CLN2-CLN3 , CLN1 , CLN1-CLN2 , CLN1-CLN2-CLN3 , CLN1-CLN2-CLN3-SWI4 , CLN2 , CLN2-CLN3 , etc. If we include all combinations from 1 through 4 of the genes taken from the 11 total possible inputs, then the total search space for each of the 12 genes consists of approximately 10 8 rules (taking about 10 minutes on a PowerMac G4 using a single 450 MHz processor). Simulation files used to generate all the data presented here are available from the authors upon request. Authors' contributions BAS originated the concept of applying scalable (URC) fuzzy logic to modeling biological systems, implemented the scheme described within on the data set, and was the primary author. JPF conceived of the approach to use exhaustive search for biological network reconstruction. JNQ and AAQ developed and initially implemented the method of combinatorial input selection for the exhaustive network search, and AAQ contributed to the text. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Microsoft Excel spreadsheets of simulation results. See descriptive text in the workbook. Click here for file

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545212

Volume Status in Severe Malaria: No Evidence Provided for the Degree of Filling of the Intravascular Compartment

The study by Planche et al. [1] provides important new information addressing intracellular volume depletion in children with severe childhood malaria, but does not address the question of whether intravascular volume depletion (hypovolemic shock) is present. Using sophisticated methodology to determine total body water and extracellular water, they demonstrate a 6.7% deficit in total body water and an 11.7% deficit of intracellular water, providing an important indication of the volumes of fluid that may be required to optimize hydration. The data, however, do not address the degree of filling of the intravascular compartment, nor should they be used to answer the question about the state of tissue and organ perfusion. Indeed, we believe that these new data present no conflict with our previously reported findings. Using methods to study critical illness physiology that are widely employed within pediatric intensive care units for interpretation of circulatory status, we have demonstrated evidence for hypovolemia in 53 Kenyan children with severe malaria complicated by metabolic acidosis [2] . Our children were younger, had longer capillary refilling times (>3 s), lower central venous pressures (mean 2.9 cm H 2 O) and higher creatinines (>80 µmol/l): all features of compensated hypovolemic shock. Furthermore, hypotension (systolic BP < 80 mm Hg) was present in 44% of children with severe acidosis (base deficit >15). These findings also indicate important baseline differences in two cohorts of children studied. We agree that reconsideration of guidelines for acute fluid management is warranted, particularly when current recommendations await an adequate evidence base. Nevertheless, conflicting opinions on the question of volume status in children with severe malaria can be satisfactorily resolved only through prospective randomized trials that include both fluid resuscitation and control groups. While the design and conduct of such trials will involve considerable challenges, optimal fluid management will never be resolved on the basis of theoretical consideration alone.

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545212.xml

535905

Neuroanatomical organization of gonadotropin-releasing hormone neurons during the oestrus cycle in the ewe

Background During the preovulatory surge of gonadotropin-releasing hormone (GnRH), a very large amount of the peptide is released in the hypothalamo-hypophyseal portal blood for 24-36H00. To study whether this release is linked to a modification of the morphological organization of the GnRH-containing neurons, i.e. morphological plasticity, we conducted experiments in intact ewes at 4 different times of the oestrous cycle (before the expected LH surge, during the LH surge, and on day 8 and day 15 of the subsequent luteal phase). The cycle stage was verified by determination of progesterone and LH concentrations in the peripheral blood samples collected prior to euthanasia. Results The distribution of GnRH-containing neurons throughout the preoptic area around the vascular organ of the lamina terminalis was studied following visualisation using immunohistochemistry. No difference was observed in the staining intensity for GnRH between the different groups. Clusters of GnRH-containing neurons (defined as 2 or more neurons being observed in close contact) were more numerous during the late follicular phase (43 ± 7) than during the luteal phase (25 ± 6), and the percentage of clusters was higher during the beginning of the follicular phase than during the luteal phase. There was no difference in the number of labelled neurons in each group. Conclusions These results indicate that the morphological organization of the GnRH-containing neurons in ewes is modified during the follicular phase. This transitory re-organization may contribute to the putative synchronization of these neurons during the surge. The molecular signal inducing this plasticity has not yet been identified, but oestradiol might play an important role, since in sheep it is the only signal which initiates the GnRH preovulatory surge.

Background Gonadotropin-releasing hormone (GnRH), the peptide responsible for the regulation of secretory activity of the pituitary gonadotropes, is found in a diffuse neuronal system situated in the preoptic area and anterior hypothalamic area [ 1 ]. The neurons project their terminals to the median eminence where the peptide is released into the hypothalamo-hypophyseal portal vessels. The secretion of GnRH is normally pulsatile, as demonstrated by the measurement of GnRH following the direct sampling of hypothalamo-pituitary portal blood in various species such as sheep [ 2 , 3 ]. The frequency of this pulsatility is the main characteristic of this secretion and it encodes each part of the sexual cycle in the female [ 4 ]. The highest circulating concentration of GnRH is induced during the follicular phase in the female by the sequential action of the two main ovarian steroids (progesterone and oestradiol). These act within the brain to trigger a large and sustained period of GnRH release, the GnRH surge, which stimulates the preovulatory LH surge, and subsequently ovulation. The pulsatile nature of GnRH secretion, as well as the large amount of peptide released during the preovulatory surge, indicate that the activity of GnRH-containing neurons can be synchronized, and it has been demonstrated that the GnRH neurons of the preoptic area receive synaptic contacts from GnRH neurons [ 5 - 7 ]. Oestradiol, which initiates the preovulatory surge of GnRH, has been shown to be involved in the synaptic plasticity demonstrated in various neuronal populations, including those in the arcuate nucleus [ 8 , 9 ]. Moreover, in rats, an increase in the expression of the molecular markers of synaptic remodelling is observed at the time of the surge in the preoptic area where most of the GnRH neuron cell bodies are found [ 10 ]. In sheep, changes in the expression of the polysialylated form of the neural cell adhesion molecule around the periphery of the GnRH cell bodies, associated with the seasonal changes in GnRH secretion, are indicative of potential plastic changes in this neural system linked with changes in secretory activity [ 11 ]. In addition, oestradiol and progesterone have been shown to modify the morphology and staining intensity of GnRH neurons within the ovine hypothalamus [ 12 ], suggesting that ovarian steroids could elicit plastic changes in GnRH neurons during the oestrous cycle. However, very little work has been done regarding neuronal organization at the level of the GnRH cell bodies during the oestrous cycle in the ewe. The aim of this work was to study the number, distribution and potential contacts present between GnRH-containing cells in intact ewes under natural conditions, focusing particularly on the neuroanatomical organization of these neurons around the preovulatory surge when a high level of oestradiol is released by the growing follicles. Results Plasma hormones Because there are some individual variations in the response to the induction of ovulation treatment (oestrus onset, preovulatory LH surge onset, etc.), an analysis of plasma hormone concentration was carried out to confirm the allocation of animals to experimental groups according to the characteristic hormonal level previously described [ 13 ]. In group 1, as illustrated in figure 1 , progesterone and LH concentrations were low, characteristic of the late follicular phase. In group 2, progesterone remained low (less than 0.05 ng/ml) and a surge of LH was observed for 4 out 5 animals. Analysis of the LH profile concentrations showed that these animals were killed either during the ascending (n = 2) or descending (n = 2) part of the LH surge; for the remaining animal only a slight increase in LH secretion was observed at the end of the sampling period. In groups 3 and 4 a preovulatory surge of LH was clearly identified in all animals. These LH surges were followed by a clear increase in progesterone concentrations and the progesterone level was in the range of 2.5 to 4.5 at the end of the sampling period (except for one animal 1.9 ng/ml in group 4). Global analysis of plasma hormone concentrations at the last sampling time before animals were killed revealed significant differences between groups for LH (p < 0.01) and progesterone (p < 0.05). As shown in figure 1 , the LH level was higher in group 2 than for any other group (p < 0.01), and progesterone concentrations were lower in groups 1 and 2 than in groups 3 and 4 (p < 0.01). For groups 1, 2, 3 and 4, mean levels ± SEM were respectively 1.07 ± 0.12, 28.20 ± 7.08, 0.56 ± 0.03, 0.65 ± 0.06 for LH and 0.05 ± 0.0, 0.05 ± 0.0, 2.74 ± 0.10, 3.62 ± 0.26 for progesterone. However, the ewes showing no clear LH surge (group 2) or a moderate increase in progesterone concentration (group 4) were considered as slightly different from any other animal of the same group and were not used for determining the number or relative clustering of GnRH neurons. Four of the eight animals in experiment 2 were killed during the LH surge (2 during the ascending and 2 during the descending part). The 4 remaining animals were killed 4–6 hours after the end of the LH surge and were discarded from the study. GnRH-immunoreactive neurons Experiment 1 GnRH-containing neurons were distributed in the preoptic area (fig 2 ) as previously described [ 14 ]. There was some variation in the staining intensity in the same section, but it was usually strong. The black DAB/Nickel ammonium sulfate precipitate was distributed throughout the cytoplasm in the cell bodies and processes. The nucleus was never stained. GnRH-ir fibres were also observed around perikarya, presenting large varicosities. The distribution of GnRH cell bodies was homogenous between animals. GnRH-ir neurons were counted when the nucleus was clearly visible. There was no difference in the total number of GnRH-ir neurons in the experimental groups (group 1: 291 ± 38, n = 5, group 2: 347 ± 41 n = 4, group 3: 345 ± 38 n = 5, group 4: 241 ± 75 n = 4) (fig 3A ). However, the organization of the labelled neurons differed in the different experimental groups. Clusters of two or more GnRH-ir neurons in which there appeared to be close contacts between neuronal cell bodies (figs 4a,4b ) were found in the preoptic area of all groups. However, the number of clusters and the proportion of identified neurons involved in clusters varied significantly between the experimental groups. These clusters (mainly pairs of neurons) were more numerous in groups 1 (43 ± 7) and 2 (41 ± 8) at the moment of the preovulatory surge than in groups 3 (28 ± 2) and 4 (25 ± 6) in the luteal phase (p < 0.05) (fig 3B ). The percentage of clusters was higher in group 1 (15.1 ± 1.2) than in groups 2 (11.8 ± 1.0), 3 (8.3 ± 0.4) and 4 (10.7 ± 0.9) (fig 3C ). Experiment 2 In the second experiment, four additional animals were studied during the GnRH surge, and the data compared with those of the group 2 ewes in the first experiment. As shown in table 1 , there was no difference in the proportion of clusters of GnRH-ir neurons in the four animals sampled during the ascending phase of GnRH surge and the four sampled during the descending phase of the surge. Discussion Our observations indicate that the organization of GnRH-ir neurons was modified during the oestrous cycle, since more clusters were observed during the follicular phase. However, we did not observe any difference between the ascending and descending phases of LH release in group 2. Therefore, our data support the hypothesis that there are changes in communication between GnRH cell bodies prior to the initiation of the preovulatory GnRH surge. The increase in the number of clusters was not mirrored by a parallel change in the total number of GnRH cell bodies. However, as the number of clusters was relatively small compared to the GnRH cell population (constituting 10% to 15%), the putative increase in the number of GnRH-ir neurons could have been masked due to the large variations in the number of labelled neurons between animals. An alternative hypothesis is that the number of GnRH neurons remains constant, and that the variations in the number of clusters is linked to variations in the peptide level, which are consistent with the variations in the staining intensity observed in all animals. In this case, the level of GnRH would be higher in clustered neurons than in isolated neurons at the moment of the preovulatory surge, and therefore neuronal communication would be more efficient in clustered neurons. The percentage of clusters increased only in group 1, while the difference between the number of clusters in groups 2, 3 and 4 was not statistically significant. This special morphological organization may constitute an anatomical support for the synchronization of GnRH neuronal activity needed to induce the GnRH preovulatory surge. Clusters of GnRH-ir cell bodies have been previously described in sheep [ 15 ] (Lehman et al, 1986), rats and monkeys [ 16 ]. In rats they represented only 2–7% of the GnRH neuronal population [ 16 ]. In our study, the proportion of clusters was similar (10–15%) to that observed in monkeys (3–15%). However, contrary to our observation, the numbers did not change with the different hormonal conditions in the monkey [ 16 ]. This might be related to the distribution of GnRH cell bodies which differs between monkeys and sheep [ 17 ], and it is possible that the increase of clusters observed in our experiments was related to a specific GnRH neuronal population involved in the GnRH surge secretion. Indeed, Fos expression at the time of the preovulatory LH surge has been shown to be expressed in a subset of GnRH neurons in rats [ 18 ]. A neuronal re-organization of GnRH-containing neurons has also been previously described in anoestrous ewes where the neurons are ensheathed by glial processes which decrease the number of axo-somatic synaptic contacts [ 19 ]. In this latter situation, photoperiod would be the major signal to induce such a modification. Another example of the relationship between synaptic afferents and GnRH secretion has been described in the monkey where the increase of GnRH activity at puberty correlates with the decrease in synaptic afferents on GnRH-containing neurons [ 20 ]. This morphological regulation of GnRH-containing neurons, through synaptic contacts or perikaryal apposition, involves glial processes, since in both sheep and monkeys these neurons are ensheathed by numerous glial processes [ 19 , 21 ]. Steroids, mainly oestradiol, are the most potent regulators in the control of GnRH neuronal activity. Therefore, we may hypothesize that this "plasticity" results from an oestradiol effect, as has been shown in the monkey where oestradiol modulates the number of synaptic contacts on GnRH neurons [ 22 ]. In rats and monkeys, numerous studies have demonstrated that oestrogens induce synaptic plasticity in the control of gonadotropin secretion (rat: [ 23 ]; monkeys: [ 24 ]). In our study, the highest number of clusters was found during the preovulatory surge which occurred about 24 hours after the expected oestradiol increase, this delay being consistent with an oestradiol effect. There was no difference in the number of labelled neurons in the experimental groups, and this observation may indicate that the level of GnRH-immunoreactivity was stable in the neurons of the preoptic area. It has previously been demonstrated in the ewe that GnRH messenger ribonucleic acid expression changes before the onset of the oestradiol-induced luteinizing hormone surge [ 25 ], and that the staining intensity of GnRH-ir perikarya increases after oestradiol treatment, but that the number of labelled neurons does not change [ 12 , 26 ]. In addition, the variations of LH secretion induced by progesterone withdrawal are not linked to a variation in GnRH mRNA [ 27 ]. Unlike the apparent stability of the GnRH level in the perikarya, GnRH-immunoreactivity decreases in the median eminence after ovulation [ 28 ]. This information could indicate that variations in GnRH secretion arise from its release from the median eminence terminals without variations in peptides in the perikarya. Conclusions In conclusion, we have demonstrated that the morphological organization of the GnRH neurons of the preoptic area is modified during the oestrous cycle, although the overall number of GnRH-ir neurons does not change. During the preovulatory surge, the GnRH neurons are more frequently found in clusters, and the percentage of clusters is significantly higher immediately prior to the preovulatory LH surge. Actual contacts between GnRH-ir neurons cannot be determined using light microscopy, but can only be demonstrated using electron microscopy. Because oestradiol is the most powerful regulator of GnRH activity during the oestrous cycle, we can hypothesize that this plasticity may be induced by steroids. The role of this plasticity remains to be demonstrated, but it could increase interneuronal communication during the preovulatory surge. Methods All experimental procedures were carried out in accordance with authorisation A37801 of the French Ministry of Agriculture. Animals Intact adult Ile de France ewes (n = 28) from the laboratory flock, maintained under natural photoperiod were used. Two experiments were performed (one year apart) during the breeding season (between November 15 th and December 15 th ). Animals were fed daily with hay, straw and corn, and water was available ad libitum . All of the ewes had lambed at least once. Experimental design Experiment N° 1 Animals (n = 20) were killed at specific time points in the oestrous cycle. In order to synchronize oestrous cycles, animals were treated with an intravaginal progesterone-releasing device (CIDR InterAg, Hamilton, New Zealand) for 14 days. Following removal of the progesterone device, animals were treated (i.m.) with 500 UI of Pregnant Mare Serum Gonadotropin (PMSG), a treatment known to induce the LH surge and ovulation in sheep (for review see [ 29 ]). It has been shown that oestrous behaviour in this breed precedes the LH surge by 7.0 ± 1.6 hours [ 30 ]. Therefore, oestrous behaviour was recorded for each ewe every 6 hours, from 18 hours after PMSG administration until the animal was observed to be in oestrus using a ram wearing an apron. The animals were then allocated to four groups: Group 1: Animals killed 1–2 hours after oestrus, i.e. before the LH surge onset, (n = 5) Group 2: Animals killed 8–12 hours after oestrus, i.e. during the preovulatory LH surge, (n = 5) Group 3: Animals killed 8 days after the preovulatory LH surge, (n = 5) Group 4: Animals killed 15 days after the preovulatory LH surge, (n = 5) Experiment N° 2 Analysis of LH secretion (see paragraph below) revealed that animals in group 2 in experiment 1 were killed either in the ascending (n = 2) or descending (n = 2) phase of the LH surge. Therefore, in order to increase the N for each sub-group, 8 animals were synchronised as previously described (one year later, during the breeding season) and killed 12 and 20 hours (n = 4) after oestrus. For both experiments, blood samples were collected by venepuncture every 2 hours from 18 hours after PMSG administration until sacrifice (groups 1 and 2) and daily thereafter (groups 3 and 4). Hormone assays Plasma samples were assayed in duplicate for LH with 100 μl aliquots of plasma using a previously described RIA method [ 31 ]. All samples from one experiment were run in a single assay. Intra-assay coefficient of variation averaged 9% and assay sensitivity was 0.16 ± 0.05 ng/ml (4 assays) of standard 1051-CY-LH (i.e. 0.31 ng/ml of NIH-LH-S1). Progesterone was determined in a single assay after hexane extraction of 100 μl of plasma [ 32 ]. The sensitivity was 5 pg/tube and the intra-assay CV 10%. Immunohistochemistry A solution of heparin (25,000 units) was injected iv 10 min before decapitation. Both carotids were catheterised and the head was perfused with 2 litres of 1% sodium nitrite followed by 4 litres of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After perfusion, the brain was quickly removed and a block containing the whole preoptic area was isolated and post-fixed for 2 hours at +4°C in the same fixative. The blocks were immersed in 30% sucrose containing 0.1% sodium azide at +4°C for 5 days. Serial 40 μm coronal sections were obtained using a freezing microtome and stored at +4°C in PBS containing 0.1% sodium azide. Every fifth section was stained for GnRH immunoreactivity. Sections were incubated for 2 hours at +4°C in PBS containing 0.3% triton X100 and 1% H 2 O 2 and then preincubated for 30 min at room temperature in blocking solution (PBS, 6% normal sheep serum, 0.3% triton, 0.1% sodium azide). Sections were exposed to GnRH primary antiserum at a dilution of 1/10,000 in PBS containing 0.3% triton, 0.2% HSA, 0.1% sodium azide for 4 days at +4°C. After washing in PBS, sections were incubated for 3 hours at room temperature in the secondary antibody (1/500 with PBS containing 0.2% BSA). After washing in PBS, sections were incubated in rabbit peroxidase-antiperoxidase complex diluted 1/10,000 in PBS containing 0.2% BSA overnight at +4°C. After washing twice in PBS and in Tris buffer (0.05 M pH 7.6), the sections were reacted with 3,3-diaminobenzidine tetrahydrochloride (0.02%) and nickel ammonium sulfate (0.25%) in the same Tris buffer containing 0.0025% H 2 O 2 for 20 min. The reactions were stopped by rinsing the sections with Tris buffer. Sections were mounted onto gelatine-coated slides, dried, counterstained with a solution of neutral red for 5 min and mounted with a cover-slide using Depex. The characteristics and the specificity of the GnRH antibody were as previously described [ 14 ]. Data analysis The LH surge was assumed to start when LH concentration exceeded 6 ng/ml of plasma, i.e. about twice the maximum value of a pulse during the follicular phase for this breed and for this LH assay. For hormone values, a global comparison was carried out using a non-parametric ANOVA with exact general score test, and comparison between groups was carried out using the non-parametric exact permutation test for independent samples (Stat Exact Software, Cytel Software Corporation, Cambridge, MA, USA). The first observation of the sections allowed the immunoreactive area surrounding the OVLT to be determined (fig. 2 ). We selected two sections where the size of the OVLT was maximum in the third ventricle, and the two rostral and caudal sections were studied. A total of six sections at 160 μm intervals were studied for each ewe. In each section, all immunoreactive neurons for GnRH were counted using a light microscope, and groups of closely-associated neurons were noted. Total number of cells and number of clusters were analyzed by a 2-way (group and section number) ANOVA (SuperANOVA, Abacus concept, California) followed by a student-Newman-keuls post-hoc test for two by two comparisons. The percentage of cells forming clusters was calculated for each animal. It was analyzed by a one-way (group) ANOVA after arcsin transformation followed by a student-Newman-keuls post-hoc test for two by two comparisons. The results are expressed as means ± standard error of the means. Authors' contribution MB performed the immunohistochemical reaction and counted the neurones, AC and YT conceived the study, and participated in its design, coordination and in the collection of biological samples. BM participated in the final statistical analysis of results and corrected the manuscript.

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523829

New Drugs for Neglected Diseases: From Pipeline to Patients

The Drugs for Neglected Diseases Initiative is a new, public- sector organization dedicated to drug discovery

In wealthy countries, state-funded research has yielded breakthroughs in molecular biology, chemistry, and engineering. These advances have been taken up by the pharmaceutical industry and applied to drug development for a growing range of illnesses and conditions. As a result, patients have access to new drugs that are better tolerated, more specific, and more effective than old ones. In poor countries, however, millions of people have yet to experience the benefits wrought by science. The deadly infectious diseases that plague them, such as sleeping sickness, Chagas disease, and visceral leishmaniasis, fail to arouse the interest of drug developers. The Drugs for Neglected Diseases Initiative (DNDi) is a new, not-for-profit organisation set up to correct this fatal imbalance by developing new drugs for these forgotten patients. Dropped off the Radar Screen Most of the drugs still used to treat ‘neglected diseases’ were developed in colonial times. These are often expensive, difficult to administer, and hard to tolerate; several of them are also becoming ineffective because of increasing parasite resistance. Very few new alternatives have been developed in the past decades: between 1975 and 1999, 1,393 new drugs were made available to the public, but only 16 of these were meant for neglected diseases [ 1 ]. What makes the lack of drugs more difficult to accept is that scientists know an enormous amount about kinetoplastids, the organisms responsible for sleeping sickness, Chagas disease, and leishmaniasis [ 2 ]. The wealth of knowledge generated in this field could easily be used for drug development if the treatment of neglected diseases were perceived as financially attractive. But populations affected by neglected diseases have no purchasing power, so there is no financial incentive for drug companies to develop the drugs. The basic mechanics of the market-driven system are failing to help these populations. So most scientific research stops at the publication stage or falls through the gaps at different stages of the drug development pipeline ( Figure 1 ) [ 3 ]. Figure 1 The Drug Development Pipeline Because of the gaps in the development pipeline, potential new drugs for neglected diseases often stay stuck at an early stage of development. (Photos: World Health Organization/P.Virot and World Health Organization/Eric Miller) Whose Role Is It, Anyway? It is dangerous to oversimplify the causes of this situation. What share of responsibility for the world's health is borne by the pharmaceutical industry, which has the know-how and the resources for innovation? Aren't international organisations also partly responsible? After all, they are the ones who allocate major funding for health programmes and encourage research programmes. And what about public research institutions in rich countries that generate the knowledge used by industry? Governments have the power to influence their research priorities and drug development decisions, either through funding or direct involvement. Unfortunately drugs for neglected diseases are low priority for governments [ 4 ]. They tend to prioritise research with potential commercial applications instead. Responses to the Crisis All is not doom and gloom. In the past few years, there has been some movement on research and development (R&D) for neglected diseases. Despite its broad mandate and limited resources, the Special Programme for Research and Training in Tropical Diseases (TDR)—established and funded by the World Health Organization, the World Bank, and the United Nations Development Programme—can be credited with several important successes in the fight against malaria and leishmaniasis. The Medicines for Malaria Venture and the Global Alliance for TB Drug Development were set up as public–private partnerships to tackle malaria and tuberculosis. These partnerships were made possible by the fact that malaria and tuberculosis are global diseases, affecting patients in the North and South, so there was enough of a market to persuade industry to develop new drugs for these diseases. A different solution, however, was needed for diseases that are limited to tropical countries, are of no military or strategic interest to wealthy countries, and are not supported by markets or patients' organisations capable of attracting the attention of politicians. This is the kind of solution put forward by the DNDi. A Collaborative Not-for-Profit DNDi is a not-for-profit organisation designed to mobilise resources for R&D of new drugs for neglected diseases. Many people and organisations around the world share an ambition to redress the lack of new treatments for neglected diseases, and bring the benefits of science to forgotten patients. Several of them came together to create DNDi: one humanitarian organisation—Médecins Sans Frontières; five research institutions—the Oswaldo Cruz Foundation from Brazil, the Indian Council for Medical Research, the Kenya Medical Research Institute, the Ministry of Health Malaysia, and the Pasteur Institute from France; and the TDR ( Box 1 ). Box 1. From Pipeline to Patients—Some Key Organizations DNDi: http://www.dndi.org TDR: http://www.who.int/tdr Medicines for Malaria Venture: http://www.mmv.org Global Alliance for TB Drug Development: http://www.tballiance.org Oswaldo Cruz Foundation: http://www.fiocruz.br Indian Council for Medical Research: http://icmr.nic.in/home.htm Kenya Medical Research Institute: http://www.kemri.org Ministry of Health Malaysia: http://dph.gov.my/ Pasteur Institute: http://www.pasteur.fr/externe Médecins Sans Frontières: http://www.msf.org The initiative is a virtual organisation with a growing network of academic and R&D expertise at its disposal. The different players involved in DNDi are bringing their knowhow in parasitology and clinical trials, their experience treating neglected patients, and their drug manufacturing capacity. They are pooling these resources to move drugs stuck in the pipeline all the way to the patients themselves. Pharmaceutical companies have a particularly important role to play: they possess vast repositories of molecules, the means to move from development to industrial production, and highly specialised teams of researchers. Their contribution will be crucial to the success of DNDi. Matching Needs and Opportunities DNDi is a needs-driven initiative—in other words, the needs of patients suffering from neglected diseases are paramount in its search for new drugs to treat them. The organisations that make up DNDi have firsthand knowledge of these needs because they work with patients in disease-endemic countries throughout the developing world. The initiative is taking this knowledge of patient needs, matching it with opportunities in R&D, and pushing the most relevant projects through the pipeline. Ultimately, neglected patients will have access to drugs targeting their specific diseases, drugs that were designed with them specifically in mind—such as short-course, low-toxicity treatments that don't require hospitalisation, or tablets to swallow rather than injections. To identify opportunities in R&D that are both relevant to patient needs and that meet required criteria of scientific merit, DNDi is sending out calls for letters of interest to the scientific community via advertisements in journals and posted on the DNDi website ( http://www.dndi.org ). These have already pinpointed several promising projects. DNDi is also proactively contacting scientists working on infectious diseases, and surveying published literature for research of interest. In the Pipeline DNDi's project portfolio currently holds nine projects at different stages of development to address identified needs for the treatment of visceral leishmaniasis, sleeping sickness ( Box 2 ), Chagas disease, and malaria ( Figure 2 ). At discovery stage, DNDi is working on validating the kinetoplastid enzyme dihydrofolate reductase as a potential target for leishmaniasis, trypanosomiasis, and Chagas disease, and on identifying inhibitors of the kinetoplastid enzymes trypanothione reductase and protein farnesyltransferase. It is also conducting high throughput screening on whole cell trypanosomes to discover novel lead compounds. Box 2. New Drugs for Sleeping Sickness Only a few drugs exist to treat sleeping sickness, and they are toxic or difficult to administer. Melarsoprol kills one in 20 patients. Eflornithine requires four daily infusions over 14 days. Given these limited options, DNDi is focusing on identifying new compounds that can cross the blood–brain barrier to treat second stage sleeping sickness. DNDi is using high throughput screening on whole cell trypanosomes to discover novel lead compounds, and is working to identify and optimise inhibitors of the enzyme protein farnesyltransferase. The initiative is working on validating the kinetoplastid enzyme dihydrofolate reductase as a drug target. Identifying trypanothione inhibitors is also relevant to other trypanosome parasites. These are long term projects. Nifurtimox, a drug for Chagas disease, has been used to treat sleeping sickness since the 1970s in some isolated places. It has never been extended to more people because no one has studied its safety or effectiveness. DNDi will assess its short-term usefulness by conducting clinical trials on a treatment combination of eflornithine and nifurtimox. DNDi will continue to explore other short- and medium-term projects. Figure 2 DNDi Projects DNDi's project portfolio contains nine projects spread out across the drug development pipeline for the treatment of leishmaniasis, sleeping sickness, Chagas disease, and malaria. HAT, human African trypanosomiasis (sleeping sickness); VL, visceral leishmaniasis. The R&D of new drugs is time-consuming and expensive if the process starts at the early discovery stage, because of the associated risk of project attrition along the way. DNDi is therefore investing resources in several pre-development and development projects as well. These include developing fixed dose combinations of artesunate/amodiaquine and artesunate/mefloquine for use against chloroquine-resistant malaria in Africa and Asia, respectively; pushing for registration of paromomycin for use against visceral leishmaniasis in Africa; assessing combinations of existing drugs for visceral leishmaniasis; and evaluating the usefulness of nifurtimox in combination with eflornithine in the treatment of sleeping sickness. Advocacy for Change Governments can—some might say should—influence drug development choices. DNDi strongly believes that governments in both developed and developing countries should take an active interest in the R&D of new drugs for neglected diseases. In parallel to its own drug development activities, DNDi is working to raise awareness of the neglected disease crisis among key policy- and decision-makers, for instance the European Commission and the National Institutes for Health in the United States. Conclusion In the poorer countries in the world, over 350 million people are at risk from neglected diseases. Currently available treatments are inadequate or nonexistent, and new solutions are urgently needed. DNDi is working to ensure that the advances of science that have brought health and comfort to wealthy nations also benefit these neglected populations.

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212688

Functional Analysis of RSS Spacers

Based on sheer numbers, microbes should rule the world. Most don't cause disease, but those that do have the advantage of multiplying and mutating at a much faster rate than any multicellular organism can. So how does a slowly reproducing, trillion-celled organism like a human protect itself? By having the right weapon for the job—and that requires an incredibly diverse arsenal. A new study by a team of researchers from Yale University School of Medicine, Duke University Medical Center, and Mount Sinai School of Medicine demonstrates how the creation of that arsenal depends on a complex series of interactions between key genetic elements and proteins during the formation of the white blood cells called lymphocytes. Two heavy hitters of the immune system—B and T cells—each produce unique protein receptors that specifically recognize and mediate the killing of the variety of potential foreign invaders, or antigens, such as bacteria, viruses, and parasites. (B cells make immunoglobulin, or antibodies, and T cells make T-cell receptors.) But these lymphocytes are unlike other cells: instead of making proteins from genes they inherited, they custom-make their genes by recombining fragments of their genes into new configurations. This genetic reshuffling process, called V(D)J recombination, yields the diversity of molecules necessary to combat the billions of different antigens they might encounter. The V, D, and J refer to different clusters of DNA sequences that follow specific rules of recombination. While the products of recombination vary, the method does not. The fragments are spliced and then reassembled in a highly regulated process directed and controlled by a stretch of DNA (called a recombination signal sequence, or RSS) next to the gene fragment. The recombination process, the researchers show, relies on complex interactions among different parts of the signal sequences and the proteins that regulate them at key steps along the recombination pathway. Each RSS is made up of three components: the nonamer, which controls the ability of proteins to bind to the gene fragments and initiate recombination; the heptamer, which directs the splicing of the gene fragment; and the spacer, which regulates how the gene fragments are recombined. Mutations in the DNA sequence of each of the three RSS components show that all play a critical role in the ability of the gene fragments to recombine appropriately. While it has been established that spacers, as their name suggests, ensure that the space between the nonamer and heptamer is correct, the researchers show that spacers also regulate recombination activity by providing protein-binding sites along the DNA sequences that affect recombination. While the nonamer is the most important determinant of recombination, changes in the spacer, these researchers demonstrate, produced dramatic changes in the ability of the gene fragments to recombine. Past studies have shown that recombination depends on the presence and sequence of specific nucleotides, but the quality of that recombination, the researchers say, can't be understood simply by analyzing those nucleotides in isolation. Generally speaking, highly conserved sequences have functional importance. But it would be a mistake, they suggest, to think that just because a nucleotide sequence isn't highly conserved, it's not biologically important. Using a computer model to predict how different protein-gene interactions affect recombination, the researchers demonstrate that a fuller understanding of the process depends on observing how all these elements—including those that aren't highly conserved—interact throughout the recombination process.

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC212688.xml

524029

β3-adrenoceptor agonist prevents alterations of muscle diacylglycerol and adipose tissue phospholipids induced by a cafeteria diet

Background Insulin resistance induced by a high fat diet has been associated with alterations in lipid content and composition in skeletal muscle and adipose tissue. Administration of β3-adrenoceptor (β3-AR) agonists was recently reported to prevent insulin resistance induced by a high fat diet, such as the cafeteria diet. The objective of the present study was to determine whether a selective β3-AR agonist (ZD7114) could prevent alterations of the lipid profile of skeletal muscle and adipose tissue lipids induced by a cafeteria diet. Methods Male Sprague-Dawley rats fed a cafeteria diet were treated orally with either the β3-AR agonist ZD7114 (1 mg/kg per day) or the vehicle for 60 days. Rats fed a chow diet were used as a reference group. In addition to the determination of body weight and insulin plasma level, lipid content and fatty acid composition in gastronemius and in epididymal adipose tissue were measured by gas-liquid chromatography, at the end of the study. Results In addition to higher body weights and plasma insulin concentrations, rats fed a cafeteria diet had greater triacylglycerol (TAG) and diacylglycerol (DAG) accumulation in skeletal muscle, contrary to animals fed a chow diet. As expected, ZD7114 treatment prevented the excessive weight gain and hyperinsulinemia induced by the cafeteria diet. Furthermore, in ZD7114 treated rats, intramyocellular DAG levels were lower and the proportion of polyunsaturated fatty acids, particularly arachidonic acid, in adipose tissue phospholipids was higher than in animals fed a cafeteria diet. Conclusions These results show that activation of the β3-AR was able to prevent lipid alterations in muscle and adipose tissue associated with insulin resistance induced by the cafeteria diet. These changes in intramyocellular DAG levels and adipose tissue PL composition may contribute to the improved insulin sensitivity associated with β3-AR activation.

Background Dietary fatty acids are known to influence the composition of stored triacylglycerol (TAG) and membrane phospholipids (PL) in adipose tissue [ 1 ]. More recently, it was demonstrated that the lipid profile in skeletal muscle reflected dietary lipids [ 2 - 4 ]. Furthermore, the modifications of fatty acid concentrations and composition in tissue lipids induced by a high fat diet has been associated with alterations in lipid metabolism and insulin sensitivity [ 5 , 6 ]. Indeed, enrichment of membrane PL with saturated fatty acids (SFA) was able to impair insulin action in skeletal muscle and adipose tissue, whereas a higher proportion of polyunsaturated fatty acids (PUFA) improved insulin sensitivity in these tissues [ 7 - 9 ]. TAG accumulation in skeletal muscle was also correlated with the development of insulin resistance, independent to the degree of obesity [ 10 - 13 ]. Intramyocellular TAG could represent only a marker of insulin resistance whereas intracellular accumulation of long chain acyl-coenzyme A, ceramide or diacylglycerol (DAG) were reported to directly alter the insulin action [ 14 ]. Chronic activation of the β3-adrenoceptor (β3-AR), which is predominantly expressed in white and brown adipose tissue, by selective agonists exerts both anti-obesity and anti-diabetic effects in rodent models of obesity [ 15 , 16 ]. Activation of this receptor has been reported to enhance energy expenditure via stimulation of thermogenesis in brown adipose tissue [ 16 ]. The improvement in glucose homeostasis induced by β3-AR agonists appears to be a consequence of increased insulin sensitivity in peripheral tissues rather than stimulation of insulin secretion by the pancreas [ 17 ]. Although, the expression of β3-AR in myocytes is still a matter of debate [ 18 - 20 ], obese rats treated with β3-AR agonists demonstrated an improvement of insulin sensitivity in brown and white adipose tissue as well as in skeletal muscle [ 17 , 21 , 22 ]. In adipose tissue this effect is believed to be mediated by the conversion of large adipocytes into small adipocytes, which are more sensitive to insulin [ 21 ]. In skeletal muscle, it seems more likely that the effects of β3-AR agonists on insulin sensitivity are mediated by alternate indirect mechanisms. The objective of the present study was to determine whether a selective β3-AR agonist could prevent alterations in the profile of skeletal muscle and adipose tissue lipids induced with the consumption of the cafeteria diet, previously reported to induce weight gain and hyperinsulinemia [ 22 , 23 ]. The selective β3-AR agonist ZD7114 was used in this study. When administered at 1 mg/kg/day, this compound has been shown to increase thermogenesis in rodents and dogs without increasing heart rate or β2-AR-mediated effects such as tremor [ 24 ]. ZD7114 pharmacological specificity was also demonstrated in brown adipocytes and smooth muscles [ 25 , 26 ]. As expected, we observed that a chronic treatment with the ZD7114 prevented the development of excessive fat mass and hyperinsulinemia induced by this diet. These preventive beneficial effects exerted by the β3-AR agonist were associated with a reduction of muscle DAG accumulation. In adipose tissue, ZD7114 treatment was able to limit the proportional reduction of PUFA into the PL pool, induced by the cafeteria diet. Our results indicate that a β3-AR agonist prevents some cafeteria diet-induced alterations of the fatty acid profile of lipids in skeletal muscle and adipose tissue. Furthermore, we propose that these changes may contribute to the improved of insulin sensitivity observed in rats treated with a β3-AR agonist during the development of obesity. Methods Animal study Male Sprague-Dawley rats were purchased from Charles River (L'arbesles, France) at 5–6 weeks of age. Animals were individually housed in temperature-controlled rooms (22°C) with a 12-h light-dark cycle. Ten days before the beginning of the study, 30 rats were provided a normal chow diet (Kliba-Nafalg, Switzerland) and free access to drinking water. At the end of this period, rats were weighed and pre-selected for their sensitivity to weight gain (i.e. rat presenting at least 35% weight gain after a 10-day selection period). The selected rats (n = 15) were equally randomized into 3 groups. A reference group, consisted of rats fed for 60 days with a standard pellet chow diet containing 28, 57 and 15% E from protein, carbohydrate and fat, respectively (REF group). The remaining rats were fed for 60 days with a cafeteria diet composed of 30 g of a mix containing salami, cookies, cheese, sausage, chips, chocolate and almonds in a proportion of 2:2:2:1:1:1:1 and 30 g of the reference group chow diet. This mixed diet contained 26, 27 and 47% energy as protein, carbohydrate and fat, respectively. At the beginning of the dietary intervention, both groups fed the cafeteria diet received daily, by gavage (0.5 ml/100 g body weight), either the selective β-3AR agonist ZD7114 at 1 mg/kg per day (CAF-ZD group) or water alone (CAF group) until day 60. Rats from the REF group (chow diet) also received a daily gavage of water (0.5 ml/100 g body weight). Body weight and food intake were recorded daily. Rats were fasted for 8 hours before sacrifice, performed under isoflurane anaesthesia. Tissues were immediately collected, weighed, frozen in liquid nitrogen and kept at -80°C until analysed. All procedures in the study were in compliance with the ethical committee of the "Service vétérinaire du canton de Vaud". Estimation of the proportions of the different lipid classes contained in the cafeteria diet (see Discussion) was performed using the USDA Nutrient Database. Lipid fatty acid composition Adipose tissue Fatty acid composition of lipids in adipose tissue was performed by Lipomics Technologies (West Sacramento, USA). Briefly, lipids were extracted from 30 mg of frozen epididymal adipose tissue in the presence of authentic internal standards by the method of Folch et al. [ 27 ] using chloroform:methanol (2:1, by vol.). After separation of individual lipid classes by preparative thin-layer chromatography, the TAG and PL were scraped and trans-esterified; the resulting fatty acid methyl esters were then separated and quantified by capillary gas chromatography as previously described [ 28 ]. Muscle The frozen gastrocnemius muscle was thawed and thoroughly dissected under stereomicroscope to remove extramyocellular adipose tissue. Lipids from lyophilized, finely powdered, dissected muscle (50 mg) were extracted according to the method of Folch [ 27 ]. PL (19:0), TAG (17:0) and DAG (15:0) internal standards (Varian; Zug, Switzerland) were added prior to lipid extraction. The lipids were loaded on a Chromabond NH2 cartridge (Varian; Zug, Switzerland), and neutral lipids were separated from free fatty acids and PL by sequential elution with chloroform/2-Propanol (2:1), 2% acetic acid in diethylether and methanol, respectively [ 29 ]. The neutral lipids were subjected to thin-layer chromatography using hexane:diethylether:acetic acid (70:30:1, by vol.) as solvent system to separate DAG from TAG [ 30 ]. The hydrolysis of TAG into DAG during sample analysis has been assessed and represented less than 0.5% of total DAG. The fatty acids from the phospholipids, TAG and DAG were converted to their methyl esters. Fatty acid methyl-ester separation was performed by automated gas-liquid chromatography (HP 6890 series) with FID detection (280°C); authentic standard mixtures of fatty acid methyl-esters (Nu-Chek-Perp; Lowell Mutter, USA) were injected to identify fatty acid methyl-ester peaks. Results are expressed in μmol fatty acids per gram lyophilised muscle. Plasma metabolites Plasma glucose and insulin were determined with commercially available kits purchased from Sigma (Buchs, Switzerland) and Crystal Chem Inc. (Downers Grove, USA), respectively. Plasma triglycerides and free fatty acids concentrations were analysed using kits from Roche Diagnostic (Basel, Switzerland) and Wako Chemicals (Richmond, USA), respectively. Statistical analysis Comparisons of the means of the dependent variables of each group were performed using a one-way ANOVA. Results Body weight and fat mass Male Sprague-Dawley rats were fed, during 60 days, either a chow diet used as a reference group (REF group), a cafeteria diet (CAF rats) or a cafeteria diet plus a daily gavage of the β3-AR agonist ZD7114 (CAF-ZD rats). At day 60, the mean body weight of CAF rats was significantly greater than that of REF rats (Table 1 ). On the other hand, CAF-ZD rats presented a significant reduction in mean body weight when compared with CAF rats (Table 1 ). Similar effects were observed on weight gain which was 46% greater in CAF rats than in REF rats (Figure 1 ; 213.2 ± 12.2 g vs. 312.3 ± 19.2 g in REF and CAF rats, respectively; p < 0.01), and reduced by 17% in CAF-ZD rats as compared to CAF rats (258.70 ± 12.70 g in CAF-ZD rats, p < 0.05). The enhancement of body weight in CAF rats was strongly associated with the weight increase of two main deep adipose depots, confirming the obesigenic properties of the cafeteria diet. Indeed, the epididymal and retroperitoneal fat pads in CAF rats were respectively 131% and 185% heavier than those of REF rats (Table 1 ). ZD7114 treatment decreased the weight of the two adipose tissue depots by about 45%, compared to CAF rats (Table 1 ). The weights of gastrocnemius skeletal muscle, liver and heart were not different between groups (Table 1 ). As expected, CAF rats had a higher energy intake compared with REF animals (106.03 ± 7.25 vs 78.49 ± 2.96 kcal/day). However, the anti-obesity effect of ZD7114 was not attributed to a reduction of energy intake (107.70 ± 7.30 kcal/day in CAF-ZD rats). Table 1 Body weight, tissues and fasting plasma metabolites REF CAF CAF-ZD Final body weight (g) 443.78 ± 21.42 540.20 ± 16.69 * 477.82 ± 12.09 + Retroperitoneal (g) 3.73 ± 0.44 10.65 ± 0.48 ** 5.78 ± 0.23 + Epididymal (g) 4.65 ± 0.41 10.73 ± 0.43 ** 5.96 ± 0.30 + Gastrocnemius (g) 2.69 ± 0.13 2.78 ± 0.01 2.79 ± 0.03 Heart (g) 1.25 ± 0.07 1.48 ± 0.01 1.46 ± 0.02 Liver (g) 13.53 ± 0.79 16.69 ± 0.15 15.47 ± 0.12 Glucose (mmol/l) 12.80 ± 1.51 12.10 ± 1.02 14.78 ± 1.11 Insulin (μU/ml) 19.75 ± 2.05 98.04 ± 16.01 ** 43.27 ± 8.26 + Fatty acid (mmol/l) 0.47 ± 0.22 0.42 ± 0.68 0.30 ± 0.39 ** Triacylglycerol (mmol/l) 2.27 ± 0.30 4.59 ± 0.58 ** 4.11 ± 0.95 Data are the mean ± SEM. Values significantly different from those obtained in the group of rats fed a chow diet (REF) are indicated by * (p < 0.05) and ** (p < 0.01). Data measured in the group of rats treated with ZD7114 (CAF-ZD) and significantly different from those in the group of rats fed a cafeteria diet (CAF) are shown by + (p < 0.05). Figure 1 Individual weight changes. Body weight was measured in rats (n = 5) fed a chow diet (REF) or a cafeteria diet alone (CAF) or treated with 1 mg/kg/day ZD7114 (CAF-ZD) at the beginning and at the end of the different interventions (day 60). Data are represented as individual values. Glucose and insulin plasma concentrations Measurement of insulin and glucose concentrations in plasma of fasted animals showed that CAF rats presented a marked hyperinsulinemia with a 4.6 fold increase in insulin concentration as compared to REF animals whereas the glucose level was not changed (Table 1 ). ZD7114 treatment limited the hyperinsulinemia induced by the cafeteria diet, as demonstrated by the 2.3 fold decrease in plasma insulin concentrations in CAF-ZD rats compared with the CAF group (Table 1 ). No significant changes in plasma glucose concentrations were observed between these two groups (Table 1 ). Adipose tissue lipids Lipid content PL and TAG contents per gram of tissue were measured in epididymal adipose tissue of rats fed with a chow diet or a cafeteria diet treated or not with ZD7114. No significant change was observed in the concentration of PL and TAG in adipose tissue of CAF rats when compared with REF animals (Table 2 ). Furthermore, ZD7114 treatment did not affect adipose tissue lipid content of CAF rats. Table 2 Adipose tissue lipid content μmole/g tissue REF CAF CAF-ZD Phospholipids 8.29 ± 1.28 10.59 ± 1.06 8.65 ± 0.87 Triacylglycerol 2476.34 ± 68.31 2462.63 ± 41.33 2271.57 ± 122.46 Data are the mean ± SEM. REF: rats fed a chow diet; CAF: rats fed a cafeteria die, CAF-ZD: rats fed a cafeteria diet and treated with ZD7114 Fatty acid composition Analysis of the fatty acid profile shows that cafeteria diet induced an increase in the proportion of monounsaturated fatty acids (MUFA), which was compensated for by a reduction in the percentage of PUFA in both adipose PL and TAG (Figure 2 ). Changes in the proportion of MUFA in PL and TAG were mainly due to the increase of oleic acid (1.7 and 2.0 fold increase in PL and TAG, respectively; Table 3 ). Reduction in the percentage of linoleic acid was mainly responsible for the decrease in proportion of PUFA in both PL and TAG (1.6 and 2.3 fold decrease for PL and TAG, respectively; Table 3 ). The proportions of arachidonic and α-linolenic acids were also slightly decreased in PL and TAG, respectively (Table 3 ). Although the global proportion of SFA was not modified in adipose tissue PL and TAG of CAF rats when compared with REF animals, the percentage of myristic and stearic acids were respectively enhanced in PL and TAG of CAF animals. Figure 2 Proportion of the different lipid classes in adipose tissue. Saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) were measured in epididymal adipose tissue phospholipid (PL) and triacylglycerol (TAG) of rats fed a chow diet (empty bars) or a cafeteria diet (black bars) alone or treated with ZD7114 (grey bars). Data are represented as mean ± SEM, and values significantly different to data measured in rats fed the chow or cafeteria diets are indicated by * (p < 0.05), ** (p < 0.01) or + (p < 0.05), respectively. Table 3 Composition of adipose tissue lipids % mole fatty acids REF CAF CAF-ZD Phospholipids Myristic acid (14:0) 3.04 ± 0.16 3.81 ± 0.19 * 2.96 ± 0.15 ++ Palmitic acid (16:0) 28.70 ± 3.10 30.72 ± 1.83 26.22 ± 1.70 Stearic acid (18:0) 15.99 ± 1.19 16.10 ± 0.75 16.93 ± 0.50 Palmitoleic acid (16:1n-7) 1.17 ± 0.28 1.35 ± 0.31 1.16 ± 0.29 Oleic acid (18:1n-9) 10.74 ± 0.94 18.17 ± 1.60 ** 16.26 ± 1.39 * Vaccenic acid (18:1n-7) 1.24 ± 0.12 0.89 ± 0.07 * 0.98 ± 0.10 Linoleic acid (18:2n-6) 21.21 ± 1.98 13.27 ± 1.20 ** 16.41 ± 1.55 * Eicosadienoic acid (20:2n-6) 2.16 ± 1.37 0.92 ± 0.92 0.37 ± 0.37 Arachidonic acid (20:4n-6) 7.69 ± 0.73 4.47 ± 1.23 * 8.90 ± 1.35 + Triacylglycerol Myristic acid (14:0) 1.23 ± 0.03 1.33 ± 0.08 1.39 ± 0.28 Palmitic acid (16:0) 21.80 ± 0.29 21.48 ± 0.63 21.02 ± 0.65 Stearic acid (18:0) 3.35 ± 0.15 5.27 ± 0.35 ** 4.84 ± 1.68 * Palmitoleic acid (16:1n-7) 2.50 ± 0.27 2.29 ± 0.34 2.44 ± 0.46 Oleic acid (18:1n-9) 24.37 ± 1.04 48.87 ± 1.35 ** 47.09 ± 0.20 ** Vaccenic acid (18:1n-7) 1.98 ± 0.12 1.59 ± 0.12 1.73 ± 1.20 Linoleic acid (18:2n-6) 38.22 ± 1.36 16.59 ± 0.72 ** 18.37 ± 0.16 ** α-linolenic acid (18:3n-3) 2.85 ± 0.16 0.67 ± 0.06 ** 0.71 ± 0.20 ** Data are the mean ± SEM. Values significantly different from those obtained in the group of rats fed a chow diet (REF) are indicated by * (p < 0.05) and ** (p < 0.01). Data measured in the group of rats treated with ZD7114 (CAF-ZD) and significantly different from those in the group of rats fed a cafeteria diet (CAF) are shown by + (p < 0.05) and ++ (p < 0.05). Only main fatty acids are presented. CAF-ZD: rats fed a cafeteria diet and treated with ZD7114. ZD7114 treatment did not change the proportions of any lipid classes measured in adipose TAG of CAF rats (Figure 2 ). However, in PL, the percentage of PUFA was significantly increased by 1.3 fold in CAF-ZD rats compared to CAF animals. This change was essentially due to a two-fold increase in the proportion of arachidonic acid measured in CAF-ZD rats (Table 3 ). A slight reduction in the percentage of myristic acid (1.2 fold decrease) was measured in CAF-ZD rats compared to CAF animals. Skeletal muscle lipids Lipid content PL, diacylglycerol (DAG) and TAG contents were measured in the gastrocnemius of REF animals and CAF rats with or without ZD7114 treatment (Table 4 ). Figure 3 Proportion of the different lipid classes in muscle. Saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) were measured in muscle phospholipid (PL), triacylglycerol (TAG) and diacylglycerol (DAG) of rats fed a chow diet (empty bars), a cafeteria diet (black bars) alone or treated with ZD7114 (grey bars). Data are represented as mean ± SEM, and values significantly different to data measured in rats fed the chow or cafeteria diet are indicated by * (p < 0.05), ** (p < 0.01) or + (p < 0.05), ++ (p < 0.01), respectively. The cafeteria diet clearly induced TAG and DAG accumulation in the gastrocnemius, as demonstrated by the respective 3.4 and 2.0 fold increases observed in CAF vs. REF rats (Table 4 ). Chronic treatment with ZD7114 did not significantly reduce the cafeteria diet induced-accumulation of TAG in muscle. In contrast, DAG accumulation was prevented as indicated by a 1.5 fold reduction of DAG levels in CAF-ZD when compared with CAF rats (Table 4 ). Values of intramyocellular TAG and DAG obtained in the present study (TAG: between 1 to 4 μmol/g fresh muscle; DAG: between 0.5 to 1 μmol/g fresh muscle) were similar to those previously described in rat skeletal muscles [ 31 ] (TAG: between 4 to 5 μmol/g fresh muscle; DAG: between 0.5 to 2.5 μmol/ g fresh muscle). Table 4 Muscle lipid content μmole/g tissue REF CAF CAF-ZD Phospholipids 60.31 ± 4.28 65.62 ± 1.26 70.80 ± 2.77 Triacylglycerol 5.40 ± 0.98 18.63 ± 3.24 ** 15.65 ± 2.57 ** Diacylglycerol 2.07 ± 0.32 4.08 ± 0.24 ** 2.66 ± 0.25 ++ Data are the mean ± SEM. Values significantly different from those obtained in the group of rats fed a chow diet (REF) are indicated by ** (p < 0.01). Data measured in the group of rats treated with ZD7114 (CAF-ZD) and significantly different from those in the group of rats fed a cafeteria diet (CAF) are shown by ++ (p < 0.05). Fatty acid composition Fatty acid profiles were determined in muscle PL, TAG and DAG. The gastrocnemius of CAF rats, compared with the REF group, presented an increase in the percentage of MUFA and a decrease in the proportion of PUFA in both TAG and DAG and to a lesser extent in PL (Figure 3 ). Variations in the percentage of oleic and linoleic acids were, respectively, responsible for the changes in the proportions of MUFA and PUFA in muscle TAG and DAG (Table 5 ). Modifications of the proportions of PUFA observed in muscle PL of CAF rats were due to slight decreases of both linoleic (1.4 fold increase) and docosahexaenoic (22:6 n-3) acids (1.3 fold increase), whereas the percentage of arachidonic acid (20:4 n-6) was increased by 1.2 fold. Table 5 Composition of muscle lipids % mole fatty acids REF CAF CAF-ZD Phospholipids Palmitic acid (16:0) 27.87 ± 0.50 26.17 ± 0.29 26.44 ± 0.57 Stearic acid (18:0) 20.21 ± 0.64 22.37 ± 0.54 * 21.64 ± 0.58 Oleic acid (18:1n-9) 6.84 ± 0.21 9.13 ± 0.48 ** 9.12 ± 0.24 ** Linoleic acid (18:2n-6) 13.09 ± 0.65 8.98 ± 0.51** 9.02 ± 0.28 ** Arachidonic acid (20:4n-6) 11.64 ± 0.46 15.07 ± 0.53** 14.11 ± 0.22 ** Docosatetraenoic acid (22:4n-6) ND 0.61 ± 0.04 0.51 ± 0.02 Adrenic acid (22:5n-3) 2.04 ± 0.07 2.44 ± 0.18 2.10 ± 0.09 Docosahexaenoic acid (22:6n-3) 18.30 ± 0.95 15.23 ± 0.52 * 16.09 ± 0.35 Triacylglycerol Myristic acid (14:0) ND 1.66 ± 0.08 1.53 ± 0.06 Palmitic acid (16:0) 37.13 ± 2.06 25.26 ± 0.95 ** 25.15 ± 0.81 ** Stearic acid (18:0) 7.94 ± 0.31 9.36 ± 0.45 * 9.38 ± 0.65 Palmitoleic acid (16:1n-7) ND 1.51 ± 0.14 1.31 ± 0.13 Oleic acid (18:1n-9) 29.00 ± 0.94 51.36 ± 1.83 ** 50.14 ± 0.93 ** Linoleic acid (18:2n-6) 25.00 ± 1.98 10.86 ± 0.83 ** 12.47 ± 0.66 ** Diacylglycerol Myristic acid (14:0) 1.92 ± 0.09 1.75 ± 0.07 1.85 ± 0.05 Palmitic acid (16:0) 25.55 ± 0.86 22.42 ± 1.22 24.88 ± 0.62 Stearic acid (18:0) 6.71 ± 0.45 8.74 ± 0.63 * 9.71 ± 0.08 ** Palmitoleic acid (16:1n-7) 2.58 ± 0.20 1.59 ± 0.14 ** 1.26 ± 0.07 ** Oleic acid (18:1n-9) 28.90 ± 1.24 52.88 ± 1.25 ** 46.45 ± 1.61 ** ++ Nervonic acid (24:1n-9) 3.92 ± 1.93 1.24 ± 0.42 2.71 ± 0.88 Linoleic acid (18:2n-6) 26.96 ± 2.22 10.14 ± 2.04 ** 8.05 ± 0.69 ** α-linolenic acid (18:3n-3) 1.12 ± 0.18 ND ND Stearidonic acid (18:4n-3) ND ND 1.90 ± 0.41 Arachidonic acid (20:4n-6) 0.78 ± 0.06 0.46 ± 0.06 ** 0.26 ± 0.04 ** + Data are the mean ± SEM. Values significantly different from those obtained in the group of rats fed a chow diet (REF) are indicated by * (p < 0.05) and ** (p < 0.01). Data measured in the group of rats treated with ZD7114 (CAF-ZD) and significantly different from those in the group of rats fed a cafeteria diet (CAF) are shown by + (p < 0.05) and ++ (p < 0.05). Fatty acids representing more than 1% in at least one group are presented. The influence of ZD7114 on the cafeteria-induced modification of fatty acid composition in the three lipid species was evaluated by comparing CAF rats with CAF-ZD rats. While ZD7114 did not affect the fatty acid profile of either TAG or PL in muscle of CAF rats, it induced changes in the fatty acid composition of muscle DAG. Indeed, the proportion of SFA was slightly (1.2 fold increase), but significantly, elevated in DAG of CAF-ZD rats with an increase in the proportion of palmitic and stearic acids (Table 5 ). Furthermore, the proportion of MUFA in DAG of CAF-ZD rat muscles was decreased compared with DAG of CAF rats. This change was essentially attributed to a decrease in the proportion of oleic acid (Table 5 ). Stearidonic acid (18.4 n-3) was only detected in DAG accumulated in muscles of ZD-CAF rats. No differences in the proportion of PUFA between DAG stored in muscles of CAF-ZD or CAF rats were observed (Figure 3 ). Discussion The present study demonstrates that feeding the cafeteria diet to rats not only promotes weight gain, hyperinsulinemia and hypertriglyceridemia, but also accumulation of lipids in skeletal muscle. Although TAG levels per gram of adipose tissue was not affected by the cafeteria diet, this dietary intervention strongly enhanced TAG accumulation in skeletal muscle, as previously described [ 4 , 5 ]. We show that this intramyocellular accumulation of TAG was associated with an increase in skeletal muscle DAG content. This finding is in agreement with the accumulation of DAG observed in human skeletal muscle cells incubated with saturated fatty acids in vitro [ 12 ]. DAG mass was also described to be increased in skeletal muscle biopsies obtained from normal volunteers in whom insulin resistance was produced by raising FFA levels during a lipid infusion [ 32 ]. However, to our knowledge, the present study demonstrates for the first time that a high fat diet, resembling the human Western diet, was able to increase DAG storage in skeletal muscle. Although both TAG and DAG accumulations in skeletal muscle were reported to be correlated with insulin resistance [ 4 , 5 , 33 ], DAG (a precursor of TAG synthesis) is proposed to directly impair insulin sensitivity by inactivating insulin receptor activity through activation of the protein kinase C [ 34 ]. The fatty acid composition of TAG stored in muscle was previously shown to be affected by dietary lipids [ 2 ]. Interestingly, we also find that the fatty acid composition of muscle DAG was modified by the cafeteria diet. Indeed, the increase in MUFA and the reduction of PUFA proportions, measured in both DAG and TAG, reflected the difference in lipid composition of the two diets (+ 19% MUFA and -28% PUFA in cafeteria diet vs. chow diet). It is noteworthy that qualitative changes in DAG were reported to affect the activity of DAG as a secondary messenger, since specificity of its fatty acyl moieties for the activation of protein kinase C has been described [ 35 , 36 ]. Although measurements of insulin sensitivity were not directly accessed in this study, our observation suggests that diet may regulate insulin response in muscle by modifying DAG composition. Fatty acid compositions of muscle and adipose tissue PL were also modified by the cafeteria diet. More specifically, the proportion of PUFA in PL was decreased and MUFA was increased in both tissues. These differences were more pronounced in PL from adipose tissue than from gastrocnemius, indicating that membrane phospholipids in adipose tissue are more susceptible to variations in dietary composition than skeletal muscle. Interestingly, reductions in the proportion of PUFA in PL from myocytes [ 7 ] and adipocytes [ 8 ] were reported to impair insulin action. As previously reported with Trecadrine, a selective β3-AR agonist [ 22 ], chronic administration of ZD7114 prevented the excess weight gain and fat accumulation induced by a cafeteria diet. This present report demonstrates that ZD7114 treatment reduced the hyperinsulinemia elicited by the cafeteria diet (by 2.3 fold), thereby suggesting an improvement of insulin sensitivity in the treated rats. This observation was associated with a significant reduction in the accumulation of skeletal muscle DAG, and a slight, non-significant decrease in TAG. It is tempting to suggest that the decrease in muscle DAG accumulation induced by the chronic administration of a β3-AR agonist could participate in the prevention of hyperinsulinemia. β3-AR agonist treatment significantly modified the fatty acid composition of DAG, causing a 1.2 fold reduction in the proportion of oleic acid. As mentioned before, these modifications in DAG composition could directly regulate insulin action in muscles. Since the presence of a β3-AR in skeletal muscle is still a subject of debate, it is difficult to elucidate the mechanism(s) by which the β3-AR agonist lowers DAG content in muscle and modifies its fatty acid composition. Although the content and the fatty acid composition of TAG stored in adipose tissue was not affected by β3-AR agonist administration, the fatty acid profile of adipose PL was modified by the treatment. The β3-AR agonist was able to restore the proportion of PUFA to a level similar to the REF rats by increasing the percentage of arachidonic acid by 2 fold. As mentioned above, a greater percentage of PUFA in PL has been reported to enhance insulin sensitivity in adipose tissue. The changes in PL composition induced by the β3-AR agonist may be associated with the previously reported improved insulin sensitivity in adipose tissue [ 8 ], and the decrease of hyperinsulinemia measured in the present study. Conclusions Whilst the effectiveness of β3-AR agonists is limited in humans, understanding the metabolic changes affected in rodents will provide insights into mechanisms, underlying insulin responsiveness in humans. The present study demonstrates that β3-AR agonist treatment not only limits the hyperinsulinemia induced by a cafeteria diet, but also partially prevents the associated alterations in adipose and muscle lipid composition. Particularly, activation of the β3-AR limits the intramyocellular DAG accumulation and the decrease in the proportion of PUFA in adipose tissue PL. This combined action may contribute to the beneficial effect of β3-AR agonists on insulin sensitivity. List of abbreviations CAF rats : Rats fed with a cafeteria diet; DAG : Diacylglycerol; MUFA : Monounsaturated fatty acid; PKC : Protein kinase C; PUFA : Polyunsaturated fatty acid; REF rats : Rats fed with a chow diet; SFA : saturated fatty acid. TAG : Triacylglycerol; ZD-CAF rats : Rats fed with a cafeteria diet and treated with the β3-AR agonist ZD7114.

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514707

Treatment of Retinopathy of Prematurity with topical ketorolac tromethamine: a preliminary study

Background Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress to blindness from retinal detachments or severe retinal scar formation. This is a preliminary report of the effectiveness and safety of a new and original use of topical ketorolac in preterm newborn to prevent the progression of ROP to the more severe forms of this disease. Methods From January 2001 to December 2002, all fifty nine preterm newborns with birthweight less than 1250 grams or gestational age less than 30 weeks of gestational age admitted to neonatal intensive care were eligible for treatment with topical ketorolac (0.25 milligrams every 8 hours in each eye). The historical comparison group included all 53 preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000. Results Groups were comparable in terms of weight distribution, Apgar score at 5 minutes, incidence of sepsis, intraventricular hemorrhage and necrotizing enterocolitis. The duration of oxygen therapy was significantly longer in the control group. In the ketorolac group, among 43 children that were alive at discharge, one (2.3%) developed threshold ROP and cryotherapy was necessary. In the comparison group 35 children survived, and six child (17%) needed cryotherapy (Relative Risk 0.14, 95%CI 0.00 to 0.80, p = 0.041). Adjusting by duration of oxygen therapy did not significantly change these results. Adverse effects attributable to ketorolac were not detected. Conclusions This preliminary report suggests that ketorolac in the form of an ophthalmic solution can reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size.

Background Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress in some infants to partial vision loss or blindness from retinal detachments or severe retinal scar formation [ 1 ]. ROP remains as one of the most frequent cause of blindness in children, in particular in countries with infant mortality rates between 10 and 60/1000 [ 2 , 3 ]. Among 177 students attending schools for children with visual impairment in the city were this study was conducted, 107 (60.5%) had ROP [ 4 ]. The incidence of both any acute ROP, and of the more severe stages, varies inversely with gestational age at birth. ROP is unusual (except in the mildest forms) in infants of greater than 31 weeks gestation, and severe complications such as retinal detachment occur in less than one half of one percent of infants greater than 31 weeks gestation. However, more than 80% of infants less than 28 weeks gestation develop some ROP, and around 10% develop "threshold ROP. In threshold ROP more that 40% of the cases progresses to retina folds or detachment, with its consequent blindness. In this stage, ablative surgery (cryotherapy or laser photocoagulation) to the peripheral avascular retina is recommended to reduce the risk of disease progression to retinal detachment [ 5 - 7 ]. Pre-threshold stage has been linked to bad results in the visual function: reduction of visual acuity, short-sightedness, amblyopic, etc. [ 8 , 9 ]. A number of strategies have been developed to try to diminish the progress of ROP, but with limited success. These strategies include antioxidants such as vitamin E [ 10 ], D penicillamine [ 11 ] and allopurinol [ 12 ], reduction of exposure to light [ 13 ] and supplementation with oxygen [ 14 ]. The active disease appears in the premature about 4 to 8 weeks after birth. In this period, the levels of vascular endothelial growth factor (VEGF) increase in the retina, as well as other chemical mediators of inflammation such as platelets activator factor (PAF), prostaglandins (PGs), and eicosanoids which would put again under way the process of vascularization that had stopped in the period of oxidative injury. This vascularization is now degenerated and invasive [ 15 - 17 ]. In models of animal experimentation it was possible to diminish the degree of retinal neovascularization with use of indometacin [ 18 ], dexamethasone [ 19 ], rofecoxib [ 20 ], and bucillamine [ 21 ], and increased activity of cyclooxygenase 2 (COX2) was also demonstrated in vessels of neo-proliferation in retina and its inhibition decreases the neovascularization in 37% [ 20 ]. Ketorolac is a non-steroid anti-inflammatory drug (NSAID) derived from indometacin. Its mechanism of action is developed through the interruption of the synthesis of prostanoids, inhibiting the way of COX 1 and 2 in arachidonic acid metabolism; in this way the tissue levels of prostaglandin F2alfa and thromboxane B2 decrease. Its adverse effects are linked predominantly to their inhibitory action of platelet aggregation. It does not alter either the platelet count, or factors of clotting. High digestive hemorrhage is the principal adverse reaction. Nervous and cardiovascular systems are not generally affected by the use of ketorolac to habitual doses [ 22 - 24 ]. Ketorolac administered as conjunctival topical diminishes prostaglandin E2 concentration in aqueous humor, without modifying the intraocular pressure [ 24 ]. In addition it is effective in uveitis induced by tumors necrosis factor (TNF) [ 25 ]. Ketorolac is unquantifiable in plasma when administered in ophthalmic drops [ 24 ]. Ketorolac ophthalmic solution is usually used in older adults with retinal disorders. It is used to diminish the Cystoid Macular Edema that complicates the surgery of cataracts. In this pathology ketorolac has proved to be effective in diminishing the macular edema and improving the visual acuity, providing evidence that their conjunctival instillation produce effects on the most internal layers of the eye. The ophthalmic use of ketorolac only reports occasional episodes of discomfort and ocular burning [ 26 - 28 ]. The use of ketorolac ophthalmic solution in pediatrics is frequent as an analgesic in corneal abrasions, and in allergic and post surgical conjunctivitis. The FDA recognizes its indication for allergic conjunctivitis, ocular pain, post surgical ocular inflammation, ocular pruritus and photophobia [ 24 , 29 ]. On the basis of experimental evidence and physio-pathogenic rationality, we treated preterm newborns admitted to neonatal intensive care unit (NICU) of our hospital with ketorolac in ophthalmic drops with the aim of decreasing the progression and severity of ROP. This study compares this group of children treated with ketorolac with historic controls to assess the impact of this treatment on the incidence of severe ROP and adverse effects. Methods This is a preliminary report, that compares a cohort of children treated with ketorolac with historic controls that did not received such treatment. From January 2001 to December 2002 all preterm newborns with birthweight less than 1250 g or gestational age less than 30 weeks of gestational age, admitted in the NICU of the University Hospital of Maternity and Neonatology of the city of Córdoba, Argentina, were eligible for treatment with topical ketorolac. The comparison group included all preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000. None of these newborns received treatment with ketorolac. There were no differences either in treatment guidelines, equipment or in the number of physicians and nurses that took care of the patients in the two periods. All the children were examined by the same group of ophthalmologists, which have many years of experience in treating patients with ROP. The international classification for ROP [ 30 ] was used to define the stages of the disease and verified by more than one observer. During the period in which ketorolac tromethamine was used, when risk signs for ROP were identified (zone I incomplete vascularization, vessels only in zone of transition I-II or anomalous ramification and equatorial incurvation of vessels in the avascular – vascular junction) [ 31 ] treatment was started with a drop of ketorolac tromethamine (0.25 mgrs.) every 8 hours in each eye. The treatment continued until they presented signs of threshold ROP and cryotherapy was indicated, or till the resolution of the condition. Parents of these children gave their consent so that their children could receive treatment. The variables considered to assess the comparability among both groups were: birthweight, Apgar score at birth less than 6 at 5 minutes, duration of oxygen therapy, peri-intraventricular hemorrhage (PIVH) equal or greater than 3 degrees, necrotizing enterocolitis (NEC) equal of greater than II degrees, and late sepsis. The analyses included only those children that were alive at discharge, but the number of deaths was reported for both study periods. The presence of undesirable effects of ketorolac such as hemorrhages, oliguresis, local manifestations of intolerance, and conjunctival infection were analyzed. Fischer exact test was used to obtain two tailed p-values. Relative risks (RR) with 95% confidence intervals where also calculated. Adjusted analyses were performed by using Poisson regression with robust estimates [ 32 ]. Statistical analysis was carried out using the statistical software SPSS version 8.0, Stats Direct, and STATA version 8.0. Results During the analyzed period 112 eligible preterm newborns were admitted, 53 between 1999 and 2000 (historic controls) and 59 between 2001 and 2002 (ketorolac group). The number of newborns that died before discharge was similar between groups (see table 1 ). Table 1 presents the characteristic of those alive at discharge. Groups were comparable in terms of weight distribution, Apgar score at 5 minutes, incidence of sepsis, intraventricular hemorrhage and necrotizing Enterocolitis (see table 1 ). The duration of oxygen therapy was significantly longer in the control group (see table 1 ). Table 1 Characteristics of newborns treated with ketorolac and historic controls. Historic controls Ketorolac p** Year 1999 – 2000 Year 2001–2002 N = 35† N = 43† n(%) n(%) Dead before discharge* 18 (34.0) 16 (27.1) 0.54 Birthweight (g) 0.28 <= 750 4 (11.4) 8 (18.6) 751–1000 13 (37.1) 9 (20.9) 1001–1250 18 (51.4) 26 (60.59 Apgar Score < 6 at 5 minutes 10 (28.6) 11 (25.5) 0.80 Oxygen (days) 0.01 0–10 9 (25.7) 21 (48.8) 11–27 10 (28.6) 15 (34.9) >= 28 16 (45.7) 7 (16.3) Sepsis 16 (45.7) 24 (55.8) 0.50 Peri–intraventricular hemorrhage > grade III 8 (22.9) 6 (14.0) 0.38 Necrotizing Enterocolitis >= II degree 5 (14.3) 9 (20.9) 0.56 † Excludes newborns that died before discharge. * N = 53 in historic controls group and N = 59 in ketorolac group, that includes all eligible live newborn during the study period. ** P-value Fisher exact test. In the ketorolac group 45 children were alive at the first ophthalmic control, two died before discharge due to late sepsis, so 43 received treatment with ketorolac. Nineteen children were discharged from NICU with treatment indication, and continued with ketorolac up to 44 weeks of gestational age, when the ophthalmologists considered that the risk for developing ROP was very small. The incidence of threshold ROP in newborns treated with ketorolac was significantly lower (Relative Risk Reduction 86%) than in the control group (see table 2 ). Adjusting for duration of oxygen therapy did not significantly change these results (see table 2 ). Further exploration of this relationship showed that duration of oxygen therapy equal or higher than 28 days was not associated with severe ROP in this data (Relative Risk 1.04, 95%CI 0.25 to 4.51, p = 0.28). This finding explains why duration of oxygen therapy does not act as a confounder. Hemorrhages were not observed in the vitreous after treatment with ketorolac. In four cases hemorrhages in the vitreous were already present at the beginning of therapy and they disappeared after 14 days of treatment. No signs of local intolerance, or conjunctival infection were observed. We did not find hemorrhages in other organs attributable to the drug, or signs of renal failure. Treatment was not suspended in none of the cases and all preterm babies received ketorolac until its interruption due to the resolution of ROP or the indication of cryotherapy. Table 2 Incidence of threshold retinopathy of prematurity in newborns treated with ketorolac and historic controls. Only those alive at discharged included in the analyses. Historic Controls Ketorolac Relative Risk (95%CI) P-value n/N(%) n/N(%) Crude Adjusted* Threshold retinopathy of prematurity 6/35 (17.1) 1/43 (2.3) 0.14 (0.00 to 0.80) 0.041** 0.12 (0.02 to 0.92) 0.04 * Adjusted by oxygen administration using robust Poisson Regression. **Fisher exact test Discussion In this preliminary study we have shown that the incidence of severe ROP was significantly lower in very preterm newborns treated with ketorolac, compared with historic controls not receiving such treatment. These results suggest that administration of ketorolac as an ophthalmic solution might be an effective preventive strategy in patient at risk of developing severe ROP. The study was conducted on non-concurrent patient groups, and changes in the subjects risk profile or quality of care between the two study periods might have an impact in the risk of developing severe ROP. The magnitude of the effect of ketorolac on the incidence of severe ROP found in this study (from 17% in the control group to 2.3% in the ketorolac group) is large and in our opinion unlikely to be explained completely by confounding factors, although this possibility cannot be completely ruled out. To our knowledge, no significant change in the standard of care took place between the study periods, but mortality was lower during the period were ketorolac was used, although the difference was not statistically significant. The groups were comparable in terms of their birthweight distribution, Apgar score, and indicators of severe morbidity. A significant difference was found in duration of oxygen therapy, but in our data there was no association between duration of oxygen therapy and severe ROP, and adjusting for oxygen did not change the results. The principal factor involved in the genesis and severity of ROP is the injury due to O2 radicals; there were no substantial changes in the management of O2 supply to the children of our unit in both periods, the equipment used to measured O2 saturation and to administer the oxygen. The prevalence of severe ROP in our maternity was relatively high, considering that for 1998 the Vermont-Oxford Network reported an incidence of 9.5% for severe ROP and of 57.2% for ROP of any degree [ 33 ], but similar to the one reported by other units in our country and in others countries with similar development [ 34 - 36 ] Ketorolac was apparently safe. No patients in the group that received ketorolac presented oliguric, or with biochemical signs of renal failure during treatment. There were no hemorrhagic manifestations that could be attributed to ketorolac; hemorrhages observed in vitreous evolved favorably with collyrium. Neither local intolerance episodes to the drops nor purulent conjunctivitis among the treated cases were observed. The effect on ROP we have observed is compatible with an impact of Ketorolac in the active phase of neovascularization, a fact that is also observed in numerous reports in animal models of ROP with NSAIDs and steroid drugs administered systemically. The hypothesis of an inflammatory component in active ROP has been quite recently recognized by experts, extensive leukocyte adhesion was observed at the leading edge of pathological neovascularization [ 17 ]. Recent research work with inhibitors of COX2 shows that anti-inflammatory drugs apparently have an impact on the evolution of ROP [ 20 ]. Neufeld and collaborators found high plasmatic profiles of TNF and other cytokines during the phase of installation of the pre-threshold stage for ROP [ 37 ]. COX has a strong angiogenic action in the normal development of retina. The inhibition of COX2 diminishes the angiogenesis in cancer and rheumatoid arthritis [ 38 ]. The ganglion cells of retina secrete PGs that would interact with angiogenic substances as VEGF and Insulin like growth factor. The neo-vessels express a greater concentration of COX2 and inhibitors of COX2 stop the angiogenesis mediated by VEFG, which is the principal factor involved in the neovascularization and which would be regulated by the PGs secreted by ganglion cells of retina and endothelial cells [ 20 ]. The hyperoxia induces liberation of TNF with a powerful inflammatory action; dexamethasone interferes with TNF production attenuating the manifestations of retinopathy by hyperoxia [ 19 ]. Topical ketorolac is very effective in neutralizing uveitis generated by experimental infection with monoclonal TNF or bacterial endotoxins [ 25 , 39 , 40 ]. ROP is a problem that modern medicine has generated due to the survival of very small preterm children and which has been unable to solve so far. About 2% of the children with birthweight less than 1500 grams are blind because of this pathology. Current preventive interventions can prevent blindness in nearly 70% of the cases. Earlier treatment using ablation of the avascular retina in pre-threshold ROP has been proposed [ 41 ], but a preventive measure that could avoid the need of ablation would be much better. Conclusions This preliminary report provides evidence suggesting that ketorolac in the form of an ophthalmic solution can be used to reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. We cannot exclude the possibility of bias been responsible of the observed effect. On the other hand, if confirmed, these results can have important public health implications. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size to provide a definite answer to this research question. Competing interest None declared. Authors' contributions MAV and RM conceived the study. MAV, MLC, EB and RM design the study. MF carried out the revision of the clinical histories. MS carries out the ophthalmologic exams of all the cases. RM verified the ophthalmologic finds. EB carried out the statistical analysis. MAV and EB drafted the article. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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535939

Can mutations in ELA2, neutrophil elastase expression or differential cell toxicity explain sulphasalazine-induced agranulocytosis?

Background Drug-induced agranulocytosis, a severe side effect marked by a deficit or absolute lack of granulocytic white blood cells, is a rare side-effect of the anti-inflammatory drug sulphasalazine. Mutations in the human neutrophil elastase gene ( ELA2 ), causing increased intracellular concentration of this serine protease, inhibits neutrophil differentiation in severe congenital neutropenia (SCN). Since the clinical symptoms of agranulocytosis and SCN are similar, we hypothesized that it may origin from a common genetic variation in ELA2 or that sulphasalazine may affect human neutrophil elastase activity and protein expression. Methods We screened for genetic differences in ELA2 in DNA from 36 patients who had suffered from sulphasalazine-induced agranulocytosis, and compared them with 72 patients treated with sulphasalazine without blood reactions. We also performed in vitro studies of the blood cell lines HL60 and U937 after sulphasalazine exposure with respect to cell survival index, neutrophil elastase protein expression and activity. Results None of the mutations in ELA2 , which previously have been reported to be associated with SCN, was found in this material. Protein expression of human neutrophil elastase in lymphoma U937 cells was not affected by treatment with concentrations equivalent to therapeutic doses. Cell survival of lymphoma U937 and promyelocytic leukemia HL-60 cells was not affected in this concentration range, but exhibited a decreased proliferative capacity with higher sulphasalazine concentrations. Interestingly the promyelocytic cells were more sensitive to sulphasalazine than the lymphoma cell line. Conclusion Neutrophil elastase expression and ELA2 mutations do, however, not seem to be involved in the etilogy of sulphasalazine-induced agranulocytosis. Why sulphasalazine is more toxic to promyelocytes than to lymphocytes remains to be explained.

Background Sulphasalazine (SA) has anti-inflammatory, immunosuppressive and antibiotic actions, and is a component in the therapy of Crohn's disease, ulcerative colitis and rheumatoid arthritis. Bacterial enzymes in the colon split sulphasalazine into sulphapyridine and 5-aminosalicylic acid before it is absorbed. Sulphapyridine acts as a sulphonamide antibiotic, whereas 5-aminosalicylic acid is believed to be the anti-inflammatory metabolite. Common side/toxic effects are vomiting, skin rash and headache. The incidence of the hematological adverse effects associated with sulphasalazine is generally low, but the reactions can be severe and sometimes fatal. The risk of sulphasalazine-induced agranulocytosis, i.e. profoundly depressed circulating neutrophils is highest within the first three months of sulphasalazine-treatment, with a fatality rate of 6.5 % [ 1 ]. Clinical symptoms of agranulocytosis include fever, malaise and susceptibility to infections. Patients with arthritic disorders have a greater risk of developing sulphasalazine-induced agranulocytosis than patients with inflammatory bowel diseases. Severe congenital neutropenia (SCN) and cyclic neutropenia (CN) occur both as inherited and as sporadic diseases. SCN has a constant low neutrophil number if left untreated, whereas CN manifests with cyclic oscillations of neutrophil number with a 21-day cycle. Recently, diverse heterozygous mutations in ELA2 , encoding human neutrophil elastase, have been identified in a majority of the cases with CN and two-thirds of the cases with SCN [ 2 ]. In this study, we hypothesized that sulphasalazine-induced agranulocytosis, with clinical symptoms similar to congenital neutropenia, may arise from genetic variation in the human neutrophil elastase gene. We genotyped 108 sulphasalazine-treated patients for ELA2 , one third which of had experienced sulphasalazine-induced agranulocytosis. We, furthermore, tested for cytotoxic doses of sulphasalazine, and studied protein expression of human neutrophil elastase in sulphasalazine-treated blood cell lines. Methods Subjects Patients were treated with sulphasalazine (Salazopyrin, Pharmacia, Sweden) for inflammatory joint diseases and inflammatory bowel disease. The cases with sulphasalazine-induced agranulocytosis were originally collected through the Swedish Medical Products Agency's register of adverse side effects [ 3 ]. The control group had been treated with sulphasalazine without adverse effects for at least 3 months. From the original patient material consisting of 39 cases and 75 controls, DNA was available for 36 cases and 72 controls. The patient journals were studied for information concerning neutrophil differentiation in bone marrow aspirates. The study was approved by the Ethics Committee of the Medical Faculty at Uppsala University, registration number 95–200. Mutation analysis Genomic DNA was extracted from whole blood using standard techniques. Fragments covering exons 2–5 of ELA2 were amplified by PCR using primer pairs listed in Table 1 . The selection of exons 2–5 and some of the flanking intron sequences was based on previously reported mutations in cases with SCN and CN [ 2 , 4 ], as outlined in Figure 1 . Products for exon 2–5 were amplified with 1.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), activated by 15 min at 95°C followed by 4 cycles 94°C 30 sec, 65°C 30 sec, 72°C 1.5 min and 35 cycles of 94°C 30 sec, 67°C 30 sec, 72°C 1.5 min with a final extension of 10 min at 72°C. The exception was amplification of exon 5, where a 2°C lower annealing temperature was used. All primers contained a consensus M13 sequence to enable sequencing with the same primer, included in BigDye Primer sequencing kit from Applied Biosystems, Stockholm, Sweden. Applied Biosystems 310 analyzer and Sequence Analysis software was used for all sequencing. Thus, the 36 cases and 72 controls were analyzed for genetic mutations in ELA2 . Table 1 Primer sequences for PCR amplification of ELA2 exon 2–5 ELA2 target sequence Primers Exon 2 F 5'-tgtaaaacgacggccagtgggaggggacaggctccttgg-3' Exon 2 R 5'-caggaaacagctatgaccaccgggacgcggggtccgagc-3' Exon 3 F 5'-tgtaaaacgacggccagtcaggcccgtcgccggatggg-3' Exon 3 R 5'-caggaaacagctatgacctccgtcgcagcctccaccct-3' Exon 4 F 5'-tgtaaaacgacggccagtgtgacgcgctgacgatctgt-3' Exon 4 R 5'-caggaaacagctatgaccgcagtaccgggctgggagcg-3' Exon 5 F 5'-tgtaaaacgacggccagtcagtccagcttccccacctt-3', Exon 5 R 5'-caggaaacagctatgaccgacctactgaccattttcaac-3' PCR primers sequences for ELA2 exon 2–5, for following sequencing reactions with BigDye primer, Applied Biosystems. Figure 1 Outline of reported mutations in ELA2 exon-sequences in patients with severe congenital neutropenia and cyclic neutropenia Outline of mutations previously reported [2, 4] in ELA2 exons 1–5. The SNP S173 [6] is indicated as an extended arrow and represents base number 4890 in accession number Y00477 and is a base C→A substitution. Cell culture The lymphoma cell line U937 and the promyelocytic cell line HL-60 (American tissue culture collection) were cultured in Dulbeccos modified Eagles Medium, DMEM (Sigma) supplemented with 10 % fetal bovine serum (SVA, Uppsala, Sweden), L-glutamine and penicillin-streptomycin (Sigma). Western blot Equal numbers (8 × 10 6 ) of U937 cells were grown in 75 cm 2 dishes in complete medium containing 0, 125 and 250 μM sulphasalazine for 24 h. For protein isolation, cells were washed in PBS and lysed in buffer containing 1% Triton X-100, 50 mM Tris-HCl pH 8.0 and protease inhibitor cocktail (Sigma) and were kept on ice for 30 min. Lysates were centrifuged for 10 min at 10 000 × g, and protein concentration was determined using BioRad protein assay. Criterion precast gels (BioRad, Sweden) were used to perform SDS-page with 20 μg protein loaded per well. After gel transfer to a nitrocellulose membrane, the membranes were blocked over night in 5 % dry milk in TBS-Tween. Primary antibody against human neutrophil elastase (Calbiochem, Sweden) was diluted 1:1000 in 5 % dry milk in TBS-T. After 2 h incubation, and four sets of washing, a secondary antibody was added (1:5000) and blots were developed using ECL (ECL Western blotting system, Amersham, Sweden). Western blot analysis of human neutrophil elastase expression was performed twice. Elastase activity assay Cells (HL-60 and U937) treated with 0, 125, 250 and 500 μM of for 24 h were lysed with 100 μl of buffer containing 100 mM Tris-HCl pH 7.4, 1 mM MgCl 2 , 0.1 % Triton X-100. After homogenization, 300 μl of 1.4 M NaCl in 0.1 % Triton X-100 was added and samples were centrifuged at 15 000 × g, for 15 min at 4°C. The supernatants were transferred to new tubes and assayed for elastase activity using Suc-Ala-Ala-Ala-pNA (Sigma) as a substrate. For each assay we took 25 μl sample, mixed with 100 μl buffer containing 100 mM Tris-HCl pH 8.5, 1 M NaCl, 500 mM MgCl 2 and 0.1 % Triton X-100. To this, 50 μl of substrate was added, to a final concentration 1 μM. After 30 min of incubation in room temperature, absorbance was read at 405 nm and the concentration was calculated from a standard curve of elastase (Sigma). Cell survival index For the cell viability assay, we used a fluorometric microculture cytotoxicity assay (FMCA) previously described by Larsson et al [ 5 ]. Briefly, 20 000 cells/well were plated in 96-well plates (NUNC, DK) in complete medium with addition of increased concentrations of sulphasalazine (0, 125, 250, 500, 750 and 1000 μM) and incubated for 72 h in a humidified atmosphere used in regular cell culturing. All samples were plated in triplicates and three wells with cell culture medium served as blanks. As controls we had cells without additions and cells only with solvent, in this case 0.5 M NaOH, with equal molarities as in the wells with the highest sulphasalazine-concentration. At the end of the 72 h incubation period, plates were centrifuged (200 × g, 5 min) and medium was aspirated in a microtitre plate washer, washed with PBS and 100 μl of 10 μg/ml of fluorescein diacetate (Sigma, Sweden), was added. This dye exclusively binds intact cell membranes of viable cells. After 1 h incubation at 37°C, the fluorescence was read in the Fluoroscan 2 (Labsystems OY, Finland) at 480 nm excitation and 530 nm as emission. The results are presented as survival index, defined as fluorescence in test wells/ fluorescence in control wells (blank values subtracted) × 100. Thus, a low numerical value indicates high sensitivity to the cytotoxic effect of sulphasalazine. Effective concentration is defined as the concentration when 50 % of the cells are viable (EC 50 ). Statistics Two-tailed Student's t-test was used to compare subject characteristics and results from cell culture between cases and controls. Frequencies of subject characteristics male versus females was tested with Chi 2 -test with one degree of freedom, using Minitab 14. A p -value less than 0.05 was denoted with (*), p < 0.01 with (**) and was considered as statistically significant. Results Subjects The characteristics of the subjects are presented in Table 2 . The agranulocytosis cases were significantly older than the control patients ( p = 0.023). The white blood cell count (WBC) before sulphasalazine-treatment did not differ between cases and controls, nor did the dose of sulphasalazine. Bone marrow aspirates had been taken from 10 patients (cases). In all samples, the myelopoesis was seriously reduced and a maturation arrest at the promyelocyte-myelocyte stage of neutrophilic differentiation was seen. Table 2 Characteristics of subjects Cases (n = 36) Controls (n = 72) p -value Age range (median) 11–77 (55) 13–90 (47) 0.023 WBC before a 9.3 ± 4.7 8.5 ± 2.5 0.26 Dose of sulphasalazine (gram/day) 2.2 ± 0.6 2.0 ± 0.4 0.13 Male : Female 17 : 19 33 : 39 0.891 a WBC data only available for 28 cases and 67 controls. Cases are defined as the patients treated with sulphasalazine, who were diagnosed with agranulocytosis and controls were defined as patients treated with sulphasalazine without hematological side effects within the first three months of sulphasalazine treatment. The statistics of differences between cases and controls in age, white blood cell count (WBC) before sulphasalazine treatment and dose of sulphasalazine was calculated with 2-tailed Student's t-test. The frequency of males versus females was calculated with Chi 2 test, one degree of freedom. Significance level was set at 0.05. Mutation analysis None of the previously reported mutations in ELA2 was found in this material, although we found a silent single nucleotide polymorphism, called S173 [ 6 ] that corresponds to a C4890A substitution in Genbank accession number Y00477 (marked with extended arrow in Figure 1 ). The incidence of the S173 polymorphism did not differ between controls and cases, 0.31 for both, and S173 has previously been detected in healthy subjects [ 6 ]. No correlation between the S173 polymorphism and white blood count before sulphasalazine-treatment was found (Table 3 ). Table 3 White blood count (WBC), before sulphasalazine treatment, in subjects with or without the S173 polymorphism WBC Subjects with S173 (n = 34) 8.74 ± 2.51 Subjects without S173 (n = 74) 8.66 ± 3.51 White blood count (WBC) was estimated by the local physicians before starting with the sulphasalazine treatment. The average WBC in subjects with or without S173 is presented as the mean value ± SD. It was no statistical difference between the groups. Elastase expression, elastase activity and cell survival after sulphasalazine exposure to HL-60 and U937 By western blot analysis, we analyzed neutrophil elastase protein expression in U937 cells. No difference in human neutrophil elastase expression was detected after treatment with 125 and 250 μM sulphasalazine (Figure 2 ), compared to controls. The elastase activity in HL-60 and U937 cells was not affected by increasing sulphasalazine concentration, ranging from 0 to 500 μM, and expressed as elastase activity/μg protein (data not shown). For the cell survival index, each FMCA experiment was performed three times separately with similar results (inter-assay variation less than 10 %). Concentrations below 250 μM sulphasalazine did not affect the survival index of U937 and HL-60 cells (Figure 3A,3B ), but at 500 μM of sulphasalazine, the survival index of HL-60 cells decreased to a third (Figure 3A ). The U937 was only marginally affected at 500 μM sulphasalazine-concentration, but cellular survival decreased with approximately 40 % at 750 μM of sulphasalazine (Figure 3B ). The effective concentration (EC 50 ) of sulphasalazine was approximately 370 μM for HL-60 cells and 820 μM for U937 cells. Figure 2 Western blot analysis of human neutrophil elastase (hNE) expression after sulphasalazine exposure U937 cells were incubated for 24 h with 0, 125 and 250 μM sulphasalazine, followed by cell lysis and protein isolation. 20 μg of protein was applied in each lane, transferred to nitrocellulose membrane and incubated with human neutrophil elastase antibody. Figure 3 Survival index of HL-60 and U937 cells, incubated with increasing concentrations of sulphasalazine Survival index of HL-60 (A) and U937 cells (B) treated with increasing concentrations of sulphasalazine (0–1000 μM) for 72 h and measured with FMCA. Survival index, defined as fluorescence in test wells/ fluorescence in control wells (blank values subtracted) × 100. Discussion Idiosyncratic drug-induced agranulocytosis can be due to several different mechanisms of action, including immunological, toxic and genetic [ 7 , 8 ]. Toxic drug-induced neutropenia is often dose-dependent, whereas immunological and genetic causes are less related to dose. In our study, bone marrow aspirates from patients with sulphasalazine-induced agranulocytosis revealed maturation arrest of neutrophils at the promyelocyte-myelocyte stage. These findings resemble promyelocytic maturation arrest seen in severe congenital neutopenia (SCN) and cyclic neutropenia (CN) [ 9 ]. In the majority of cases with SCN and CN, germline mutations in the human neutrophil elastase gene ( ELA2 ) are implicated as the primary abnormality [ 2 , 4 ]. The focus of this study is therefore on the human neutrophil elastase gene as a possible cause of sulphasalazine-induced agranulocytosis. We found a coding synonymous polymorphism in ELA2 , which, however, was equally represented among cases and controls. Heterozygous mutations in ELA2 act in a dominant manner, interfering with sub-cellular trafficking of neutrophil elastase, and leading to an accumulation of neutrophil elastase in the cytosol [ 10 ]. For normal neutrophil cell maturation, the proliferative action of the granulocyte colony stimulating factor (G-CSF) is necessary [ 11 ]. When G-CSF is exposed to active elastase enzyme in vitro, G-CSF is rapidly cleaved and rendered inactive [ 11 ]. In theory, SCN and CN are caused by an accumulation of neutrophil elastase, leading to an inactivation of G-CSF and a negative feedback on granulopoiesis, which causes neutropenia. Other proteins, connected to expression and transportation of human neutrophil elastase, have also been linked to SCN disease. In canine cyclic hematopoieses, lack of the intracellular transport protein AP3β causes accumulation of canine neutrophil elastase in the cytosolic compartments [ 12 ], and mutations in ELA2 may disrupt the AP3β-recognition site [ 13 ]. Furthermore, mutations in the proto-oncogene GFI1 , a transcriptional repressor of ELA2 , causes over-expression of neutrophil elastase in mice, thus, making them neutropenic [ 14 ]. During maintenance therapy with sulphasalazine, trough serum sulpha concentration is on average approximately 100 μM at the Department of Clinical chemistry and pharmacology, Uppsala University hospital. To avoid toxic effects, trough serum concentration of sulpha should stay below 600 μM [ 15 ]. Our in vitro data suggest a decreased cell survival of sulphasalazine at concentrations around 500 μM. Interestingly promyelocytic leukemia HL-60 cells were more sensitive to sulphasalazine than lymphoma U937 cells, with EC 50 values of 370 μM and 820 μM, respectively. Human neutrophil elastase expression in lymphoma U937 cells did not differ after sulphasalazine at 125 and 250 μM, indicating that human neutrophil elastase production is not affected by sulphasalazine at subtoxic levels. Conclusions In conclusion, neutrophil elastase does not appear to be involved in the etiology of sulphasalazine-induced agranulocytosis. No causative ELA2 mutations were found, and therapeutic concentrations of sulphasalazine did not increase the expression of human neutrophil elastase. High concentrations of sulphasalazine were toxic to white blood cells in vitro; however, there is no evidence that this toxicity is mediated through human neutrophil elastase. Promyelocytic cells were more sensitive to sulphasalazine than lymphoma cells, and the reason for this difference may also explain sulphasalazine-induced agranulocytosis. Competing interests The author(s) declare that they have no competing interests. Authors' contributions AJ carried out the molecular genetic studies, participated in the sequence alignment, drafted the manuscript and carried out the in vitro experiments. MW participated in the design of the study and performed the statistical analysis. HM conceived the study, and AJ, MW and HM participated in its design and coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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Rubia cordifolia, Fagonia cretica linn and Tinospora cordifolia exert neuroprotection by modulating the antioxidant system in rat hippocampal slices subjected to oxygen glucose deprivation

Background The major damaging factor during and after the ischemic/hypoxic insult is the generation of free radicals, which leads to apoptosis, necrosis and ultimately cell death. Rubia cordifolia (RC), Fagonia cretica linn (FC) and Tinospora cordifolia (TC) have been reported to contain a wide variety of antioxidants and have been in use in the eastern system of medicine for various disorders. However, their mechanism of action was largely unknown. We therefore selected these herbs for the present study to test their neuroprotective ability and the associated mechanism in rat hippocampal slices subjected to oxygen-glucose deprivation (OGD). Methods Hippocampal Slices were subjected to OGD (oxygen glucose deprivation) and divided into 3 groups: control, OGD and OGD + drug treated. Cytosolic Cu-Zn superoxide dismutase (Cu-Zn SOD), reduced glutathione (GSH), glutathione peroxidase (GPx), nitric oxide (NO) was measured as nitrite (NO 2 ) in the supernatant and protein assays were performed in the respective groups at various time intervals. EPR was used to establish the antioxidant effect of RC, FC and TC with respect to superoxide anion (O 2 .- ), hydroxyl radicals ( . OH), nitric oxide (NO) radical and peroxynitrite anion (ONOO) generated from pyrogallol, menadione, DETA-NO and Sin-1 respectively. RT-PCR was performed for the three groups for GCLC, iNOS, Cu-Zn SOD and GAPDH gene expression. Results All the three herbs were effective in elevating the GSH levels, expression of the gamma-glutamylcysteine ligase and Cu-Zn SOD genes. The herbs also exhibited strong free radical scavenging properties against reactive oxygen and nitrogen species as studied by electron paramagnetic resonance spectroscopy. In addition all the three herbs significantly diminished the expression of iNOS gene after 48 hours which plays a major role in neuronal injury during hypoxia/ischemia. Conclusions RC, FC and TC therefore attenuate oxidative stress mediated cell injury during OGD and exert the above effects at both the cytosolic as well as at gene expression level and may be an effective therapeutic tool against ischemic brain damage.

Background It is generally believed that a major portion of post-traumatic neuronal necrosis after brain injury does not result from diffuse primary injury, but rather from a secondary process. The injury appears to trigger a cascade of molecular events that lead to gradual vascular and neuronal tissue degeneration, thus destroying the anatomical substrate necessary for the neurological recovery. A large body of evidence obtained from a wide variety of experimental studies of acute CNS injury strongly suggest that various reactive oxygen species (ROS) and nitrogen species (RNS) have been implicated in the progressive secondary degeneration that follows the injury [ 1 - 3 ]. ROS and RNS have been associated with secondary injury that amplifies the magnitude of final neuronal damage. Both biochemical analyses and studies with transgenic mice has shown that ROS/RNS production persists for many hours after the initial insult. This offers a potential therapeutic window for pharmacologic intervention of clinical relevance. Several classes of pharmacologic mimetics of superoxide dismutase/catalase have been synthesized. Evaluation of these catalytic antioxidants in laboratory models of acute brain injury has shown both robust neuroprotection and a prolonged therapeutic window at doses apparently devoid of neurotoxicity [ 4 , 5 ]. Another important aspect determining extent of oxidative stress mediated post-reperfusion injury subsequent to ischemia is the antioxidant status of the affected tissue as it is of great importance for the primary endogenous defence against free radical attack. Various types of antioxidant enzymes like Cu-Zn superoxide dismutase (SOD), peroxidases such as glutathione peroxidase (GPx), catalase have been reported to be neuroprotective [ 5 , 6 ]. Rubia cordifolia (RC), Fagonia cretica linn (FC) and Tinospora cordifolia (TC) are tropical herbs and have been extensively used in the treatment of various types of haematological, hepatic, neurological and inflammatory conditions [ 7 ]. The antioxidant and anti-inflammatory and immuno-modulatory properties of RC and TC has also been well documented [ 8 - 11 ]. Although RC has been reportedly used as an Ayurvedic medication in a wide variety of conditions, reports regarding the use of FC and TC are not available. In light of the aforementioned properties of RC and TC and relatively scant studies on FC we hypothesized that these herbs may overcome the oxidative stress mediated injury during ischemic neuronal injury via modulating the antioxidant pool of the cells. In order to test this hypothesis we devised a two pronged strategy in the present study to evaluate the effect of the drugs on the status of antioxidant enzymes such as GPx and Cu-Zn SOD, the levels of reduced glutathione (GSH), the principal redox regulator of the cell, and the status of nitric oxide (NO) generation the in the hippocampal slices subjected to oxygen-glucose deprivation (OGD). Methods Reagents All reagents unless stated otherwise were obtained from Sigma Chemical Co. USA, Merck, India and Loba Chemie, India. All animals used were as per the institutional animal ethics committee approval. Preparation of hippocampal slices Method for preparation of slices was similar to those of Taylor & Weber[ 12 ]. Normal New Zealand male Wistar rats weighing between 180–220 gm. were anaesthetized with ether and decapitated, whole brain was removed and placed in ice-cold oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM) NaCl – 125, KCl – 3.5, CaCl 2 – 2.0, MgSO 4 – 1.0, NaHCO 3 – 26, Na 2 HPO 4 – 1.25 and D-glucose – 10 mM. The chilled brain was removed and the hippocampal regions were dissected, slices of 0.6 mm were obtained on a modified Stadie – Riggs microtome and were immersed in oxygenated aCSF between 22–28°C for 120 min to allow the tissue to get stabilized. Induction of in vitro oxygen-glucose deprivation (OGD) hypoxic ischemia Hippocampal Slices were subjected to OGD (oxygen glucose deprivation) [ 12 ]. (Taylor + Weber) by suspending the slices in D-Glucose deficient aCSF equilibrated with 95% Nitrogen gas and 5% CO 2 , until the partial oxygen pressure was less then 20% as that of normoxic aCSF (pO 2 was measured using a blood gas analyzer and was found to be ≤ 35 mm Hg). Slices were incubated in oxygen glucose deficient aCSF for 30 min at 37°C. Transferring the slices to reperfusion chamber containing aCSF started reperfusion. The extent of injury was estimated by assaying LDH release in the medium using an LDH kit. Study groups Hippocampal slices were divided into 3 groups of OGD for present study. a) The overall control group consisted of the slices immediately stabilized for 2 hours at 22–26°C in normoxic aCSF. b) The experimental group consisted of stabilized slices subjected to OGD without any drug treatment. c) The experimental treated group consisted of OGD slices reperfused in the medium (aCSF) containing 1) 2 mg/ml concentration of RC, TC and FC for 30 min. 2) Ascorbic acid and Reduced glutathione equivalent to their content in RC, FC and TC for 30 min. Biochemical assays In all the above study groups the following biochemical parameters were monitored. 1) Free Cu-Zn superoxide dismutase (Cu-Zn SOD) was assayed using the method of Beyer et al[ 13 ]. 2) Reduced Glutathione (GSH) was assayed by the method of Teitz [ 14 ]. 3) Glutathione peroxidase (GPx) was assayed by the method of Paglia et al. [ 15 ]. 4) Nitric oxide (NO) was measured as nitrite (NO 2 ) in the supernatant by the method of Green et al. [ 16 ]. 5) Protein was estimated by the method of Lowry et al. [ 17 ]. Electron Paramagnetic Resonance (EPR) measurement EPR (Magnettech X-band Miniscope MS-100, Berlin Germany) was used to establish the antioxidant effect of RC, FC and TC with respect to superoxide anion (O 2 . ), hydroxyl radicals ( . OH), nitric oxide (NO) radical and peroxynitrite anion (ONOO) generated from pyrogallol, menadione, DETA-No and Sin-1 respectively. All the free radical donors (100 μM – 500 μM) were incubated in phosphate buffered saline (pH 7.4, 37°C) containing the spin-trap, Tempone-H (1 mM), oxidation of which generates 4-oxo-tempo with a characteristic three-line EPR signal centred at 3365 G. Development of this signal was monitored for 60 min from addition of the oxidising species and compared to parallel incubations containing RC, FC and TC (10 or 50 μM, n = 3). The amplitude of the first line of the spectrum was measured; data are expressed in arbitrary units. The EPR parameters for these experiments were as follows: Microwave frequency – 9.4 GHz; microwave power – 20 mW; modulation frequency – 100 kHz; modulation amplitude – 1500 mG; centre field 3365 G; sweep width 50 G; sweep time 20 sec; No. of passes – 1; receiver gain 3E1. Reverse Transcriptase Polymerase chain reaction (RT-PCR) GCLC (Glutamyl – cysteinyl ligase catalytic subunit), iNOS (inducible nitric oxide synthase) and Cu-Zn SOD mRNA was isolated using TriZOL reagent from the hippocampal slices and reverse transcribed to study the effect of RC, FC and TC on the expression status of the above genes in OGD untreated slices after a period of 24 and 48 hours post OGD. Gene expression in treated groups was studied after 24 hours. GAPDH served as the house-keeping gene. After an initial reverse transcription, the cDNAs obtained were amplified using the following respective primers and PCR conditions: GCLC was amplified by 32 thermal cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 mins followed by an extension at 72°C for 10 mins using primers: for ' 5'gtggtactgctcaccagagtgatcct and rev ' 5'tgatccagtaactctgggcattcaca. iNOS was amplified at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min for a total of 27 cycles followed by a 10-min extension at 72°C using primers: for '5'-gtgttccaccaggagatgttg-3' and rev ' 5'-tggggcagtctccattgcca-3'. Cu-Zn SOD was amplified at 94°C for 45 s, 56°C for 30 s, and 72°C for 45 s for a total of 23 cycles followed by a 10-min extension at 72°C using primers: for '5'-tctaagaaacatggcggtcc-3' and rev'5'-cagttagcaggccagcagat-3'. GAPDH was amplified using 20 thermal cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min 30 s, followed by final extension for 10 mins at 72°C, primer used were for' 5'ccacccatggcaaattccatggca and rev 5'tctagacggcaggtcaggtcaacc. Results Intracellular levels of GSH is differentially modulated by RC, FC and TC Figure 1 depicts the effect of the three herbs RC, FC and TC on the intracellular GSH levels in treated rat hippocampal slices post glutamate toxicity. A significant elevation in the GSH level was recorded in untreated controls as compared to the stabilized group (p < 0.001). Where as RC and FC did show an increase in the GSH levels as compared to TC. Figure 1 Effect of RC, FC and TC on the cytosolic GSH levels during OGD in rat hippocampal slices. * p <0.001 compared to stabilized, and + p < 0.001 compared to OGD. Results expressed are a mean of 6 independent experiments ± SEM. Effect of RC, FC and TC on GCLC gene expression Figure 2 shows the effect of RC, FC and TC on GCLC (Glutamyl – cysteinyl light chain) RT-PCR gene expression, all the three herbs were found to be inducers of the gene expression as compared to the untreated controls. Expression of housekeeping gene GAPDH was unaltered in all the lanes. Figure 2 a: Representative GCLC mRNA expression in relation to GAPDH in control, OGD and OGD + drug treated rat hippocampal slices. Lane 1 – Control, Lane 2 – OGD 24 hr, Lane 3 – OGD 48 hr, Lane 4 – OGD + FC, Lane 5 – OGD+TC, Lane 6 – OGD+RC. b: Percentage densitometric expression of GCL mRNA expression in relation to GAPDH in control, OGD and OGD + drug treated rat hippocampal slices. * p < 0.01 and ** p < 0.001 versus control, + p < 0.01 versus * & ** and ++ p < 0.001 versus * & **. Results expressed are a mean of 6 independent experiments ± SEM. RC, FC and TC directly scavenge free radicals RC, FC and TC contain polyphenols, a class of compound that can directly interact with electrophillic species and thus can act as a direct scavenger of free radicals. To test this hypothesis we employed EPR spectroscopy to study the interaction of RC, FC and TC with certain free radicals such as O 2 .- , . OH, NO and ONOO using specific donors. EPR spectroscopy revealed that addition of RC, FC or TC (10 μg/ml) to the O 2 .- generator, pyrogallol (100 μM; n = 3, Fig 3a ) or the . OH generator, menadione (500 μM; n = 3, Fig 3b ), NO generator, DETA-NO (100 μM; n = 3, Fig 3c ) and ONOO generator Sin-1 (100 μM; n = 3, Fig 3d ) in the absence of a spin trap failed to generate a spin signal over a 60 min period. This indicated, either the herbs interacted with the free radicals covalently and therefore yielded no paramagnetic signals or did not interact at all. However, 3-line spin signals characteristic of formation of the stable radical 4-oxo-tempo developed in a time-dependent manner when the same oxidant generating substances were incubated with the recognised spin trap, tempone-H (1 mM). Co-incubation of RC, FC and TC with pyrogallol (100 μM) in the presence of tempone-H caused a significant inhibition of spin signal development over a 60 min time period (p < 0.001, 2-way ANOVA – results of Bonferroni post-hoc analyses and non-linear curve fits are shown on the figure; n = 3). Figure 3 Non-linear curve fit analysis of the effect of RC, FC and TC on scavenging free radicals such as A) O 2 - , B) OH . , C) NO and D) ONOO - generated by pyrogallol, menadione, DETA-NO and SIN-1 respectively. The reference curve fit is depicted in the respective groups. Results expressed are a mean of 3 independent experiments ± SEM. Intracellular levels of GPx are increased by RC, FC and TC Figure 4 depicts the effect of the three herbs RC, FC and TC on the intracellular GPx levels in treated rat hippocampal slices post glutamate toxicity. A significant rise in the GPx level was recorded in treated slices as compared to the stabilized and control group (p < 0.001). Here too RC and FC show an increase in the GPx levels as compared to TC. Figure 4 Effect of RC, FC and TC on the glutathione peroxidase levels in rat hippocampal slices. + p < 0.01 versus stabilized and ++ p < 0.001 versus OGD. Results expressed are a mean of 4 independent experiments ± SEM. Cu-Zn SOD gene expression and enzyme level is upregulated by RC, FC and TC Figure 5 depicts the effect of the three herbs RC, FC and TC on the expression and activity levels of intracellular Cu-Zn SOD in stabilised, untreated and treated rat hippocampal slices post OGD. A significant drop in the SOD level was recorded in untreated OGD slices as compared to the stabilized group (p < 0.001). A significant rise in the levels of both SOD gene expression and the enzyme activity was observed for RC, FC and TC (p < 0.001). Figure 5 Effect of RC, FC and TC on Cu-Zn SOD gene expression and cytosolic free levels during OGD in rat hippocampal slices. RT-PCR pictograph of Cu-Zn SOD is shown aligned to the corresponding groups. * p < 0.001 versus stabilized, + p < 0.001 versus OGD. Results expressed are a mean of 6 independent experiments ± SEM. RC, FC and TC and exogenous antioxidants decrease nitric oxide generation and iNOS gene expression The effect of exogenously added antioxidants namely ascorbic acid and reduced glutathione equivalent to their concentrations found in three herbs RC, FC and TC on nitric oxide generation in comparison to the three herb extracts is shown in figure 6 . All the three herbs along with the exogenously added GSH and Vit C significantly inhibited NO 2 generation in the treated OGD slices. The decrease in the level of NO 2 was found to parallel a decrease observed for the expression of the iNOS gene in the same group of the hippocampal slices (Figure 7 ). Figure 6 Effect of RC, FC and TC and amounts of ascorbic acid and GSH equivalent to that present in the herbs (RC*, FC* and TC* respectively) on NO 2 generation in hippocampal slices. + p <0.001 versus control (100%) and ** p < 0.001 versus OGD. Results expressed are a mean of 3 independent experiments ± SEM. Figure 7 Effect of RC, FC and TC on the iNOS gene expression in rat hippocampal slices subjected to OGD and OGD+RC/FC/TC. Lane 1 – Control, Lane 2 – OGD 24 hr, Lane 3 – OGD 48 hr, Lane 4 – OGD + FC, Lane 5 – OGD+TC, Lane 6 – OGD+RC. *p < 0.05 vs control, **p < 0.001 vs control, +p < 0.001 vs **. Results expressed are a mean of 6 independent experiments ± SEM. Discussion Ischemic cells are known to be under oxidative stress and hence experience oxidative injuries like membrane alterations, lipid peroxidation, increased ionic influx, especially Ca and Na etc. [ 18 - 20 ] elevated levels of O 2 - , NO, NO 2 , decreased activity of Ca 2+ Mg 2+ ATPase and Na + /K + ATPase [ 21 ]. In the present study we have reported the protective effects of RC, FC and TC during OGD insult to rat hippocampal slices. GSH was protected from depletion during the insult and this protection could be correlated to an elevation in the expression of the g-GCS gene. The peroxide scavenging enzyme, glutathione peroxidase (GPx) activity was also significantly restored by the three plant extracts. Treatment of the OGD-hippocampal slices with pure vitamin C and GSH in proportions equivalent to that found in the extracts exhibited a parallel effect on NO generation as to that found with the drugs. RC, FC and TC effectively reduced free radical levels by mechanisms involving increased expression of Cu-Zn SOD, decreased expression of iNOS and simultaneous scavenging of the free radicals such as O 2 - , OH . , NO and ONOO. Overall, RC, FC and TC exhibit potential cytoprotective ability in rat hippocampal slices subjected to OGD. In addition to its role as an antioxidant, the GSH status of a cell is critical for various other biological events that include transcriptional activation of specific genes and modulation of redox-sensitive signal transduction and hence pro-inflammatory processes during cerebral ischemia [ 22 ]. GSH also plays a crucial role in the regulation of expression of several redox-sensitive antioxidant and anti-inflammatory genes [ 23 ], processes which are aggravated especially, post-ischemic insult as a result of reperfusion of white blood cells to the injured area [ 24 ]. As a result there is a rapid loss of reducing equivalents of the cell and hence an onset of oxidative stress. The oxidative stress further leads to the upregulation of expression of a wide variety of pro-inflammatory cytokines, including adhesion molecules, all of which contribute to tissue injury, apoptosis/necrosis [ 25 , 26 ]. Therefore maintenance of GSH pool and other antioxidant levels is critical to cell survival and adaptation to the ischemic injury [ 27 ]. In response to the battery of free radicals generated during ischemia, the cells initially neutralize the oxidative challenge via GSH mediated antioxidant mechanisms. However, a rapid decline in the levels of GSH soon follows which ultimately leads to tissue injury. Therefore it is imperative that any therapeutic intervention should be able to cater to this deficiency observed during ischemia/OGD. Our results show that RC, FC and TC were able to reverse the GSH levels, which was significantly depleted during OGD. This restorative/protective activity of RC, FC and TC could be partly attributed to their native antioxidant contents (GSH = 8.33 ± 0.5, 10.26 ± 0.55 and 6.94 ± 0.49, Vit C = 27.52 ± 0.93, 32.99 ± 1.03 and 41.86 ± 0.68 and Polyphenols = 18.33 ± 2.02, 11.88 ± 1.33 and 21.00 ± 1.26 mg/g extract respectively). However, it was not clear at this juncture as to how much of these antioxidants are actually bio-available, an area which is out of scope of the present study. On the other hand RC, FC and TC may exert such a restorative effect by increasing the synthesis of GSH in the cells. To test this hypothesis we studied the expression status of γ-GCS gene during OGD in both untreated and treated hippocampal slices. RC, FC and TC showed a positive inductive effect on the γ-GCS gene expression after 48 hours. Although significant, it is to be noted that the three drugs exhibited partial differences in their response towards the expression of the GCLC gene expression and in restoration of the GSH levels. This indicates that the drugs may act via other mechanisms that may have a sparing effect on the GSH levels. Scavenging of the free radicals is one such mechanism whereby depletion of GSH is prevented [ 28 , 29 ]. In order to test the later hypothesis we examined whether or not RC, FC and TC could directly interact with the free radicals such as O 2 - , OH . , NO and ONOO using pyrogallol, menadione, DETA-NO and Sin-1 as respective donors. Electron paramagnetic resonance study has revealed a significant scavenging effect of the three drugs on the free radicals chosen for the study. The results were more pronounced for OH . and NO radicals as depicted by the non-linear curve fit analysis. The effects on O 2 - , and ONOO radicals were also found to be quite promising. Therefore, direct scavenging of the free radicals is an important mechanism by which the drugs may exert their cytoprotective effect, not only by sparing GSH utilization by free radicals but also preventing the free radical mediated tissue damage. GSH depletion can also be brought about by its abnormal redirection towards neutralisation of the oxidants. Within the normal metabolic course of the cell, reduction of the prostanoid hydroperoxides to their respective hydroxides, is catalysed by the enzyme glutathione peroxidase (GPx), which requires GSH as one of the co-substrates for the reaction [ 30 , 31 ]. This is basically a protective mechanism of the cell against the harmful lipid peroxides generated during their synthesis and oxidative stress. In the present study we have recorded a significantly decreased GPx activity in OGD hippocampal slices as compared to the controls. The observed decrement may be attributed to the depleted GSH levels either due to increased utilisation and/or diminished activity of γ-GCS. Furthermore, diminished GPx activity, at least in part, indicates cellular accumulation of the lipid hydro peroxides, which can potentially turn on a chain reaction wherein more unsaturated lipids become targets for further peroxidative tissue injury. The ability of RC, FC and TC to enhance the GPx activity is therefore an important finding since one of the protective mechanisms of the herbs under study might be mediated via upregulation of the GPx activity. This effect may further be explained in view of the fact that the herbs themselves contain an appreciable amount of GSH and ascorbate. A large body of evidence suggests that another important intracellular enzyme Cu-Zn SOD is found to be neuroprotective in nature along with GPx and GSH [ 32 , 33 ]. We observed a significant drop in the cytosolic free levels of Cu-Zn SOD in the untreated OGD slices as compared to the stabilized and the three herbs RC, FC and TC significantly restored the levels of the antioxidant enzyme. However, it was not clear at this juncture as to the mechanism involved in such a restoration. Oxidative stress is known to induce the synthesis of Cu-Zn SOD as a defensive mechanism aimed at containing the oxidant levels generated during inflammatory/ ischemic conditions [ 34 ]. Cu-Zn SOD is one of the first lines of defense against free radicals such as O 2 - and NO and acts as a direct scavenger of these extremely potent hazardous species. We therefore investigated whether the antioxidant properties exhibited by the three herbs also involve their modulatory effect on the expression of the Cu-Zn SOD expression. The increased expression of the Cu-Zn SOD gene in RC, FC and TC treated OGD slices clearly confirm the positive modulatory effect the three drugs have on the cellular antioxidant system. Increased expression of Cu-Zn SOD in response to these herbs is a crucial finding especially since this enzyme is directly implicated in the scavenging of not only O 2 - but also the more potent toxicant ONOO [ 35 ]. Thus the three herbs show potent antioxidant properties via modulating the expression of the antioxidant genes such as GCLC and Cu-Zn SOD. The mechanism of antioxidant and neuroprotective effects reported above for RC, FC and TC are new findings. However, as to what components of the drugs bring about such cell-protective effects could not be fully ascertained. Preliminary analysis has revealed that all the three herbs have significant amounts of GSH and Vit C and another important antioxidant, polyphenol. In addition inductively coupled plasma spectroscopic analysis have also revealed the presence of important trace elements in the three herbs (personal observations (Zn- 13.81, 18.19 and 14.94, Cu- 3.16, 9.46 and 13.79, Vd- 30.00, 18.55 and 26.00, Se- 1.79, 3.33 and 0.28 and Mo- 0.39, 1.64 and 1.15 ppm in RC, FC and TC respectively). We therefore thought that the three herbs exert their ameliorative properties due to their antioxidant and trace element contents. In order to test this hypothesis we investigated the effect of GSH and Vit C on NO generation in OGD treated and untreated hippocampal slices. The amount of GSH and Vit C used were equivalent to that found in the three herbs. Our results indicate that GSH and the Vit C components of the herbs are at least partly responsible for the attenuation of NO generation in the treated OGD slices. The effects of RC, FC and TC were almost similar to those recorded for GSH and Vit C. NO is an important neurotransmitter in the brain and also can be rendered harmful due to unprecedented generation during hypoxic/ischemic/inflammatory conditions [ 20 , 36 ]. In addition, NO can combine with O 2 - to form a more toxic ONOO, which is extremely deleterious to the cells [ 37 ]. Hence our observation of the ability of the herbs to attenuate NO generation is not only new but also is indicative of the possible mechanism by which these herbs might exert their protective functions. It is interesting to note that RC, FC and TC also increase Cu-Zn SOD expression thus contributing to the decrease in ONOO formation due to an increased scavenging of O 2 - . Furthermore the three herbs were found to repress the expression of the iNOS gene, which is considered to be an important damaging factor during hypoxia/ischemia [ 37 ]. Thus the decrease in generation of NO by RC, FC and TC is not only due to direct scavenging by the herbs (fig 3 ) but also via the transcriptional modulation of the iNOS gene, which is induced during OGD. Conclusions In conclusion, RC, FC and TC exert cell/neuroprotective properties via preventing the depletion and increasing GSH levels by inducing GCLC expression, by reducing oxidant levels via direct scavenging, decreasing iNOS expression and by increasing the antioxidant gene Cu-Zn SOD. Further protective ability may be attributed to the enhanced activity of GPx brought about by the herbs. The antioxidant contents of the herbs appear to be important components for the observed effects. However, further investigations are required to ascertain the role of individual constituents in the efficacy of the above described properties of the herbs in order to ascribe potential pharmacological applications to these herbs. Competing interests Authors do not have any competing interest with anyone whatsoever. Authors' contributions AKR – This work is a part of AKR's PhD thesis and he has done all the experiments, data collection and processing and manuscript preparation. MGM – He is co-supervisor for AKR and was involved in the ideology, manuscript preparation and providing facilities for the work. SKB – He is the supervisor for AKR and was involved in the main ideology and study design of the work, manuscript preparation and performing EPR studies and training AKR for the respective techniques. Pre-publication history The pre-publication history for this paper can be accessed here:

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555954

Patient-Reported Outcome and Quality of Life Instruments Database (PROQOLID): Frequently asked questions

The exponential development of Patient-Reported Outcomes (PRO) measures in clinical research has led to the creation of the Patient-Reported Outcome and Quality of Life Instruments Database (PROQOLID) to facilitate the selection process of PRO measures in clinical research. The project was initiated by Mapi Research Trust in Lyon, France. Initially called QOLID (Quality of Life Instruments Database), the project's purpose was to provide all those involved in health care evaluation with a comprehensive and unique source of information on PRO and HRQOL measures available through the Internet. PROQOLID currently describes more than 470 PRO instruments in a structured format. It is available in two levels, non-subscribers and subscribers, at . The first level is free of charge and contains 14 categories of basic useful information on the instruments (e.g. author, objective, original language, list of existing translations, etc.). The second level provides significantly more information about the instruments. It includes review copies of over 350 original instruments, 120 user manuals and 350 translations. Most are available in PDF format. This level is only accessible to annual subscribers. PROQOLID is updated in close collaboration with the instruments' authors on a regular basis. Fifty or more new instruments are added to the database annually. Today, all of the major pharmaceutical companies, prestigious institutions (such as the FDA, the NIH's National Cancer Institute, the U.S. Veterans Administration), dozens of universities, public institutions and researchers subscribe to PROQOLID on a yearly basis. More than 800 users per day routinely visit the database.

Review In clinical research it has become increasingly common to assess the patients' perspective of their symptoms and their impact on their daily life as a tool for determining treatment and a means of evaluating the outcome of the treatment chosen [ 1 , 2 ]. The added value of measuring Patient-Reported Outcomes (PRO) starts to be recognized by key players in the field of clinical research [ 3 ]. How patients perceive their health, and the impact of their treatment on their life can provide insight to clinicians previously unavailable [ 4 - 6 ]. However the successful application of PRO studies is dependant on the selection of the appropriate questionnaires for a given application [ 7 , 8 ]. They must be selected according to the domains they measure and the populations and pathologies for which they are designed. Practical issues, such as the availability of different translations, copyrights, and access to instruments are also major criteria in the choice of instruments. The search for the most appropriate instruments is hindered by the substantial increase of PRO questionnaires developed in the past ten years. A recent search on PubMed, matching "quality of life" and "questionnaires" shows a striking growth of 450% between the last two decades (Figure 1 ). Figure 1 PubMed Search: Number of references with Quality of Life-AND-questionnaire from 1966 to 2004 In order to facilitate the selection process the project of a Patient-Reported Outcome and Quality of Life Instruments Database (PROQOLID) was initiated by Mapi Research Trust in Lyon, France. Initially called QOLID (Quality of Life Instruments Database), the project's purpose was to provide all those involved in health care evaluation with a comprehensive and unique source of information on PRO and Health-Related Quality of Life (HRQOL) measures available through the Internet. In collaboration with Dr. Marcello Tamburini (Director, Unit of Psychology, National Cancer Institute, Milan, Italy), the developer of the QLMed.org web site, PROQOLID was launched at the beginning of 2002. PROQOLID was created by the systematic collection of over 470 validated HRQOL and PRO instruments and their subsequent ordering into categories (e.g. pathologies, conditions, population). Through the structured presentation of synthesized, reliable and constantly updated data on PRO instruments, the PROQOLID database aims to present an overview of existing PRO instruments including all relevant and updated information on each. By providing this information the PROQOLID database facilitates access to the instruments and their developers and eases the process of selecting the instrument appropriate for a given application. Instruments can be chosen through a powerful interactive search function that will allow for the rapid selection of an appropriate instrument. What follows is an overview of the proper usage of the PROQOLID web site through a series of FAQ's collected directly from users of the web site. The PROQOLID database can be accessed on the Internet at . Access What are the differences in access to PROQOLID's database between members and guests? The access to PROQOLID is organized in two levels. The guest or free level is available to all visitors at no charge. This level provides brief information for each instrument, including • Full name of the instrument and acronym • Author(s) • Objective • Pathology • Disease • Type of instrument: Coping, Disability/physical functioning, Health status, Psychosocial/psychological, Quality of life, Satisfaction, Social functioning, Symptom/functioning, Utility, Work • Population: Adolescent, Adult, All, Caregivers, Female, Geriatrics, Male, Pediatrics, Terminal patients • Mode of administration: Caregiver-administered, Interviewer-administered, Nurse-rated, Physician-rated, Proxy-administered, Self-administered, Telephone-administered • Number of items • Original language • List of existing translations • Existence of a database: Yes / No • Time recall Since 1995, Mapi Research Trust has been a non-profit organization promoting the use and development of Patient Reported Outcomes. In an effort to accomplish this, a significant part of PROQOLID has been made accessible to all users free of charge. In order to further develop and improve the database and provide additional information on the available instruments, Mapi Research Trust requests a financial participation in order to access PROQOLID'S advanced (or members') level. By subscribing to PROQOLID, you are supporting the continuous collection and update of this unique PRO resource. Membership options are available for pharmaceutical or commercial companies, non-profit organizations, universities, individual academic researchers and students. Benefits to members include a greater degree of practical information on the instruments and, when available, includes the review copy of the instrument, its translations and the user manual, most of them in PDF format. Detailed information of the advanced level available include for each instrument • Name of the instrument (full and abbreviated) • Name and contact information of the Authors • Contact person for information on, or permission to use, the instrument in its original language • Copyright information • Detailed conditions of use (e.g. fee, written permission, user agreement etc.) • Review copy of the original instrument (when possible). Depending on the author's wish, original instruments may be used under specific conditions such as an access fee or signed agreement • Bibliographic references of the original instrument • Contact person for information on, or permission to use the translations • Review copy of the available translations (when possible) • Bibliographic references of the available translations (when possible) • Dimensions covered by the instrument • Time for completion • Age range • Scoring: response options, available scores, weighting, score direction and Minimal Important Difference (MID) or Minimal Clinically Important Difference (MCID) • Existence of a user manual and copy of the user manual (when possible) • Link to the PRO database identification form, when available • Methodology of development • Internal consistency reliability • Related websites • Other bibliographic references Users Who uses PROQOLID? The web site is available to anyone having an interest in the development, availability and use of Patient-Reported Outcomes (PRO). Through the power of the Internet the PROQOLID project intends to provide this information to the world. Every major pharmaceutical company, non-profit organizations such as the US Food and Drug Administration, the NIH's National Cancer Institute, the Veterans Administration as well as dozens of Universities, researchers and students worldwide subscribe to the advanced level of PROQOLID on a yearly basis. The PROQOLID database is routinely visited by over 800 users per day, thereby educating clinicians, researchers, students, and the world about the availability and proper usage of PRO instruments. Content How are the instruments organized in the PROQOLID database? The PROQOLID database was created in an effort to provide a means to facilitate the search process for and provide more efficient searches of any given PRO instrument. By organizing instruments in the PROQOLID database by several easy to understand categories, both time and energy are saved by the user. The different categories can be located on the Search page of the web site or by accessing directly from the tool bar at the top of the page. The different categories are as follows: Alphabetical The purpose of the Alphabetical list is to provide an overview of all existing PRO instruments. Over 1000 instruments are listed in alphabetical order according to their abbreviated name (or acronym). Some of the instruments are only listed, and these are displayed in standard font. For the remaining instruments access is available by simply clicking on the green link containing the abbreviated name of the instrument. Instruments can be accessed through an interactive letterbox at the top of the page. For example if the instrument begins with "D", simply click on the "D" at the top of the page and all instruments beginning with that letter will be displayed alphabetically. Generic Instruments The generic instruments are listed by alphabetic order on a separate web page. Pathology/disease A specific web page is dedicated to each pathology, and the instruments are listed either as generic instruments of the pathology or as disease-specific. The classification is structured based on Medline's Medical Subject Headings (MeSH) to ensure that the concepts are widely accepted. Please note that some diseases may be part of several pathologies. For example the disease "dementia" is part of both the pathologies "Neurology" and "Psychiatry/Psychology". Population The web page lists the instruments as they apply to specific populations including Adolescent, Adult, All, Caregivers, Female, Geriatrics, Male, Pediatrics, and Terminal patients. Author's name Instruments are grouped alphabetically according to the author's name, and as in the alphabetical list a letterbox is provided at the top of the page. Search engine You may search for instruments according to 10 criteria, including the name of the instrument, the pathology, the population or the available languages. The various criteria may be crossed referenced using the following Boolean Operators: AND, OR, NOT. How many instruments are contained in PROQOLID? The PROQOLID database was developed and is updated in close collaboration with the instruments' developers. It currently describes over 470 PRO instruments according to a structured format. The list is currently growing at the rate of fifty instruments per year. The database also includes review copies of 350 original instruments and 350 translations, most of them in PDF format. In order to determine the available translations simply access the instrument on the web site and all existing translations are conveniently listed. Also available through PROQOLID are over 125 associated User Manuals, and the description of 80 separate PRO databases. A fifth update of the whole database is underway and will include new information for each questionnaire on the reproducibility (or test-retest reliability) and clinical validity. In addition the PROQOLID website contains links to 150 external Internet resources relevant to the field of PRO usage and development. A general question and answer section on PRO is also included as well as on line full text articles on the development, validation, and linguistic adaptation of many of the PRO instruments published in medical journals. What are the instruments criteria to be eligible for inclusion in PROQOLID? To be eligible for inclusion in the database an instrument must be the subject of a publication that describes its development and/or validation. There is no charge to authors who wish to insert their instruments in PROQOLID nor are authors paid for their participation in this program. The Mapi Research Trust in Lyon, France determines ultimate decision for inclusion. Search How does the Search Engine work? Besides the search by categories of instruments listed above (i.e. alphabetical list, generic, pathology/disease, population and author's name), an interactive search engine is included in the PROQOLID web site. All of the instruments contained on the web site can be accessed from this location. Searches can be made by: • Abbreviated Name • Full Name • Author • Pathology • Disease • Type of Instrument • Population • Mode of Administration • Inclusion of a PRO Database • Language In an effort to increase the effectiveness of each search the ability exists to include up to nine (9) sub-parameters per search with the stipulation of "AND" or "OR". For example one could enter Author "=" Anderson J "OR" Author "=" Anderson R "AND" Type of Instrument "=" Quality of Life, "AND" Language "=" French, or any other combination that would suit the users needs. Through this function a user is able to drastically narrow the number of instruments displayed in the results window, thereby saving time and effort. If questions exist on the functioning of the search engine, or any questions about the PROQOLID database a contact page is provided with names and e-mail addresses for Mapi Research Trust and individual site managers in both Europe and North America. Additionally a video including a demo of PROQOLID can be seen on the home page. Update Who maintains the PROQOLID database? Mapi Research Trust has maintained the PROQOLID database for three years. The information contained in the website for each instrument is updated at least once a year in collaboration with the instruments' developers and over fifty new instruments are added to the website each year. Mapi Research Trust has listened to the needs of the Pharmaceutical Industry, Industry Regulators, health care professionals, and patients. With the passing of time the organisation has developed into an intricate team of professionals whose single goal is to define and unite the various requirements of each of these groups in order to provide better communication and understanding of each groups needs. PROQOLID achieves to translate these objectives into a concrete application which ultimate goal is the improvement of the patients' quality of life and health outcomes. Conclusion The Patient-Reported Outcome and Quality of Life Instruments (PROQOLID) database aims to present an overview of existing PRO instruments. PROQOLID currently describes more than 470 PRO instruments in a structured format. It includes review copies of over 350 original instruments, 120 user manuals and 350 translations. Most are available in PDF format. The database is updated in close collaboration with the instruments' authors on a regular basis. Fifty or more new instruments are added annually. By providing this information the PROQOLID database facilitates access to the instruments and their developers and eases the process of selecting the most appropriate instrument for a given application. Instruments can be chosen through a powerful interactive search function. The PROQOLID database can be accessed on the Internet at . Authors' contributions MPE conceived the database and participated in its implementation and helped to draft the manuscript. CA helped in the conception of the database and drafted the manuscript. LLP has contributed in the design, coordination and follow up of the database. All authors read and approved the final manuscript.

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545206

Are Anticapsular Antibodies the Primary Mechanism of Protection against Invasive Pneumococcal Disease?

Background Antibody to capsular polysaccharide has been the basis of several vaccines that offer protection against invasive disease from Streptococcus pneumoniae . The success of such vaccines has led to the inference that natural protection against invasive pneumococcal disease is largely conferred by anticapsular antibody. If this is so, one would expect that the decline in disease from different serotypes would vary significantly, and that the appearance of substantial concentrations of anticapsular antibodies would coincide temporally with the decline in age-specific incidence. Methods and Findings Using incidence data from the United States, we show that, on the contrary, the decline in incidence with age is quite similar for the seven most important serogroups, despite large differences in exposure in the population. Moreover, only modest increases in antibody concentration occur over the second and third years of life, a period in which serotype-specific incidence declines to less than 25% of its peak. We also present detailed data on the distribution of antibody concentrations in Israeli toddlers, which are consistent with the United States findings. The same conclusion is supported by new data on age-specific incidence in Finland, which is compared with published data on antibody acquisition in Finnish toddlers. Conclusion We suggest some additional studies of the mechanisms of protection that could distinguish among potential alternative mechanisms, including acquired immunity to noncapsular antigens, maturation of nonspecific immune responses, or changes in anatomy or exposure.

Introduction The protective effects of antibody to pneumococcal capsular polysaccharides have been appreciated since the development of serum therapy, in which passively transferred, serotype-specific antipneumococcal serum reduced mortality from pneumococcal pneumonia by half [ 1 ]. The development of pneumococcal polysaccharide vaccines for adults [ 2 ] and the efficacy of pneumococcal polysaccharide–protein conjugate vaccines in infants and children [ 3 , 4 ] have confirmed that active immunity to the polysaccharide can provide excellent protection against invasive disease from pneumococci of the same serotype, and in some cases protection against cross-reacting serotypes within the same serogroup. While the ability of passive or vaccine-induced anticapsular antibodies to protect against pneumococcal disease is clear, less is known about the natural development of immunity to pneumococcal disease in unimmunized persons. In unimmunized populations, the incidence of invasive disease follows a well-known age distribution, peaking in the first 2 y of life, declining by more than an order of magnitude by the second and third decades of life, and then rising at an accelerating pace, with incidence in persons over 70 y approaching that in infants [ 5 ]. The reason for the decline in incidence has not been conclusively determined, yet it is often suggested that the acquisition of anticapsular antibodies plays a critical role in this decline [ 6 , 7 ]. Indeed, it has been proposed that the human immune system sees each serotype of Streptococcus pneumoniae as a distinct, independent pathogen [ 8 ]. The hypothesis that protection from invasive pneumococcal disease is caused by the acquisition of anticapsular antibodies directed against each of the pneumococcal serotypes yields two simple predictions about the age-specific epidemiology of pneumococcal disease. First, it predicts that the age-specific timing of the decline in invasive disease should be different for different serotypes: those that are rare, poorly immunogenic, or both should decline later in life than those that are common and immunogenic. Second, it predicts that protection against invasive disease from a given serotype should coincide temporally with the acquisition of anticapsular antibody to that serotype, both at an individual level and at a population level. We tested these predictions using data from the United States, Finland, and Israel. Methods United States Dataset Incidence of invasive pneumococcal disease was measured in eight sites around the United States participating in the Centers for Disease Control and Prevention's Active Bacterial Core Surveillance between 1994 and 1999. The data used here are restricted to those periods during which serotyping was routinely performed: 1994–1999 for the Georgia site, 1995–1999 for the Minnesota site, and 1998–1999 for all other sites [ 5 ]. Data were not available on the timing of anticapsular antibody acquisition in these same populations, but we compared the timing of the decline in pneumococcal disease against previously published data on age-specific prevalence of anticapsular antibody levels greater than 0.2 mcg/ml [ 9 ]. Israel Dataset Antibody concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (with absorption by cell wall polysaccharide but not by 22F polysaccharide) in blood samples that were obtained from 130 toddlers at enrollment and at approximately 12 and 24 mo after enrollment in a double-blind, controlled trial of a nine-valent pneumococcal conjugate vaccine. The toddlers analyzed for this study were those in the control group, which received meningococcal C conjugate vaccine; the details of the trial [ 10 ] were previously described. Preliminary analyses of these data confirmed previous findings [ 11 ] that ELISA measurements were highly correlated (and therefore likely revealed cross-reactions) for all pairs of serotypes, except for type 14, for which correlations were minimal, consistent with previous findings of little cross-reaction. For this reason, we chose to analyze age trends only in serotype 14 antibodies. Finland Dataset Mandatory reporting from all microbiological laboratories in Finland to the National Register of Infectious Disease ( http://www3.ktl.fi/stat/ ) identified all blood and cerebrospinal fluid isolates of S. pneumoniae obtained in the years 1995–2001. Incidence within 6-mo age groups was calculated using population denominators obtained from Statistics Finland (Helsinki, Finland). Since the primary purpose of examining incidence in Finland was to compare age-specific rates against published distributions of antibody concentrations for the same age groups [ 12 ], we restricted our attention to serotype 14 and serogroup 6, for which subsequent investigations suggested antibody measurements in Finland were relatively unaffected by cross-reactions [ 13 ] (ELISA measurements for published data from Finland used a special type 6B polysaccharide that was found to minimize cross-reactions [ 13 ]). Results United States Findings Figure 1 shows the age-specific incidence of invasive pneumococcal disease, by capsular serogroup, obtained from population-based active surveillance in the United States prior to the introduction of the conjugate vaccine. Figure 2 shows age-specific incidence by type of infection, for the same age range. Figure 1 Age-Specific Incidence of Invasive Pneumococcal Disease in the United States by Serogroup, Based on Data from Active Bacterial Core Surveillance Serogroups 4 and 23 are shown only up to 48 mo, after which incidence is less than 1/100,000 person-years. All serogroups besides those in the heptavalent vaccine are shown combined as non-vaccine serogroups (NVG). Figure 2 Age-Specific Incidence of Invasive Pneumococcal Disease in the United States by Disease Type, Based on Data from Active Bacterial Core Surveillance Meningitis incidence is plotted only up to 30 mo, after which it remains at or below 1/100,000 person-years. “Pneumonia” indicates bacteremic pneumonia, while “bacteremia” indicates nonfocal bacteremia. “Total” includes other invasive diagnoses. Incidence peaks between the ages of 9 and 15 mo, and falls in an approximately parallel fashion thereafter, for each of the seven most important serogroups (which are those included in the seven-valent conjugate vaccine) and for the remaining serogroups put together. The same pattern is observed for both pneumonia and bacteremia. For each serogroup, incidence by age 24 mo is approximately half that in the peak age group, and by 36 mo, incidence for each serogroup has fallen to 10%–25% of its peak. The consistent timing of the pattern across multiple serogroups argues for a common mechanism, rather than for independent acquisition of immunity to each serogroup as a separate event. Since most individuals do not suffer from invasive pneumococcal disease in this age range, carriage or mucosal disease (otitis media) from pneumococci may be the immunizing event for anticapsular antibodies in the general population [ 12 ] (although in principle immunity to some serogroups could be generated in response to cross-reacting antigens from other bacterial species or other sources [ 14 ]). Different serogroups have vastly different frequencies among pneumococci isolated from carriage [ 12 , 15 , 16 , 17 ] and otitis media [ 12 , 18 ]; for example, serogroups 4 and 18 and the non-vaccine serogroups are isolated far less commonly than several of the other pneumococcal types identified in Figure 1 . One could postulate that these differences in frequency of carriage are offset by differences in immunogenicity; however, there is little evidence that serotypes 4 or 18C are more immunogenic than other, far more common serotypes [ 4 , 15 ]. One could also postulate that the frequency of isolation of serotypes from carriage depends on duration as well as incidence, so that the serotypes for which carriage appears rare are simply carried for a shorter duration. While the data to address this speculation are limited, the duration of carriage of types 4 and 18C seems to be comparable to that of other, more frequently carried serotypes [ 15 , 16 ]. Thus, the most parsimonious interpretation of the data on the timing of the decline in age-specific susceptibility is that one or more common mechanisms are responsible for the decline in disease from all serotypes. Testing the second prediction against data is hampered by the fact that, to our knowledge, no study has characterized the age-specific distribution of antibody concentration in a large population using the currently accepted methodology, which includes absorption with both cell wall polysaccharide and serotype 22F polysaccharide [ 13 , 19 ]. Analyses by Soininen and colleagues have found that antibodies measured by standard ELISA in unimmunized children are highly cross-reactive between different serotypes, and that cross-reactive antibodies lack opsonophagocytic function and often appear in the absence of any documented exposure to a given capsular serotype. As a result, age-specific antibody concentration data for any given serotype are “contaminated,” to a greater or lesser degree, by cross-reactive antibodies with other specificties. The most important exception to this problem occurs for antibodies to serotype 14, for which cross-reaction is minimal [ 11 ]. A recent publication describes the age-specific proportion of children in the United States with anti-type-14 polysaccharide antibody concentration exceeding the putative protective concentration of 0.2 μg/ml ( Figure 3 of [ 9 ]). At 12 mo, 90%–95% of the population falls below this level, and at 24 mo, 80%–85% remains below it—despite a 40%–50% drop in disease incidence from 12 mo to 24 mo. At 36 mo, 75% of children remain below the putative protective level, although by this age incidence has fallen more than 80% from its 12-mo peak. In summary, if the 0.2-μg/ml concentration were truly the threshold for “protection,” the 20%–30% reduction in the unprotected population between ages 12 and 36 mo would be inadequate to explain the 90% decline in disease incidence. Clearly, 0.2 μg/ml is not a precise dividing line between being “protected” and “unprotected,” a threshold that (if it exists) may vary by serotype, but given the available data, there is reason to doubt that anti-type-14 antibody alone is responsible for the decline in disease in this age range. Figure 3 Box-Whisker Plot of Anti-Type-14 Polysaccharide Antibodies in Israeli Toddlers, by 6-Mo Age Groups Central boxes indicate median and 25th and 75th percentiles; whiskers indicate upper and lower adjacent values. Israel Findings To assess whether the limitations of the United States antibody described above—i.e., the availability of only one cutoff point for antibody concentrations—might be providing an incomplete picture of the distribution of antibody levels by age, we examined an additional dataset from Israeli toddlers. For the reasons described above, we examined only antibodies to serotype 14, for which the distribution of concentrations by age is shown in Figure 3 . These data indicate that between the ages of 12–17 and 36–41 mo, the median antibody concentration increases by about 2-fold. These data are broadly consistent with those published for the United States; antibody levels rise very gradually, though detectably, during the second and third years of life. It is difficult to believe—albeit not impossible—that the dramatic declines in disease incidence over these years are explained simply by this small rise in antibody concentrations. Finland Findings Incidence of serogroup 6 and serotype 14 invasive pneumococcal disease by 6-mo age groups in Finland, shown in Figure 4 , is broadly similar to that found in the United States, albeit with lower absolute incidence for both serogroups. Peak incidence occurs in the 12–17-mo age group, and incidence declines to 25%–30% of its peak rate by 24–29 mo of age. This decline in incidence may be compared against the cumulative distributions of antibody concentrations in Finnish toddlers shown in Figure 2 of [ 12 ]. Between ages 12 and 24 mo, there is a discernible increase in the concentration of antibodies in the population, but the median concentration increases by only about 2-fold in this period. Moreover, the proportion of the population with antibody concentration below any particular threshold that may indicate protection changes little in this period. For example, the proportion of the population with anti-type-14 antibody concentrations less than 0.2 μg/ml declines from approximately 55% to approximately 40%, and the proportion with less than 0.5 μg/ml is reduced from about 95% to about 80%. Similar patterns are seen in the Finnish antibody data for type 6B [ 12 ]. Thus, as in the Israeli data, only a very small shift in the distribution of type-specific anti-polysaccharide antibody concentration is observed during the second year of life, yet incidence of invasive disease from the serotypes in question declines substantially. Figure 4 Age-Specific Incidence of Invasive Pneumococcal Disease Caused by Serogroups 6 and 14 in Finland, Based on Active Laboratory-Based Surveillance Discussion We have assessed two lines of epidemiological evidence, analyzed ecologically, that bear on the role of anticapsular polysaccharide antibody as the determinant of protection against invasive disease that develops during the second and third year of life. The simultaneous and approximately parallel nature of the decline in disease incidence for the seven most important serogroups in the United States suggests that one mechanism, rather than seven independent mechanisms, account for the declines in invasive disease from these serogroups. Moreover, only a slight increase in anticapsular antibody concentration is measurable in Finnish, United States, and Israeli toddlers during the same age range. As we discuss below, each of these lines of evidence is subject to caveats, but we believe that, taken together, these observations make a strong case for the importance of one or more factors other than acquisition of anticapsular antibodies in the development of protection against pneumococcal disease. There are several possible candidates for mechanisms that could explain this age-related decline in pneumococcal disease. These include the following: acquisition of antibodies or cellular immune responses to noncapsular pneumococcal “species” antigens; age-related changes in host biology that are not related to acquired immunity, such as maturation of the innate immune system or changes in anatomy or receptors for pneumococcal attachment; changes in other risk factors, such as exposure; or changes related to other microorganisms, including changes in the resident flora or changes in the incidence of viral infections. Systemic antibodies to several pneumococcal protein antigens, which are conserved across pneumococcal strains and serotypes, develop following pneumococcal carriage and otitis media and are present by the beginning of the second year of life [ 20 , 21 ]. In both Finland [ 20 ] and Kenya [ 21 ], there is an increase in the concentration of antibodies to the pneumococcal proteins pneumolysin and pneumococcal surface protein A over the first two or more years of life. In Kenya, antibodies to another conserved protein, pneumococcal surface adhesin A, showed similar distributions in the first, second, and subsequent years of life, while in Finland, levels of these antibodies were already high (equivalent to adult levels) in the first year of life, and increased above these levels in the second year. In mice, either passively transferred human serum IgG against pneumococcal surface protein A or vaccine-induced antibodies to pneumococcal surface protein A and/or pneumolysin are protective against invasive disease. Such data are consistent with the hypothesis that antibodies to these, or perhaps other, conserved pneumococcal proteins are in part responsible for the decline in invasive disease in the second and subsequent years of life. A number of investigators have tested the hypothesis that antibodies to the pneumococcal teichoic acid, known as cell wall polysaccharide (CWPS), are capable of protecting individuals against pneumococcal invasive disease. While studies in animals [ 22 ] and humans [ 23 ] have failed to find a protective effect of antibodies to CWPS or its components, a recent study showed that passive transfer of human IgG against phosphorylcholine, a component of CWPS, could protect mice against invasive pneumococcal infections [ 24 ]. Notably, such antibodies might be elicited by a number of bacteria in addition to pneumococci, such as Haemophilus influenzae, which also produce phosphorylcholine. We are unaware of studies on the timing of acquisition of anti-CWPS antibodies. We have recently shown that mice that are exposed thrice at weekly intervals to intranasal colonization with encapsulated pneumococci are protected against subsequent carriage, that this protection is effective for heterologous as well as homologous capsular types, and that it is effective even in MuMT mice, which lack the ability to produce antibodies (Malley R, Trzcinski K, Srivastava A, Thompson CM, Anderson PW, et al., unpublished data). We have also shown that intranasal immunization with unencapsulated, killed pneumococci protects against nasopharyngeal colonization, in a fashion that is independent of antibody but requires CD4+ T cells at the time of challenge. The relevance of cellular immune mechanisms in protecting humans against pneumococcal colonization or disease is not known. Another candidate for a factor that may be changing with age is susceptibility to viral infections, especially influenza, which may predispose to pneumococcal colonization [ 25 ] or disease [ 26 , 27 ]. Recent evidence from clinical trials of pneumococcal conjugate vaccines shows that the vaccines can reduce the incidence of infections such as bronchiolitis that are usually associated with viruses [ 28 ] and of documented, virus-associated pneumonia [ 27 ]. These findings raise the possibility that the decline in pneumococcal disease with age reflects, in part, a decline in the incidence or severity of viral infections, so that fewer such infections lead to secondary pneumococcal disease. Exposure to pneumococci probably changes in some fashion over the first 5 y of life. However, for changes in exposure to account for the sharp drop in disease incidence following the first birthday, it would be necessary for exposure also to drop severalfold per year over this age range. Studies of pneumococcal carriage do show gradual changes in the prevalence and serotype composition of the nasopharyngeal flora in these years, but the prevalence of carriage changes much more gradually than the incidence of invasive disease [ 29 ]. We are not aware of data that bear strongly on the plausibility of other possible mechanisms for the age-related decline in pneumococcal disease, such as changes in anatomy, physiology, receptor expression, or resident bacterial flora. However, factors other than antibody—such as innate or acquired cellular immune responses, age-related anatomical changes, or changes in exposure to pneumococci—cannot be ruled out, and more than one factor may be involved. Indeed, the peak of pneumococcal meningitis incidence in the 3–6-mo age group (see Figure 2 ) suggests that the mechanism of protection against meningitis may differ from those against pneumonia and bacteremia. Although we suggest that anticapsular antibody is not primarily responsible for the age-specific decline in invasive pneumococcal disease, there is no question that the capsule is an important virulence factor that interacts with the innate and acquired immune system in a number of ways. It is clear that the pneumococcal capsule interferes with various host clearance mechanisms [ 30 ]. It would be unsurprising if different capsular types were differentially effective in permitting pneumococci to evade phagocytosis and other host defenses [ 31 ] (M. Melin, H. Jarva, S. Meri, and H. Käyhty, unpublished data). If this were the case, then one could envision that certain capsular types might in fact follow a different age-specific incidence. In particular, recent analyses suggest that serotypes 1 and 5 have relatively stable incidence over a range of age groups (W. P. Hausdorff, D. R. Feikin, and K. P. Klugman, unpublished data). The evidence adduced here is subject to several limitations. With respect to the relative timing of acquisition of protection against different serotypes, one could postulate that because some of the most common pneumococcal serotypes, such as 6B, 19F, and 23F, are also among the least immunogenic [ 12 ], the effective exposure of the immune system is more consistent across serogroups than it appears from serogroup frequency alone. However, this pattern is not general; for example, serotype 14 is both very common and highly immunogenic [ 12 ]. With respect to the absolute timing of protection relative to the acquisition of antibody, one could argue that low levels of anticapsular antibody, perhaps of low affinity, may be present and even active at levels below those that can be reliably detected by current assays, or that B cell memory may be present and protective at an earlier age than that at which high levels of antibody are measurable. Inferences about protective antibody concentrations from animal studies and from concentrations achieved by vaccines suffer from several uncertainties. Making allowances for all of these limitations, we nonetheless believe the data suggest that mechanisms other than anticapsular antibody are primarily responsible for the age-specific decline in pneumococcal invasive disease that starts at the age of 1 y. The likelihood that mechanisms other than anticapsular antibody confer immunity to pneumococcal disease has important implications with respect to vaccine design. As experience with conjugate pneumococcal vaccines in children unfolds, it is becoming increasingly clear that such a strategy suffers from several limitations, including the possibility of serotype replacement (already confirmed in several clinical trials), a modest effect on nasopharyngeal colonization, limited serotype coverage, cost, and difficulties in production that have led to shortages since licensure. A better understanding of the mechanisms that underlie natural immunity to pneumococcus could pave the way for the development of more effective, species-specific pneumococcal vaccines. Patient Summary Background Streptococcus pneumoniae is a common bacterium that lives in the upper respiratory tract of many children, and some adults. The bacterium generally causes no harm in healthy individuals, but in some circumstances it can cause mild infections, such as ear infections, or more severe ones, such as lung infection (pneumonia), bloodstream infection (bacteremia), or infection of the lining of the brain (meningitis). These more severe forms, called invasive pneumococcal disease, occur especially in children, elderly people, and others with weakened immune systems. The bacterium exists in different versions, or serotypes. The different versions of the bacterium each have a different outer shell (the so-called bacterial capsule). Scientists have developed vaccines against Streptococcus pneumoniae that protect against the most common serotypes. These vaccines consist of a cocktail made up of material from the capsules of the most common serotypes. This material causes the body's immune system to produce antibodies that can fight Streptococcus pnemoniae and protect vaccinated individuals against disease caused by the common serotypes. In many developed countries vaccination is recommended for all children and elderly people. Why Was This Study Done? Most people get exposed to many different versions of the bacterium over the course of their lives. These encounters cause little or no disease in most people, and the risk of disease declines sharply and remains low through middle age, before climbing again in the elderly. Based on experience with vaccines, scientists have thought that this “natural” protection that develops with age was also based on antibodies against the bacterial capsule. The authors of this study wanted to test whether this was actually true. What Did the Researchers Do? If in the healthy population protection against invasive disease is in fact due to anticapsular antibodies, one can make certain predictions about the frequency of invasive disease among certain age groups. The researchers tested those predictions against actual disease records from the United States, Israel, and Finland. What Did They Find? The actual records did not match the predictions very well, suggesting that natural protection against invasive pneumococcal disease is not based on anticapsular antibodies alone. What Does This Mean? These results suggest that there are elements of natural protection against invasive pneumococcal disease that we do not understand yet. Moreover, these elements seem to involve more general protection against various forms of the bacterium rather than individual protection against particular serotypes. What Next? Given the importance of the disease, we should try to understand all elements of natural protection. Such understanding might help researchers develop better vaccines to prevent invasive pneumococcal disease, and maybe even improve treatment of patients who have become ill. More Information Online World Health Organization information page on pneumococcal vaccines: http://www.who.int/vaccines/en/pneumococcus.shtml United States Centers for Disease Control and Prevention factsheet on pneumococcal vaccine: http://www.cdc.gov/nip/publications/VIS/vis-PneumoConjugate.pdf Health Canada information on pneumococcal vaccine: http://www.hc-sc.gc.ca/english/iyh/medical/pneumococcal.html Information for health-care providers from the United Kingdom Nation-al Health Service: http://www.prodigy.nhs.uk/guidance.asp?gt=Immunizations%20-%20pneumococcal PneumoADIP Web page on childhood pneumococcal disease: http://www.pneumoadip.com/

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524175

Single nucleotide polymorphisms in the apolipoprotein B and low density lipoprotein receptor genes affect response to antihypertensive treatment

Background Dyslipidemia has been associated with hypertension. The present study explored if polymorphisms in genes encoding proteins in lipid metabolism could be used as predictors for the individual response to antihypertensive treatment. Methods Ten single nucleotide polymorphisms (SNP) in genes related to lipid metabolism were analysed by a microarray based minisequencing system in DNA samples from ninety-seven hypertensive subjects randomised to treatment with either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the β 1 -adrenergic receptor blocker atenolol for twelve weeks. Results The reduction in blood pressure was similar in both treatment groups. The SNP C711T in the apolipoprotein B gene was associated with the blood pressure response to irbesartan with an average reduction of 19 mmHg in the individuals carrying the C-allele, but not to atenolol. The C16730T polymorphism in the low density lipoprotein receptor gene predicted the change in systolic blood pressure in the atenolol group with an average reduction of 14 mmHg in the individuals carrying the C-allele. Conclusions Polymorphisms in genes encoding proteins in the lipid metabolism are associated with the response to antihypertensive treatment in a drug specific pattern. These results highlight the potential use of pharmacogenetics as a guide for individualised antihypertensive treatment, and also the role of lipids in blood pressure control.

Background Hypertension is a complex trait caused by multiple environmental and genetic factors interacting through the cardiac, vascular and endothelial systems. Several drug classes with different mechanisms of action, including inhibitors of the renin-angiotensin-aldosterone system (RAAS), calcium channel blockers, adrenergic receptor blockers and diuretics, are available for treatment of hypertension. However, the response to antihypertensive treatment is highly variable between individuals, which makes it difficult to predict the efficacy of a specific drug in the individual patient [ 1 - 3 ]. Currently, there are no clinically useful biochemical or metabolic markers for predicting the individual responses to antihypertensive treatment [ 4 - 6 ]. Twin studies have estimated that as much as half of the variability in blood pressure levels between individuals is due to genetic factors [ 7 , 8 ]. Based on the abundance of single nucleotide polymorphisms (SNPs) in the human genome [ 9 ], it can be expected that one or more SNPs occur in each of the genes encoding components of the blood pressure regulating systems, and that they are the genetic factors influencing individual blood pressure levels. Coding SNPs affecting the function of enzymes and receptors in pathways of blood pressure regulation, or regulatory SNPs, affecting the expression levels of genes, are likely to explain part of the variability of the response to antihypertensive treatment. Hence, these functional SNPs, or other SNPs inherited in linkage disequilibrium with them, could be potential pharmacogenetic markers for predicting the response to a certain drug, and thus guide the selection of the optimal drug for each individual patient [ 10 - 12 ]. The RAAS and the sympathetic nervous system play key roles in blood pressure regulation. We have earlier shown that polymorphisms in the angiotensin converting enzyme gene [ 13 ] and a SNP in the aldosterone synthase gene [ 14 ] are related to changes in blood pressure during treatment with the angiotensin II receptor blocker irbesartan, whereas two SNPs in the angiotensinogen gene were associated to the reduction in blood pressure by the β 1 -adrenergic receptor blocker atenolol [ 15 ]. Dyslipidemia with high levels of serum triglycerides and free fatty acids, and elevated serum cholesterol levels and low levels of high-density lipoprotein cholesterol are common in hypertensive patients. Association has been found between disturbance in lipid metabolism and hypertension, but so far no attempts have been made to relate variables reflecting lipids, or the genes involved in lipid metabolism, to the individual response to antihypertensive treatment. We have recently developed a microarray based minisequencing system for parallel genotyping of multiple SNPs in blood pressure regulating candidate genes [ 16 ]. Here we analysed the relationships between the genotypes of SNPs in the apolipoprotein A-IV, apolipoprotein A-V, apolipoprotein B-100, low density lipoprotein receptor, hepatic lipase and lipoprotein genes and reductions in blood pressure in hypertensive patients randomised to monotherapy with either irbesartan or atenolol. We found that SNP alleles in the apolipoprotein B gene and the low density lipoprotein receptor gene were associated to the antihypertensive response after twelve weeks of treatment. Methods Study population DNA extracted from blood samples from 97 hypertensive patients from the double blind parallel group "Swedish Irbesartan Left Ventricular Hypertrophy Investigation versus Atenolol" (SILVHIA) trial [ 17 ] were analysed. Men and women above the age of 18, having primary mild to moderate hypertension and left ventricular hypertrophy were enrolled in the trial and randomised to receive either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the β 1 -adrenergic receptor blocker atenolol once daily as monotherapy. The dose was doubled after six weeks if the diastolic blood pressure was ≥ 90 mmHg. Blood pressure was measured by trained nurses using a mercury sphygmomanometer, after the patients had rested for at least 10 min in the seated position. Left ventricular hypertrophy was defined as left ventricular mass index of > 131 g/m 2 for men and > 100 g/m 2 for women, assessed by echocardiography. The data presented relates to the change in blood pressure after 12 weeks of treatment. For details on the SILVHIA trial, see Malmqvist etal [ 17 ]. Baseline characteristics for the patients are presented in Table 1 . The study was approved by the ethics committees of all participating centres of the SILVHIA trial and that of the Medical Faculty of Uppsala University. Table 1 Characteristics of the hypertensive patients in the two treatment groups. Irbesartan group 2 Atenolol group 2 Number of patients 48 49 Age (years) 54 ± 8 54 ± 8 Gender (proportion females) 37% 31% Height (m) 1.74 ± 0.09 1.73 ± 0.09 Weight (kg) 83 ± 15 82 ± 14 Smokers trial start (%) 29 18 Baseline fs-cholesterol (mM) 6.1 ± 1.0 5.8 ± 1.1 Baseline fb-glucose (mM) 5.7 ± 3.1 5.2 ± 2.5 Pre-treatment SBP 1 (mmHg) 164 ± 18 160 ± 20 Pre-treatment DBP 1 (mmHg) 104 ± 7 103 ± 8 Change in SBP at 12 weeks (mmHg) -16 ± 20 -11 ± 16 Change in DBP at 12 weeks (mmHg) -9.0 ± 11 -12 ± 7.7 1 Systolic blood pressure (SBP) and diastolic blood pressure (DBP) 2 Data are mean ± SD SNP markers and genotyping procedure In our previous study [ 16 ], 98 SNPs were selected from the NCBI (dbSNP, ) and the SNP Consortium (TSC, ) databases and validated in a pooled DNA sample representing the Swedish population. A subset of these SNPs located in genes involved in lipid metabolism and that were polymorphic in the Swedish population were included in the study presented here, together with one additional SNP in the apolipoprotein A-V gene. See Table 2 for information on the SNPs. Table 2 Investigated polymorphisms given as gene names, acronym and GenBank accession number. Gene name and acronym 1 dbSNP ID 2 Amino acid alteration SNP name 3 Apolipoprotein A-IV rs5092 Thr/Thr A1449G APOA-IV; J02758 Apolipoprotein A-V rs662799 Promoter C31455T APOA-V; AC074203 Apolipoprotein B-100 rs1801701 Arg/Gln G10108A † APOB; M19828 † ; M19810 § rs1367117 Thr/Ile C711T § Low density lipoprotein receptor rs688 Asn/Asn C16730T LDLR; AF217403 rs5925 Val/Val C2000IT Lipase, hepatic rs6083 Asn/Ser A110G LIPC; M35429 Lipoprotein rs328 Ser/Term C9040G LPL; AF050163 rs312 Intron G7315C rs314 Intron A7360G 1 Gene name and acronym, GenBank accession number for the sequence used in the design of primers for the PCR and minisequencing reactions. 2 SNP identification number in the NCBI SNP database dbSNP, . 3 Corresponding to the nucleotide position in the gene sequence referenced in the first column and the nucleotide variation in the coding strand. Fragments comprising the SNPs were amplified in multiplex PCR described previously [ 16 ]. A microarray based minisequencing single nucleotide primer extension assay, in which one or two of four ddNTPs labelled with the fluorophore Tamra (Perkin Elmer Life Sciences, Boston, MA, USA) were incorporated by the Thermo Sequenase™ DNA-polymerase at each SNP site. The incorporated ddNTPs were detected using a fluorescence scanner, and the fluorescence signals were extracted. A signal intensity fraction, obtained by dividing the fluorescence signal intensity for allele 1 with the sum of the fluorescence signal intensities for allele 1 and allele2, was used to assign the individual genotypes. The SNP APOA-V C31455T was genotyped using a microtiter plate minisequencing assay with tritium detection [ 18 ]. Statistical analyses Analysis of covariance (ANCOVA) with each SNP as factor, baseline blood pressure as covariate and the change in blood pressure as response, was performed. The analyses were performed by treatment group and blood pressure measurement (systolic and diastolic blood pressures). Correction for multiple testing was performed by calculation of critical p-values corresponding to a nominal type I error of 5% using a permutation test [ 19 ]. Two tailed significance levels were used. Results and discussion We explored possible associations between individual genotypes of ten SNPs and reduction in systolic and diastolic blood pressure as response to treatment with atenolol or irbesartan (Figure 1 ) in samples from the SILVHIA trial [ 17 ]. In the irbesartan group, a change in systolic blood pressure appeared to be related to genotype for the SNPs ApoA-IV A1449G, ApoA-V C31455T and ApoB C711T. In the atenolol treatment group, presence of the C-allele of the SNP LDLR C16730T was associated to the reduction in systolic blood pressure. Figure 1 Effect of SNP genotype on the change in blood pressure after 12 weeks of treatment for the ten SNPs. For each of the SNPs, the pattern of change in blood pressure related to genotype is illustrated for the systolic blood pressure (SBP) and diastolic blood pressure (DBP) in separate panels. In each panel, the mean change in blood pressure is shown for the SNP genotypes in the atenolol treatment group on the left, and the corresponding results in the irbesartan treatment group are given on the right. The error bars corresponds to the standard error of the mean. The p-values indicating significance for the APOA-IV A1449G, APOA-V C31455T, APOB C711T and LDLR C16730T SNPs are given in the corresponding panels. The number of individuals of each genotype is shown above the bars in each panel. Correction for multiple testing using a permutation test [ 19 ] yielded critical p-values of 0.004 and 0.007 for systolic blood pressure after atenolol and irbesartan treatment, respectively, and 0.006 and 0.007 for the diastolic blood pressure, corresponding to the significance level of p = 0.05. After the permutation test, carriership of the C-allele of the C711T SNP in the apolipoprotein B gene remained significantly associated to the reduction in systolic blood pressure (p = 0.004) in the irbesartan treatment group (Figure 1 ) while the individuals homozygous for the T-allele showed no reduction in systolic blood pressure. The same pattern of response related to genotype was seen for diastolic blood pressure, although it did not reach statistical significance. In the atenolol treatment group, the SNP C16730T in the low density lipoprotein receptor gene showed a trend of association to the reduction in systolic blood pressure. Presence of the C-allele was related to blood pressure reduction (p = 0.006), while the individuals homozygous for the T-allele (n = 9) actually showed an increase in systolic blood pressure (Figure 1 ). A similar response pattern was not seen for the diastolic blood pressure during atenolol treatment (p = 0.44) (Figure 1 ). There were 39 carriers of the favourable C-allele of the APOB C711T SNP in the irbesartan treatment group. The average reduction in systolic blood pressure for these individuals was 19 mmHg, compared to 0 mmHg for the individuals lacking this allele. In the atenolol treatment group, the individuals carrying the favourable C-allele of the SNP LDLR C16730T showed an average reduction of 14 mmHg in systolic blood pressure compared to an increase of 7.5 mmHg for the individuals homozygous for the T-allele. The SNP C711T in the apolipoprotein B gene is located in the coding region of the gene, and alters a threonine residue to an isoleucine residue in the protein. This SNP is located in the amino-terminal part of the enzyme and has been suggested to affect the dimerisation of apolipoprotein B and low density lipoprotein during cholesterol transport [ 20 ]. The C16730T SNP in the LDLR gene results in a synonymous amino acid change, however this SNP could be in linkage disequilibrium with another functional SNP potentially influencing the response to drug treatment. Irrespectively if the tested SNPs are actually functional, our findings imply a potential connection between lipid metabolism and response to antihypertensive treatment. We have recently found circulating apolipoprotein B to be the most powerful predictor of endothelium-dependent vasodilation of the commonly used markers of cholesterol metabolism [ 21 ]. It is not evident, however, why apolipoproteins predict the response to irbesartan, and not to atenolol treatment, as these drugs appear to improve endothelium-dependent vasodilation to a similar extent [ 22 ]. Lipid abnormalities, commonly seen in hypertension, have been considered to be connected to the blood pressure level by the common denominators obesity and insulin resistance. Other studies have suggested a more direct effect of lipids in blood pressure control, as infusion of the fat emulsion Intralipid together with heparin increases blood pressure in healthy subjects [ 23 - 25 ]. This effect is more pronounced in the normotensive subjects with a family history of hypertension [ 26 ]. It has also been shown that an acute elevation of free fatty acids alters heart rate variability, an index of cardiac autonomic nervous system balance [ 27 ], suggesting that lipid metabolism may be involved in the regulation of cardiovascular autonomic tone. Thus our results that indicate involvement of components of lipid metabolism in the response to antihypertensive treatment are supported by cross-sectional epidemiological studies. In our earlier exploratory study, 74 SNPs with a minor allele frequency over 5%, including nine of the SNPs analysed here were tested as predictors of blood pressure regulation in the SILVHIA study samples using a multiple regression model [ 16 ]. The main aim of this study was to establish the microarray-based genotyping system. Analysis of twenty-eight SNPs from this panel that are located in genes from the renin-angiotensin aldosterone system identified a SNP in the aldosterone synthase gene (CYP11B2 T267C) and two SNPs in the angiotensinogen gene (AGT G1218A and T1198C) that appeared to be associated to blood pressure reduction [ 14 , 15 ]. A limitation in these previous studies was that correction for multiple testing was not applied, whereas in the current study we used a permutation test. A remaining weakness in our study is the small number of samples available for analysis, which does not allow detection of small to medium size gene effects, and results in uncertain estimation of the the magnitude of the effects detected. Moreover, in a small study there is the risk of a non-representative group of patients with respect to gender, age, and genotype distribution. Despite these limitations, we detected a significant effect of the SNP C711T in the apolipoprotein B gene and the SNP C16730T in the low density lipoprotein receptor after correction for multiple testing. The pharmacogenetically interesting results from our study need to be replicated in other studies. As the C711T SNP in the apolipoprotein B gene predicted response to treatment with irbesartan, and the C16730T SNP in the low density lipoprotein receptor gene appeared to predict response to atenolol treatment, our results point at possible use of SNPs in genes encoding components of lipid metabolism in pharmacogenetic panels for selecting the optimal drug for each patient. To our knowledge our study is the first one to investigate the relationship between polymorphisms in genes involved in lipid metabolism and the response to antihypertensive treatment. Competing interests The authors declare that they have no competing interests. Authors' contributions UL performed the development of genotyping technology, genotyping lab work, interpretation of data and had a substantial role in writing the manuscript. LL provided clinical expertise, participated in selection of candidate genes and contributed to writing. LK provided clinical expertise, established a database of the SILVHIA phenotypes, and in writing. LB performed the statistical analysis. TK provided the SILVHIA samples and contributed to writing the manuscript. A-CS contributed by planning and supervision of the project, and to writing the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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529323

In Times of Stress, Mutate Early and Often

For a human, the normal response to stress is to reduce it through some purposeful action, be it indulging in chocolate or calling in sick, at a rate which we can vary to fit the circumstances. For a strain of bacteria faced with stress, the choice is often more stark: it must mutate or die. Among evolutionary theorists, an important question has been whether the rate of mutation is fixed, or instead can adaptively increase in response to stress, thereby increasing the likelihood of a favorable mutation. Something like this latter possibility was envisioned by Darwin, but fell out of favor among some neo-Darwinists, for whom a steady rate of mutation was more in keeping with their overall model of evolutionary gradualism. This debate is taken up in a new study in this issue by P. J. Hastings and colleagues, who examined the mechanism by which Escherichia coli lacking the ability to digest lactose, called lac − mutants, regain that ability when presented with lactose as their only food source. It has been known for some time that the reversion of lac − mutants to a lac + state can be achieved by either of two genetic events: amplification, which creates numerous copies of the nonfunctional lac gene, and point mutations, which give rise to functional versions of the gene (many non-useful mutations also occur; thus, there is no directed mutation, in keeping with standard Darwinian evolution). According to the gradualist view, amplification precedes mutation, and the rapid appearance of lac + cells is explained by a normal mutation rate acting on multiple copies of the gene. In contrast, according to the hypermutation view, amplification and mutation are independent events, and lac + cells arise quickly because the mutation rate has increased. While some results from previous studies have supported the gradualist interpretation, the experiments of Hastings et al. show that hypermutation is the most plausible explanation. A variety of procedural improvements allowed them to analyze more individual cells at an earlier stage of colony development. For instance, they analyzed colonies composed of as few as one hundred cells, rather than the ten thousand cells in prior experiments, and even nascent colonies at the two-cell stage. Sectored colonies of lac + and lac − E. coli The study produced clear evidence that point mutations arise very early in the development of lac + colonies, before amplification can account for the number of lac + revertants observed. Amplification is not only independent from mutation, but occurs relatively late under starvation. The researchers found that amplification, but not point mutation, requires the presence of a particular DNA polymerase, further strengthening the case that amplification need not precede mutation. They also showed that amplification by itself does not induce a so-called SOS response. The SOS system includes a group of genes that cause an increase in mutation in response to stress, and one hypothesis arising from the gradualist model was that amplification turned on the SOS response. Based on their data, the authors reject the strict gradualist model for the adaptive mutation mechanism in the Lac system. They propose that amplification and hypermutation are independent responses to stress, each of which increases the likelihood of adaptive change. They also suggest that a stress-induced increase in the rate of point mutations may have implications for a variety of mutation-related phenomena, from tumor formation to development of resistance to antibiotics and chemotherapeutic drugs.

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529445

A common genetic factor underlies hypertension and other cardiovascular disorders

Background Certain conditions characterised by blood vessel occlusion or vascular spasm have been found to cluster together in epidemiological studies. However the biological causes for these associations remain controversial. This study used a classical twin design to examine whether these conditions are linked through shared environmental exposures or by a common underlying genetic propensity to vasospasm. Methods We investigated the association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease in twins from a national register. Phenotype status was determined using a questionnaire and the genetic and environmental association between phenotypes was estimated through variance components analysis. Results Responses were obtained from 2,204 individuals comprising 525 monozygotic and 577 dizygotic pairs. There was a significant genetic contribution to all four traits with heritabilities ranging from 0.34 to 0.64. Multivariate model-fitting demonstrated that a single common genetic factor underlies the four conditions. Conclusions We have confirmed an association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease, and shown that a single genetic factor underlies them. The demonstration of a shared genetic factor explains the association between them and adds weight to the theory of an inherited predisposition to vasospasm.

Background A number of conditions characterised by blood vessel occlusion and/or vascular spasm have been found to be associated in both clinical and epidemiological studies. These include hypertension (HPT), Raynaud's phenomenon (RP), migraine (MIG) and coronary artery disease (CAD) [ 1 - 5 ]. Whether these associations are the consequences of a shared environmental risk factor or represent an underlying propensity to develop the conditions through a common biological mechanism remains controversial. The exploration of the genetic and environmental relationships underlying these conditions is one approach to resolving the biological basis for the association. We have examined the association between these phenotypes in a classical twin study conducted in a large sample. This approach allows us to assess whether the association between HPT, RP, MIG and CAD is explained by a common genetic or environmental aetiology. Methods Subjects and methods Subjects for this study were twins enlisted with the St Thomas' UK Adult Twin Registry [ 6 ]. These monozygotic (MZ) and dizygotic (DZ) twin volunteers have been recruited since 1992 using twin registers and successive national media campaigns. For historical reasons most enrolled twins are female. This well studied population is sent regular questionnaires for self-completion concerning a wide range of health issues. Questions relating to HPT, MIG, RP and CAD respectively were contained within large questionnaires sent to the twins in 1998 and 2000. The questions were non-consecutive and respondents were unaware of the hypothesis being tested. The questionnaires also included standard questions relating to zygosity assignment [ 7 ]. In addition, fifty-four percent of the respondents had their zygosity assigned with certainty by multiplex DNA fingerprinting using variable tandem repeats on samples taken on previous attendances at the Twin Unit. Classification of HPT, RP, MIG AND CAD Classification of the traits HPT, RP, MIG and CAD was based on standard, validated criteria where possible. HPT was classified by asking about a doctor's diagnosis of "high blood pressure" when not pregnant. Questions to determine presence of migraine were based on the UCSD Migraine Questionnaire [ 8 ] (including at least five episodes of unilateral, pulsating headache over the previous year, duration four to seventy-two hours, noise and light sensitivity). RP was classified by confirming digital sensitivity to cold and required the respondent to have had recurrent episodes of colour change involving at least two colours [ 9 ]. CAD was classified either by respondents having a doctor's diagnosis of heart disease or angina, a previous heart attack or heart operation; or by answering affirmatively to questions taken from the Rose Angina questionnaire [ 10 ]. Analysis Traits were defined categorically as present or absent using the definitions listed above. Phenotypic associations between the four traits were assessed using odds ratios and 95% confidence intervals (CI) corrected to take into account the paired nature of the data. The odds ratios were adjusted for possible confounding by age, smoking and body mass index (BMI). Evidence for a genetic contribution to each trait was examined by estimating casewise concordance [ 11 ]. This measures the probability that the co-twin of an affected twin also expresses the trait. Higher casewise concordance in MZ pairs compared to DZ twin pairs indicates a genetic effect. Where data suggested a genetic influence, a quantitative measure of genetic contribution was estimated using structural equation modelling (Mx software [ 12 ]). This standard approach to twin analysis assumes an underlying model in which the observed phenotypic correlation among twins is explained by latent additive genetic influences (A) (which have a correlation of 1 in MZ twins and 0.5 in DZ twins), common environmental influences (C) (having a correlation of 1 in both MZ and DZ twins) and the random environment (E) (uncorrelated among twins). Analysis proceeds on the assumption that the observed categorical phenotype is accounted for by a continuous underlying liability to the trait [ 13 ]. Correlation in underlying liability is used as the basis for measuring the association between variables in the modeling. The significance of shared genetic and environmental factors was tested by stepwise deletion in a sequence of models containing the variance components (A, C and E) and assessing the deterioration in chi-squared for the fit of the model. Heritability was estimated from the size of the additive genetic contribution to the final selected model. Modeling was then extended to consider the genetic and environmental associations between all four variables simultaneously [ 13 ]. Three multivariate models were considered:- (1) the Cholesky decomposition (Figure 1 ) which includes four independent genetic and environmental factors. The first factor loads on all the traits, the second factor loads on all traits except the first, the third loads on all traits except the first two etc. This provides the fullest potential explanation of the data Figure 1 The Cholesky AE model. Diagram representing the Cholesky AE model, in which additive genetic (A) effects are shown loading on to the four traits: the unique environment (E) would load similarly (2) the independent pathway model (Figure 2 ) is a submodel of the Cholesky model and considers the data to be explained by a single shared genetic and shared environmental factor Figure 2 The independent AE pathway model. Diagram of the independent AE pathway, in which a single genetic and single unique environment factor loads on to each of the four traits, as well as factors specific to each trait (3) the common pathway model (Figure 3 ) which considers a single shared latent phenotype, determined in turn by latent genetic and environment factors. Figure 3 The common pathway model. Diagram of the common factor pathway in which single genetic and unique environment factors load on to the traits via a phenotypic latent variable The suitability of the common and independent pathway models may be determined by comparing the model's Akaike information criterion (AIC) with that of the fullest model, the Cholesky. The AIC represents the balance between model fit and the number of parameters (parsimony), with lower values of AIC indicating the most suitable model. Parameter estimates from the most appropriate model were used to calculate the genetic and environmental correlation between variables. Results Complete data for analysis were available from 1,102 pairs of female twins, comprising 525 MZ and 577 DZ pairs. The mean (± SD) age of the twins was 48.5 (± 8.2) years. The prevalence of each trait is shown in Table 1 . Table 1 Prevalence of the traits by zygosity and the p value of the difference between zygosities trait MZ prevalence (%) n = 1050 DZ prevalence (%) n = 1154 p HPT 131 (12.5) 181 (15.7) 0.052 RP 111 (10.6) 131 (11.4) 0.584 MIG 264 (25.1) 266 (23.1) 0.290 CAD 71 (6.8) 76 (6.6) 0.873 n represents number of twins, MZ monozygotic, DZ dizygotic, HPT hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease Four of the six combinations of traits showed significant phenotypic association after adjusting for age, smoking and BMI (odds ratios, Table 2 ). HPT was clearly not associated with RP but there was a suggestion of association of HPT with migraine (significantly so if adjusted for age and smoking only, data not shown). Since smoking status, age and BMI did not influence the size of the odds ratios, these variables were not included in the multivariate modeling. Concordance data and the results of univariate modeling confirmed a significant heritable component to all 4 traits (Table 3 ). Multivariate model fitting showed that for each of the three models tested, a model containing genetic (A) and unique environmental (E) factors provided the best explanation of the data (Table 4 , in bold): incorporation of a shared environmental factor (C) offered no significant improvement in the fit of any model. The independent AE pathway model (Figure 2 ) offered the best explanation of the data, suggesting that a single common genetic factor loads on the four traits HPT, RP, migraine and CAD. A unique environmental factor loads similarly on the traits. Using this model, genetic and unique environmental correlations between the four traits were calculated (Table 5 ). Overall, the genetic correlations were greater than the environmental correlations, suggesting a greater role for the genetic factor than the unique environmental factor. Table 2 Odds ratios (95% confidence interval) of unadjusted and adjusted (for age, smoking and BMI) cross-trait associations HPT RP MIG CAD HPT adj RP 0.88 (0.59–1.31) adj 0.99 (0.64–1.54) MIG 1.31 (0.99–1.73) 1.62 (1.21–2.17) adj 1.31 (0.95–1.80) 1.68 (1.23–2.3) CAD 2.85 (1.96–4.16) 2.03 (1.29–3.17) 2.19 (1.53–3.13) adj 2.41 (1.56–3.73) 2.27 (1.42–3.65) 1.77 (1.19–2.63) Calculation of odds ratios included a correction factor for familial clustering. Unadjusted odds ratios are shown above and adjusted below, for each combination of phenotypes. BMI represents body mass index, HPT hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease. Table 3 Casewise concordance rates by zygosity and heritability estimates of the four traits twin type total pairs discordant pairs (+/-) concordant pairs with trait (+/+) casewise concordance (95% CI) heritability (95% CI) HPT MZ 525 73 29 0.44 (0.34–0.55) 0.64 (0.49–0.79) DZ 577 135 23 0.25 (0.17–0.34) RP MZ 525 75 18 0.32 (0.21–0.44) 0.46 (0.30–0.63) DZ 577 111 10 0.15 (0.07–0.24) MIG MZ 525 144 60 0.46 (0.38–0.53) 0.43 (0.30–0.55) DZ 577 186 40 0.30 (0.23–0.37) CAD MZ 525 55 8 0.23 (0.10–0.35) 0.34 (0.13–0.55) DZ 577 72 2 0.05 (0.00–0.12) Casewise concordance rates (95% confidence intervals, CI) are shown for each trait by zygosity. An estimate of trait heritability (95% CI) is also given. HPT represents hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease. Table 4 Results of the model fitting: comparison of the 3 models model chi 2 df p AIC CHOLESKY ACE 32.95 30 0.33 -27.05 AE 33.35 40 0.76 -46.65 CE 65.82 40 0.01 -14.18 INDEPENDENT ACE 38.98 36 0.34 -33.02 AE 40.82 44 0.61 -47.18 CE 72.03 44 0.01 -15.97 COMMON FACTOR ACE 49.68 42 0.19 -34.32 AE 49.68 47 0.38 -44.32 CE 81.49 47 0.00 -12.51 For each submodel, the chi 2 statistic, the degrees of freedom (df) the probability (p) and the Akaike information criterion (AIC) are shown. AIC is used to evaluate the fit, with the best fitting submodel shown in bold in each case. Table 5 Genetic correlations (bold type, below in table) and unique environmental correlations (above) of the traits HPT RP MIG CAD HPT 0.29 0.01 0.17 RP 0.20 0.00 0.05 MIG 0.23 0.36 0.00 CAD 0.35 0.56 0.65 HPT represents hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease Discussion This is the first study to examine the role of genetic and environmental factors in explaining the association between HPT, RP, migraine and CAD. The results suggest that all four variables share a heritable basis. These conditions have been shown to be associated with one another and individually each is known to have a genetic basis. The data presented here confirm these previous findings and suggest that shared environmental factors such as diet and lifestyle do not contribute to their expression. In view of the nature of these phenotypes, we speculate that the shared genetic component leads to a predisposition to vasospasm. Indeed, a 'vasospastic phenotype' to account for their co-occurrence has been postulated by others [ 14 ]. The demonstration of a single genetic component lends weight to such a theory. A number of considerations should to be taken into account when interpreting these results. Self administered questionnaires were used for trait ascertainment, introducing the possibility of recall bias. In the present study efforts were taken to minimise recall bias: when surveyed, the twins were unaware of the hypothesis being tested; the questions relating to vascular phenotypes were included non-sequentially and were amongst many other questions in two questionnaires mailed at different times; and the twins completed questionnaires separately with no knowledge of their co-twin's responses. Furthermore there is no reason to suspect differential rates of recall in MZ when compared to DZ twins, hence any effects of recall should not have biased our estimates of genetic influence. Some conditions, such as RP, are notoriously difficult to diagnose regardless of the method employed [ 15 ] and it is possible that some subjects have cardiac valve rather than coronary artery disease. No attempt was made to differentiate primary RP and essential HPT from their secondary forms. However, questionnaire diagnoses were based on standard methods [ 8 - 10 ]. Despite these limitations, the traits' prevalences are in keeping with the findings of others for RP (9.6%[ 3 ], 5% and 16.8% according to geographical area sampled [ 16 ]), migraine (20% [ 17 ]) and CAD (8% [ 18 ]). In addition, heritability estimates are consistent with published findings for MIG (49–58% [ 19 ]), blood pressure (40–70% [ 20 ], 57% [ 21 ]) and CAD (15–30% [ 18 ]). Taken together, these observations suggest that our twins are representative of the adult female population and do not simply reflect a 'healthy volunteer' sample. The assumptions underpinning twin studies themselves may be questioned. Unequal sharing of the family environment by MZ and DZ twins has been raised as a concern, but this has been shown not to be the case [ 22 ]. In addition, twins from this cohort have been shown to be similar to singletons with respect to anti-hypertensive drug use, blood pressure and other phenotypes [ 23 ]. The proposed inverse relationship between birth weight and HPT [ 24 ] and CAD [ 25 ] could potentially bias a study of twins and cardiovascular disease. The explanation for this relationship is still debated, but maternal environment has been suggested to be the main influence on adult blood pressure [ 26 ]. As we have demonstrated that the shared environment makes no significant contribution to the vasospastic phenotype, this is not a likely source of error. It is clear that genetic factors play an important role in all four conditions. The demonstration that they are heritable is consistent with numerous reports of clustering in twin and family studies conducted in a range of settings, including a Dutch kindred with an autosomal dominant condition characterised by vascular retinopathy, migraine and Raynaud's phenomenon [ 27 ]. The detection of a common, genetically determined mechanism that contributes to these conditions is important because it points to an intermediate phenotype, vasospasm, and provides a possible focus for future studies. As with all chronic diseases and traits, the difficulty is in establishing which genes are responsible. Vascular tone is controlled at many levels, including local soluble mediators and neurotransmitters. Genes proposed through the study of the individual phenotypes include the beta2-adrenergic receptor gene in hypertension [ 28 ]; the muscle acetylcholine- and serotonin receptor genes in RP [ 29 ]; and a G protein subunit polymorphism and endothelial nitric oxide gene polymorphism in CAD [ 30 ]. A gain of function mutation in a vasoconstrictor or a loss of function mutation in a vasodilator may predispose to vasospasm: many of these genes deserve further consideration with the identification of a common genetic factor underlying HPT, migraine, RP and CAD. In summary, this twin study has identified phenotypic associations between four vascular conditions and shown that the association is explained by a single common genetic factor. These findings are consistent with the proposed 'vasospastic phenotype' and suggest that studies of genes controlling vascular tone will help to define the genetic basis of these conditions. Competing interests The author(s) declare that they have no competing interests. Authors' contributions FMKW analysed the data and drafted the manuscript. LFC designed the questionnaires, analysed the data and assisted with the manuscript. TDS collected the twins on the Twin Register and assisted with the manuscript. AJM conceived of the study, collected the twins on the Twin Register, analysed the data and co-drafted the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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526776

The use of warfarin in veterans with atrial fibrillation

Background Warfarin therapy is effective for the prevention of stroke in patients with atrial fibrillation. However, warfarin therapy is underutilized even among ideal anticoagulation candidates. The purpose of this study was to examine the use of warfarin in both inpatients and outpatients with atrial fibrillation within a Veterans Affairs (VA) hospital system. Methods This retrospective medical record review included outpatients and inpatients with atrial fibrillation. The outpatient cohort included all patients seen in the outpatient clinics of the VA Connecticut Healthcare System during June 2000 with a diagnosis of atrial fibrillation. The inpatient cohort included all patients discharged from the VA Connecticut Healthcare System West Haven Medical Center with a diagnosis of atrial fibrillation during October 1999 – March 2000. The outcome measure was the rate of warfarin prescription in patients with atrial fibrillation. Results A total of 538 outpatients had a diagnosis of atrial fibrillation and 73 of these had a documented contraindication to anticoagulation. Among the 465 eligible outpatients, 455 (98%) were prescribed warfarin. For the inpatients, a total of 212 individual patients were discharged with a diagnosis of atrial fibrillation and 97 were not eligible for warfarin therapy. Among the 115 eligible inpatients, 106 (92%) were discharged on warfarin. Conclusions Ideal anticoagulation candidates with atrial fibrillation are being prescribed warfarin at very high rates within one VA system, in both the inpatient and outpatient settings; we found warfarin use within our VA was much higher than that observed for Medicare beneficiaries in our state.

Background Warfarin therapy is highly effective for the prevention of ischaemic stroke in atrial fibrillation [ 1 ]. Despite the accepted benefit of warfarin therapy, several reports have indicated that warfarin therapy is underutilized even in ideal anticoagulation candidates with atrial fibrillation. Most studies have reported rates of use between 13–60% [ 2 - 8 ]. For example, a national study of inpatient Medicare beneficiaries with atrial fibrillation demonstrated that approximately 55% of patients were discharged on warfarin [ 9 ]. Many of the previous studies about the use of warfarin in atrial fibrillation have focused on the prescription of warfarin on discharge from an acute hospitalization. Since some patients may be discharged from the hospital with a plan to begin warfarin therapy as an outpatient, these prior studies may have underestimated the use of warfarin for patients with atrial fibrillation. The Veterans Affairs (VA) Healthcare System is a useful setting for studying the use of warfarin therapy in both the inpatient and outpatient arenas because the electronic medical record contains prescription medication data as well as the inpatient and outpatient medical records (including progress notes, laboratory data, radiology reports, and other consult reports). The objective of this study was to examine the use of warfarin in both inpatients and outpatients with atrial fibrillation within a VA setting. Specifically, we used the same methodology as the Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation [ 10 ], so that we could compare rates of warfarin use in ideal anticoagulation candidates with atrial fibrillation from one VA system to those in the private sector. Methods We assembled two retrospective cohorts of patients to evaluate both inpatients and outpatients with atrial fibrillation. The medical records of both the inpatients and the outpatients were reviewed to confirm the diagnosis of atrial fibrillation, to identify any exclusion criteria, and to determine if patients were being prescribed warfarin. Diagnosis of atrial fibrillation Using the criteria developed by the Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation [ 10 ], a physician's documentation of the diagnosis of atrial fibrillation was required for inclusion (electrocardiogram data were not used to make the diagnosis of atrial fibrillation). For the outpatients, Physicians' Current Procedural Terminology (CPT) codes were used to identify potential patients with a diagnosis of atrial fibrillation. For the inpatients, the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) discharge diagnosis code (427.31) was used to identify potential patients with a diagnosis of atrial fibrillation. Chart review was used to confirm the diagnosis for both the inpatients and the outpatients; a physician's documentation of atrial fibrillation in a progress note, a consult note, the discharge summary, or the problem list was needed for confirmation. Medical record abstraction was conducted by two of the authors (KR, SK) using standard definitions. All of the exclusions were reviewed by three of the authors (KR, SK, DMB), a sample of the charts of patients with an exclusion criteria was re-abstracted (DMB), and any disagreements were resolved by consensus. Cohort descriptions The outpatient cohort included all of the patients seen in the outpatient clinics of the VA Connecticut Health Care System during the month of June 2000 with a diagnosis of atrial fibrillation. Outpatient clinics include both primary care and subspecialty clinics. Some of these clinics have a particular interest in the care of patients with atrial fibrillation such as cardiology and anti-coagulation clinics, however, most of the clinics do not have such a special interest (e.g., mental health, physical therapy, dermatology, endocrinology). The inpatient cohort included all patients discharged from the VA Connecticut Health Care System West Haven Campus with any discharge diagnosis of atrial fibrillation (primary or secondary diagnosis) during the period of October 1, 1999 through March 31, 2000. Some of the patients in the inpatient cohort were readmitted during our study period; this report includes data from individual patients for their first hospital stay. Exclusion criteria The Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation project developed a set of exclusion criteria to identify ideal candidates for warfarin therapy; we used this exclusion criteria for the current study. Patients were excluded if they met one or more of the following: current sinus rhythm; bleeding disorder; endocarditis or pericarditis (within 6 months); seizures; intracranial hemorrhage; intracranial surgery or biopsy; lone atrial fibrillation; dual chamber pacemaker; alcohol or drug abuse; allergy to warfarin; hepatic failure; schizophrenia or active psychotic disorder; comfort care or terminal illness with life expectancy less than 6 months; un-repaired intracranial aneurysm; extensive metastatic cancer; brain cancer; malignant hypertension; peptic ulcer disease; hemorrhage; documentation that the patient refused warfarin therapy; prior complication or allergy related to past use of warfarin; or physician documentation of a rationale for not prescribing warfarin, including risk for bleeding, risk for falls, mental status impairment, liver disease, arthritis requiring non-steroidal anti-inflammatory medications or aspirin, pending surgery or other invasive procedure, terminal illness, patient's inability to obtain necessary blood work, or history of patient's non-adherence to warfarin [ 10 ]. Patients in intermittent or paroxysmal atrial fibrillation were included in the study, however, patients who were noted to be in current sinus rhythm and for whom atrial fibrillation was not a current problem were not included. For example, a patient with atrial fibrillation in the setting of an acute myocardial infarction or post-coronary bypass grafting for whom the atrial fibrillation was not a current medical problem was not included. Warfarin prescription For all of the patients, warfarin prescription was determined from the medical record. Patients receiving warfarin from the VA pharmacy were readily identified from the VA pharmacy component of the medical record. Patients receiving warfarin privately were identified from the progress notes. For inpatients, warfarin prescription was evaluated at the time of discharge from the hospital. For outpatients, warfarin prescription was evaluated at the time of examination of the medical record. Each outpatient medical record was examined in detail to determine the presence or absence of warfarin prescription. For those outpatients patients in whom this determination was difficult or if the data collector had a question about the patient, then the medical record was examined again by three of the authors to determine the presence or absence of warfarin prescription. This study received Institutional Review Board approval. Statistical analysis Student's t-tests were used to compare dimensional variables, and Fisher Exact and chi-square tests were used to assess binary variables. Two-sided p-values <0.05 were considered to be statistically significant. Exact binomial 95% confidence interval were calculated for the proportions of patients using warfarin in the inpatient and outpatient cohorts. The SAS System software release 6.12 (Cary, N.C.) was used for data analysis. Results Outpatient cohort A total of 538 patients from the VA Connecticut outpatient clinics were identified as having a diagnosis of atrial fibrillation (age: 74.0 years mean ± 8.3 standard deviation; 529 [98%] men). Of these, 73 patients had one or more contraindication to anticoagulation (Table 1 ). Of the 465 eligible patients, 455 (98%; 95%CI 96–99%) were prescribed warfarin. Table 1 Exclusion Criteria* Characteristic Inpatients Outpatients N = 212 N = 538 N (%)† N (%)† Sinus rhythm 32 (15) 42 (8) Death 21 (10) 1 (0.2) History of gastrointestinal hemorrhage 18 (8) 6 (1) Fall risk 14 (7) 4 (0.7) Pacemaker 4 (2) 9 (2) Lone atrial fibrillation 4 (2) 1 (0.2) Terminal illness 4 (2) 0 (0) Patient refused 3 (1) 6 (1) History of intracranial hemorrhage 3 (1) 3 (0.6) Transfer to outside facility 3 (1) 0 (0) Multi-infarct dementia in comfort care patients 2 (0.9) 0 (0) Warfarin held for procedure or surgery 2 (0.9) 0 (0) Warfarin allergy 1 (0.5) 0 (0) Failed to comply with warfarin protocol, warfarin stopped 1 (0.5) 1 (0.2) History of seizures 1 (0.5) 0 (0) Elective admission to begin sotalol and discontinue warfarin 1 (0.5) 0 (0) Previous bleeding on warfarin 0 (0) 1 (0.2) *These exclusion criteria were taken directly from the Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation. 10 † Note: some patients had more than one reason for not being prescribed warfarin. The ten patients who were not prescribed warfarin did not differ from those who received warfarin with respect to age (mean age ± standard deviation; no warfarin: 76.6 ± 11.5, warfarin: 73.9 ± 8.1; p = 0.3). Among the ten patients who were not prescribed warfarin: one was a dialysis patient who received his medical care primarily from private physicians outside of the VA, he was eventually placed on warfarin; the medical record of one 90-years-old patient, who also received the majority of his health care from private physicians, indicated that his private physician had elected not to prescribe anticoagulation "because of age"; one patient had a history of alcohol use; and in the remaining 7 patients there was no documentation of a reason for why the warfarin had not been prescribed (3 of the 7 patients were receiving the majority of their medical care outside of the VA). Inpatient cohort A total of 212 individual patients were discharged with a diagnosis of atrial fibrillation (age: 72.9 years mean ± 9.9 standard deviation; 211 [99.5%] males). During the admission these 212 patients, 17 died during their hospitalization, 3 were transferred to a facility outside of the VA Connecticut Healthcare System, and 77 had one or more contraindication to anticoagulation. Of the 115 remaining eligible patients, 106 (92%; 95%CI 86–96%) were discharged on warfarin. The nine patients who were not prescribed warfarin did not differ from those who were prescribed warfarin with respect to age (mean age ± standard deviation; no warfarin: 76.4 ± 7.4, warfarin: 72.6 ± 7.9; p = 0.2). To determine if some of the eligible inpatients who were not discharged on warfarin later received warfarin in the outpatient setting, we examined the outpatient records of the nine inpatients who were not discharged on warfarin: 5 died; 3 no longer receive care at our medical center (and no medication data were available); and for 1 patient, the medical record stated that he was offered warfarin therapy but that he refused to accept it. Similarly, we evaluated a sample of 50 eligible patients who had been discharged on warfarin therapy and examined their warfarin use post-discharge: 7 no longer receive care at our medical center (and no medication data are available); 4 were taken off of warfarin (3 because they were cardioverted as outpatients, and for 1 patient the warfarin was discontinued after an episode of bright red blood per rectum); and 2 patients have died. Among the eligible inpatients 12/115 (10%) had history of prior stroke or transient ischemic attacks; 3/12 (25%) were not prescribed warfarin on discharge. No reasons were documented for why these patients were not given warfarin. Unique patients There was overlap between the inpatient and outpatient cohorts such that a total of 722 unique patients were identified among the 212 inpatients and the 538 outpatients. Discussion We found high rates of warfarin prescription in ideal anticoagulation candidates with atrial fibrillation treated within this VA system. A total of 561 of 580 ideal anticoagulation candidates (97%) were prescribed warfarin: 98% of ideal outpatient anticoagulation candidates and 92% of ideal inpatient anticoagulation candidates. These rates of warfarin use for atrial fibrillation are substantially higher than those reported previously from private sector academic and community hospitals. For example, as part of the Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation, medical records were reviewed from a random sample of Medicare beneficiaries, hospitalized during the period 1998–1999, with any discharge diagnosis of atrial fibrillation from each state [ 9 , 10 ]. The exclusion criteria for the Medicare medical record review were the same as those used for the current study and were developed to select a cohort of atrial fibrillation patients who were "ideal" candidates for oral anticoagulation because they do not have any contraindications to oral anticoagulation [ 10 ]. Therefore, one would expect that the rates of warfarin use would be higher in ideal anticoagulation candidates than in a general population of patients with atrial fibrillation. Overall, the rate of warfarin prescription for ideal anticoagulation candidates with atrial fibrillation patients by state ranged from 31–65%, with a median of 55% in the Medicare study [ 9 ]. In Connecticut, 57% of eligible atrial fibrillation inpatients were discharged on warfarin [ 9 ]. Therefore, the inpatient rates observed in the current study of 90–92% are much higher than those observed for Medicare beneficiaries using similar methodology. We report the anticoagulation rates from one VA healthcare system, and our findings may not be generalizable to other VA healthcare systems or to non-VA hospitals. Specifically, our results may not be generalizable to women with atrial fibrillation. Furthermore, given that the out-patient cohort for this study was obtained from a one-month sample, we may have selected patients who are more likely to be seen at an out-patient clinic, and therefore, our findings may also have limited generalizability to atrial fibrillation patients who do not require or who do not have access to regular out-patient clinical care. Although we have found higher rates of warfarin use than most of the previous studies in this area [ 2 - 9 ], our findings are similar to those reported by Gottlieb and Salem-Schatz [ 11 ] who found that 78.8% of atrial fibrillation patients in an HMO setting were receiving warfarin. Our findings are also similar to those reported by Bradley et al. from another VA health care system [ 12 ]. Bradley et al. demonstrated that 89% of patients without a contraindication to anticoagulation were prescribed warfarin [ 12 ]. Several possible factors might account for the high use of warfarin in VA hospitals. First, the actual rates of warfarin prescription may be higher in VA facilities where anticoagulation clinics are well established, the electronic medical record ensures that all services (primary care and consult services) have access to a patient's medical record, the staff has academic affiliations, and the VA culture embraces quality improvement and medical error reduction initiatives. Within the VA Connecticut Health Care System, pharmacist-directed anticoagulation clinics are available at two sites, in West Haven and Newington, Connecticut. Veterans who choose to obtain warfarin from the VA pharmacy are usually followed at one of these two anti-coagulation clinics. Veterans can elect to purchase warfarin from private pharmacies and have their anticoagulation intensity monitored privately (usually by their private internist or private cardiologist). Second, the higher rate may result from data collection differences between studies. Specifically, given the comprehensive VA electronic medical record, VA-based researchers may be able to identify more contraindications for anticoagulation. No quality improvement projects to increase the use of warfarin for patients with atrial fibrillation in the VA Connecticut Healthcare System were initiated during or immediately prior to the study period. A limitation of the current study is that we were unable to determine the specific reasons for why such a high rate of warfarin use was observed. The retrospective nature of this study permitted us to evaluate clinical practices without altering physicians' behavior. However, this retrospective chart review may have limitations. First, we may not have identified those patients who are prescribed warfarin by private practitioners and obtain their warfarin from non-VA pharmacies. This would result in even higher rates of warfarin prescription than we have reported. Second, we assembled our cohort using diagnosis codes for atrial fibrillation and did not use electrocardiographic data. Those patients who had atrial fibrillation by electrocardiogram, but who were not identified as having atrial fibrillation by their clinicians, would not have been included in this study. Because such patients are unlikely to receive warfarin our estimates of warfarin use are higher than would have been observed if we had used electrocardiography to identify atrial fibrillation patients. While many studies of the use of warfarin for atrial fibrillation have also assembled cohorts using diagnosis codes and not electrocardiographic data, the VA-based study by Bradley et al. used electrocardiographic criteria and their findings are similar to ours [ 12 ]. Third, some may argue that we excluded patients who would benefit from anticoagulation. For example, patients with atrial fibrillation and numerous other risk factors for stroke might benefit from anticoagulation despite the presence of a contraindication to anticoagulation such as a risk for falls. We chose to use the exclusion criteria developed for the Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation [ 10 ] so that we could compare the rates of warfarin prescription observed within one VA system to those seen for Medicare beneficiaries. Fourth, this study includes a total of 722 unique patients. Although some studies of warfarin use in patients with atrial fibrillation have included similar numbers of patients (e.g., N = 635 in the study of Medicare beneficiaries with ischemic stroke and atrial fibrillation by Brass, et al) [ 13 ], many studies have included much larger sample sizes (e.g., N = 11,699 in the study of Medicare beneficiaries with new-onset atrial fibrillation) [ 14 ]. Often, the studies with the largest sample sizes were secondary analyses of existing administrative datasets [ 14 ]. Future studies should be directed at evaluating the use of oral anticoagulation in veterans with atrial fibrillation using national VA data where both large sample sizes and nationally representative sampling are possible. Conclusion We conclude that high rates of adherence to treatment guidelines regarding the use of anticoagulation in patients with atrial fibrillation can be achieved. Our experience, and that of Bradley et al., indicates that high rates of warfarin use can be achieved across at least two VA settings [ 12 ]. Competing interests The authors declare that they have no competing interests. All of the authors are employed by the VA Connecticut Healthcare system. Dr. Bravata is supported by an Advanced Research Career Development Award from the Department of Veteran Affairs Health Services Research & Development Service. Authors' contributions All of the authors contributed to this manuscript, participated in the research design and manuscript preparation. Two of the authors (SK, KR) conduct the data collection. Three of the authors (DMB, SK, KR) reviewed the data and conducted the analyses. Pre-publication history The pre-publication history for this paper can be accessed here:

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Pharmacokinetic-pharmacodynamic modelling of the cardiovascular effects of drugs – method development and application to magnesium in sheep

Background There have been few reports of pharmacokinetic models that have been linked to models of the cardiovascular system. Such models could predict the cardiovascular effects of a drug under a variety of circumstances. Limiting factors may be the lack of a suitably simple cardiovascular model, the difficulty in managing extensive cardiovascular data sets, and the lack of physiologically based pharmacokinetic models that can account for blood flow changes that may be caused by a drug. An approach for addressing these limitations is proposed, and illustrated using data on the cardiovascular effects of magnesium given intravenously to sheep. The cardiovascular model was based on compartments for venous and arterial blood. Blood flowed from arterial to venous compartments via a passive flow through a systemic vascular resistance. Blood flowed from venous to arterial via a pump (the heart-lung system), the pumping rate was governed by the venous pressure (Frank-Starling mechanism). Heart rate was controlled via the difference between arterial blood pressure and a set point (Baroreceptor control). Constraints were made to pressure-volume relationships, pressure-stroke volume relationships, and physical limits were imposed to produce plausible cardiac function curves and baseline cardiovascular variables. "Cardiovascular radar plots" were developed for concisely displaying the cardiovascular status. A recirculatory kinetic model of magnesium was developed that could account for the large changes in cardiac output caused by this drug. Arterial concentrations predicted by the kinetic model were linked to the systemic vascular resistance and venous compliance terms of the cardiovascular model. The kinetic-dynamic model based on a training data set (30 mmol over 2 min) was used to predict the results for a separate validation data set (30 mmol over 5 min). Results The kinetic-dynamic model was able to describe the training data set. A recirculatory kinetic model was a good description of the acute kinetics of magnesium in sheep. The volume of distribution of magnesium in the lungs was 0.89 L, and in the body was 4.02 L. A permeability term (0.59 L min -1 ) described the distribution of magnesium into a deeper (probably intracellular) compartment. The final kinetic-dynamic model was able to predict the validation data set. The mean prediction error for the arterial magnesium concentrations, cardiac output and mean arterial blood pressure for the validation data set were 0.02, 3.0 and 6.1%, respectively. Conclusion The combination of a recirculatory model and a simple two-compartment cardiovascular model was able to describe and predict the kinetics and cardiovascular effects of magnesium in sheep.

Background The effective use of some drugs can be limited by their adverse effects on the cardiovascular system, particularly when they are used intravenously in relatively high doses. There have been many studies documenting the cardio-vascular effects of drugs. Similarly, many mathematical models of the cardiovascular system, of varying complexity, have been presented in the literature [ 1 , 2 ]. In pioneering work, models of the cardiovascular system have been linked to pharmacokinetic models of volatile anaesthetic disposition [ 3 - 5 ]. These kinetic-dynamic models have since been developed into mannequin based anaesthesia simulators, which now have a pivotal role in the training of anaesthetists. This approach has been facilitated by the fact that models of volatile anaesthetic disposition have traditionally been physiologically based (e.g. using representations of tissue:blood partition coefficients and blood flows for individual organs or groups of organs). It is therefore possible to equate blood flow in the cardiovascular model to blood flow in the pharmacokinetic model. Nevertheless, a limiting factor in the implementation of this approach is the availability of experimental data on concentration-effect relationships [ 5 ]. In contrast, for traditional ("non-volatile") drugs, there have been very few instances in which kinetic models of a drug have been linked to cardiovascular pharmacodynamic models. The work of Francheteau et al. is an important exception [ 6 ], but even this early work was restricted to analysis of only one aspect of the cardiovascular system (i.e. accounting for heart rate mediated control of blood pressure but not Frank-Starling control of cardiac output). However, it is clear this approach has the potential to provide a more rational basis for designing dose regimens of cardio-active drugs, and could provide insight into strategies for controlling their cardio-vascular effects. It maybe possible to predict a priori the cardiovascular consequences of, for example, a change in dose regimen of a drug. There are a number of difficulties in implementing this approach for traditional drugs. One problem is that most drugs do not cause changes in one single cardiovascular variable (such as blood pressure) that can be described in the usual manner using a simple semi-empirical dynamic model (e.g. an E max model). Rather, a number of cardiovascular variables may be altered simultaneously in a manner that is complex and interrelated. Thus, any dynamic model used must account for these intrinsic relationships between cardiovascular variables. Another problem is that changes in the cardiovascular system (in particular blood flow distribution) invariably alter the kinetics of the drug under study. Therefore, the kinetic model of the drug must be able to account for the effects of blood flow changes on the disposition of the drug. This requires the kinetic model to have a physiological basis, and importantly excludes the widely used mamillary compartmental pharmacokinetic model. The general aims of this study were threefold. First, to develop a simple dynamic model of the cardiovascular system that was of sufficient complexity to account for the major mechanisms by which drugs can alter cardiovascular variables. Second, to examine whether recirculatory kinetic models [ 7 ] have sufficient physiological basis to account for drug related blood flow changes. Third, to examine approaches for identifying the important concentrations (and their sites in the body) that can be used to link the kinetic and dynamic models. The specific aim was to use previously published data collected using a chronically instrumented sheep preparation [ 8 , 9 ] to develop a kinetic-dynamic model for the cardiovascular effects of magnesium. Magnesium is given intravenously to treat a number of diseases, including pre-eclampsia. It relaxes smooth muscles in blood vessels thereby lowering systemic vascular resistance, with a consequent decrease of mean arterial blood pressure and increase in cardiac output. It provides a useful drug for initial analysis as its kinetics and dynamics are relatively simple and well understood. The overall hypothesis of this work is that it is possible to construct a faithful model of the cardiovascular effects of drugs such as magnesium. While doing so requires more assumptions and estimates of parameter values than normally associated with semi-empirical pharmacokinetic-pharmacodynamic modelling, a physiological approach greatly increases the utility of the resulting models. It is proposed that the general methods presented here could be applied to the development of similar models for other drugs with acute cardiovascular effects. Methods General rationale With respect to devising a pharmacodynamic model of the cardiovascular system, the important steps are: 1. Identifying which cardiovascular variables (e.g. heart rate, blood pressure) are important. This depends on the drug and the intended use of the model, but it is proposed that there is a minimum set of variables that is needed for a basic description of cardiovascular status. 2. Devising a way of conveniently presenting the output of the dynamic model for a range of cardiovascular variables for comparison with data. 3. Identifying a cardiovascular model of the appropriate complexity. Ideally the model must be of the minimum complexity that includes the cardiovascular variables identified above, and the major sites of action of the drugs. 4. Identifying which parameters of the cardiovascular model can be estimated by curve-fitting, and which require prior estimates or measurements of physiological values. Most cardiovascular models are stiff numerical systems with many parameters, and only a small number can be estimated by curve-fitting the data in the traditional way. With respect to the pharmacokinetic model, there remains one crucial step: 5. Constructing a kinetic model with a physiological basis that is sufficiently realistic to describe and predict the concentration of the drug in the key target organs controlling the cardiovascular system. On first principles, these could be expected to include: a. the myocardial concentrations when the drug has a direct myocardial effect (e.g. causes myocardial depression); b. the CNS concentrations when the drug affects the cardio-respiratory control centre of the brain; c. the arterial blood concentration when the drug affects baro-receptors or smooth muscle in the walls of the arterial vascular system; d. the venous blood concentration when the drug affects smooth muscle in the wall of the capacitance vessels of the venous vascular system. It is known that these concentrations can follow different time-courses, particularly after bolus administration or a change of infusion rate [ 10 , 11 ]. However, it may not be necessary to know the time-course of these concentrations for every drug, depending on its mechanism of action. Data sets and software The data used to construct the model were collected in the same laboratory using a conscious chronically instrumented sheep preparation and have been published previously [ 8 , 9 ]. This facilitated the model building process, as the effect of differences in species and measurement methods could be discounted. Data set 1 [ 9 ] (for model development) was a detailed set of cardiovascular measurements made after the administration of 30 mmol of magnesium over 2 min to 5 sheep. Measurements included arterial and coronary sinus (effluent from the heart) magnesium concentration, cardiac output, mean arterial blood pressure, heart rate, an index of myocardial contractility (Maximum positive rate of change of left ventricular Pressure, dp/dt) and an index of filling pressure (Left ventricular end diastolic pressure) and myocardial blood flow. These were made until 25 min after the start of administration. Data set 2 [ 8 ] (for model validation) was a less comprehensive set of cardiovascular measurements made after the administration of 30 mmol of magnesium over 5 min to 5 sheep (not the same sheep as Data set 1). Measurements included arterial magnesium concentration, cardiac output, and mean arterial blood pressure, and were made until 25 min after the start of administration. The blood pressure data for one animal in this data set was excluded, as it was idiosyncratically low. The time-course of the data averaged across sheep were used for all modelling – the resultant model therefore represents the response of the average sheep. Inter- and intra-animal variability were not considered, although it is noted that the final model may provide insight into sources of kinetic and dynamic variability for later study. The software used was the Scientist for Windows program (Version 2.01, Micromath, Salt Lake City, Utah, USA), predominantly for curve-fitting. The R language, Version 1.9.0, [ 12 ] was used for graphical data analysis, data handling and simulations. Coding the same model in the two different programs provided a useful check for errors. For the least squares curve-fitting, the best fit was determined as that with the highest Model Selection Criteria (MSC) and without non-identifiable parameters. The MSC is essentially an inverse Akaike Information Criterion scaled to compensate for data sets of different magnitudes (Scientist for Windows manual, Micromath, Salt Lake City, Utah, USA), and is calculated as follows where w i is a weighting term, p is the number of parameters and n is the number of data points: All data points were weighted equally. A parameter was arbitrarily defined as non-identifiable if the standard deviation of the parameter returned by the fitting program was greater than the parameter estimate (i.e. the coefficient of variation was greater than 100%). A model with non-identifiable parameters means that the data do not contain sufficient information to estimate the parameter with precision. The symbols used throughout have been based on standards for the pharmacokinetic literature. Unfortunately, the use of C for both concentration (in pharmacokinetics) and compliance (in cardiovascular physiology) creates of conflict for this paper. To avoid confusion, CPL will be used for compliance here. Pharmacodynamic model of the cardiovascular system Identification of important cardiovascular variables The choice of the cardiovascular variables used in the model is clearly dependent on the drug under study and the intended purpose of the model. However, we propose that a minimum of 7 fundamental cardiovascular variables is sufficient for most pharmacological purposes. These variables are shown with their default unit of measurement in the model: Central venous pressure (CVP, mmHg), Myocardial contractility (CNT, L mmHg -1 ), Stroke volume (SV, L), Heart rate (HR, min -1 ), Cardiac output (CO, L min -1 ), Systemic vascular resistance (SVR, Resistance units, RU) and mean arterial blood pressure (MAP, mmHg). This choice of variables requires several assumptions: Assumption 1 All variables are time averaged in that beat to beat variation is ignored (e.g. mean arterial blood pressure is used rather than systolic and diastolic blood pressures). Assumption 2 That the function of the left and right side of the heart is the same, and there are no abnormalities in the pulmonary vasculature so that the heart-lung system can be treated as one pump. Assumption 3 Long time-scale events such as fluid shifts and renal mechanisms controlling blood pressure are ignored. Furthermore, this choice of variables is dictated by several fundamental relationships between the variables. Firstly, that myocardial contractility is a proportionality constant between CVP pressure and stroke volume (the volume of blood pumped with each beat of the heart). CVP * CNT ≈ SV    ...(2) Mathematically, CNT must therefore have the units of volume / pressure. However, contractility is difficult to measure in vivo, and that there are a number of surrogate measures including dp/dt. These can also be used with appropriate scaling factors. Assumption 4 That there are no factors affecting the relationship between myocardial fibre length (the true determinant of stroke volume) and central venous pressure (e.g. changes in myocardial compliance). CVP is therefore used as an easily measured index for myocardial fibre length – the assumption is that the two are related using a scaling factor. Left ventricular end diastolic pressure is also as an alternative index when data are presented as percent change from baseline. The second relationship is that between stroke volume and heart rate to give the cardiac output (the volume of blood pumped by the heart per unit time): SV * HR = CO    ...(3) The last relationship is that between cardiac output and systemic vascular resistance to approximate the mean arterial blood pressure. CO/SVR ≈ MAP    ...(4) This is because MAP usually greatly exceeds the CVP: CO/SVR = MAP-CVP    ...(5) SVR therefore has the units of pressure over flow. In this paper, the resistance units (RU) are therefore mmHg L -1 min. Presentation of relationships between cardiovascular variables The effect of a drug on one or two variables can usually be summarised on a plot of the variable (drug effect) against time. However, it is more difficult to summarise the dynamic effect of a drug on the cardiovascular system for the following reasons: First, the large number of variables required in the summary, where the seven described above could be considered a minimum. Second, the fixed inter-relationships (e.g. Eqns. 2–4) between these variables that should be revealed by the summary (e.g. if SV increased by 25% and all else remains the same, then CO should also increase by 25% (Eqn. 3)). Third, usually an analysis requires comparing one cardiovascular state (e.g. pre-drug) with another (e.g. post-drug), or examining the time-course of drug effects. It is proposed that a "cardiovascular radar plot" with a modified logarithmic, normalised scale is an efficient means of limiting these problems. An example radar plot is shown and described in Fig. 1 . Radar plots are particularly useful for visually testing whether a model of the cardiovascular system behaves appropriately for all 7 key cardiovascular variables when challenged with a particular drug or physiological circumstance. It is particularly useful to see if the pattern of changes is internally consistent. For example, in Fig. 1 it is clear that magnesium dropped SVR, but the drop in MAP was not great as expected because there was a baroreceptor mediated increase in heart rate. Figure 1 An example of a cardiovascular radar plot. A cardiovascular radar plot of the effect of magnesium (n = 5 sheep) on the cardiovascular system under baseline conditions and for a number of time-points until 25 min after the intravenous administration of magnesium. A scale for each of the 7 key cardiovascular variables radiates from the centre of the plot. LVEDP (left ventricular end diastolic pressure) is a surrogate for CVP; both should change proportionally. The data are transformed and the scale constructed so that 3 is the baseline (pre-drug) value. Thus, the blue line for the baseline data is a ring passing through 3 for each variable. Baseline conditions therefore have a characteristic equilateral 7 sided shape. The full scale is structured as follows: 1 one quarter baseline 2 half baseline 3 baseline 4 twice baseline 5 4 times baseline This scale has the property that for an equivalent increase or decrease in a cardiovascular variable compared to baseline, the line will move an equal distance in or out from the baseline value. It can be seen that following magnesium there was a drop in SVR with a baroreflex increase in HR to compensate for the drop in blood pressure. LVEDP also dropped, but with minimal change in contractility. Drugs that affect the cardiovascular system via different mechanisms produce plots with characteristic shapes, which can be recognised with experience. Note: The order of the variables on the radar plot has been chosen to account for key relationships between the variables, with CVP (as given by LVEDP) as the most fundamental variable at the top: In an anti-clockwise direction the following relationships or approximations hold: CVP * CNT ≈ SV SV * HR = CO CO/SVR ≈ MAP Cardiovascular model – Structure and parameter estimation There are many published models of the cardiovascular system of various levels of complexity and intended for various tasks [ 1 ]. However, in this paper, the cardiovascular model was constructed progressively from first principles, with adaptations and increases in complexity as dictated by the requirements of the modelling process and the data. This ensured the model was the minimum that was needed for the task at hand. In vivo, the cardiovascular system has two major control systems; control of cardiac output via the Frank-Starling mechanism, and control of blood pressure via baroreceptor control of heart rate. These were added progressively to the model. A simple Frank-Starling model A simple model of the Frank-Starling mechanism was developed (Fig. 2 ) assuming the blood is predominately in two pools – the arterial and venous vasculature. The two pools are connected by a pump (the heart) moving blood from the venous to arterial side for which the rate of pumping is proportional to the venous pressure. Blood flows from the arterial to venous side through a passive resistance (the SVR). The pressure in each pool is a function of the compliance in the pool. Compliance (CPL) governs the relationship between volume and pressure: Figure 2 A simple Frank-Starling model of the cardiovascular system. A simple two compartment model of the circulation, with control of the cardiac output via the Frank-Starling mechanism. When the heart is not pumping, the pressures on the venous (P v ) and arterial (P a ) sides of the circulation are equal (the mean circulatory pressure (MCP = P v = P a ) is approximately 7 mmHg). The unstressed volumes of the venous (V v 0) and arterial sides (V a 0) are governed by the relative compliance of the venous and arterial pools (CPL v and CPL a , respectively). If the pumping action of the heart is initiated, a fraction of the blood (dV) moves from the venous to the arterial side thereby increasing arterial pressure and decreasing venous pressure. The pressure gradient causes blood to flow from the arterial side to the venous side (at a rate given by the venous return, CO R ). This depends on the pressure gradient (P a -P v ) and the systemic vascular resistance (SVR). Pressure = Volume/Compliance    ...(6) The solution to the simple Frank-Starling model can be found algebraically, but for consistency is shown in Additional File 1 as differential equations. Central to the Frank-Starling model is the concept of cardiac function curves – usually given as the pressure in each pool as cardiac function (contractility) is increased from zero to a normal value. These curves are useful for finding appropriate initial estimates for blood volume, arterial and venous compliance, and systemic vascular resistance. To achieve the physiologically plausible cardiac function curve shown in Fig. 3 , blood volume was set at 3.5 L [ 13 ]. Given that in a normal (50 kg) sheep the baseline cardiac output is approximately 6 L/min and mean arterial blood pressure is 100 mmHg (Table 1 ), baseline systemic vascular resistance is therefore 100/6 ≈ 17 RU. The remaining unknowns of this system (kc, CPL a , CPL v ) were chosen to duplicate the following behaviour (Fig. 3 ) which is consistent with measurements in this species: When CNT is zero (i.e. the heart is not pumping) then dV is zero and the mean circulatory pressure (MCP) is approximately 7 mm Hg. When CNT is such that the cardiac output is approximately 6 L min -1 , then MAP and CVP are approximately 100 and 2 mmHg, respectively (Table 1 ). In practice, it was found easier to express the arterial compliance (CPL a ) as the ratio of arterial compliance to venous compliance (C ratio ). Figure 3 Cardiac function curves for the Frank-Starling model. A summary of the behaviour of the simple Frank-Starling model. The relationship between cardiac function (contractility) and the arterial and venous pressures matches well that reported in many textbooks. The venous compliance CPL v was 0.45, and the ratio of CPL v /CPL a was 15. Table 1 Baseline (pre-drug) cardiovascular variables. A set of target values that was representative of the sheep studied in our laboratory was compiled from previous measurements and literature values as indicated. A set of parameter values for the final (Constrained-Frank-Starling-Baroreceptor) was derived (Table 2) that produced an internally consistent model that closely replicated these target values (also shown for comparison). Variable Name Target Value Target value origin Model derived Value Units V blood Blood volume 3.5 literature [13] 3.5 L CVP Central venous pressure 2.00 unpublished previous measurements and literature [20] 2.00 mmHg CPL v Venous compliance 0.45 inferred from V blood & CVP 0.46 L mmHg -1 MAP Mean arterial pressure 100 previous measurements [21] 100.9 mmHg SVR Systemic vascular resistance 17.00 calculated from CO & MAP 17.0 RU CO Cardiac output 6 previous measurements [21] 5.8 L min -1 HR Heart rate 100 previous measurements [21] 98.3 beats min -1 SV Stroke volume 0.06 calculated from CO & HR 0.059 L CNT Contractility 3000 previous measurements [9, 21] 3000 mmHg sec -1 S1 Sympathetic tone – chronotropy 1 scaling factor only 1 dimensionless S2 Sympathetic tone – Contractility 1 scaling factor only 1 dimensionless Frank-Starling and Baroreceptor model The control of arterial blood pressure via baroreceptor control of heart rate was added to the simple Frank-Starling model, as shown in Fig. 4 . The arterial pressure set point (MAPset) was used to calculate the difference between the actual and set pressure (MAPdelta). This pressure difference was used to change heart rate with a gain given by "HRgain". When HRgain is zero, the model reduces to the simple Frank-Starling model. As HRgain is increased, the more heart rate is adjusted to defend changes in arterial pressure. A value of 3 was initially used for HRgain. The resultant cardiac function curve for this model is shown in Fig. 5 , and the equations for the model are shown in Additional File 2 . Figure 4 A Frank-Starling-Baroreceptor model of the cardiovascular system. The Frank-Starling model of the circulation from Fig. 2 combined with baroreceptor control of arterial blood pressure (P a ) via changes in heart rate (HR). MAP set is the set point of the control system, and HR gain is the gain of the control system that operates on the difference between the actual and set arterial blood pressures (MAP delta ; Eqn. 7). The right side cardiac output term is expanded to include the role of myocardial contractility (CNT), stroke volume (SV) and heart rate (HR). "kc" is a conversion factor to adjust for the index used to measure myocardial contractility (Eqn. 8). Strictly, myocardial contractility is the proportionality factor between Pv and stroke volume (SV = CO R /HR). However, it is often quantified using indirect indices, such as maximum positive change of ventricular pressure (dP/dt max ). The value of kc will depend of what index of contractility is used (see Eqn. 8). Figure 5 Cardiac function curves for the Frank-Starling-Baroreceptor model. A summary of the behaviour of the Frank-Starling-Baroreceptor model. The relationship between cardiac function (contractility) and the arterial and venous pressures matches well that reported in many textbooks. However, the vascular volumes show the majority of the blood in the arterial compartment, which is at odds with the fact that the majority of the blood under baseline conditions is in the venous vessels. Furthermore, no constraints have been placed on the model so that unrealistic values (e.g. large negative pressures) can be achieved in some circumstances. Constraining the model to increase physiological plausibility The final version of the model introduced a number of constraints to increase its physiological plausibility. These were: 1) Assuming that under baseline conditions that approximately 1/3 of the total blood pool is in the arterial system. 2) That the intercepts of the pressure-volume "curves" for the venous and arterial compartments were linear such that both curves gave the mean circulatory pressure (MCP) at the unstressed volumes (V v 0 and V a 0, see Fig. 6 ). 3) That the venous pressure could not be less than zero, and that the arterial pressure could not be less than the MCP. 4) That heart rate was constrained to be between 0 and 220. 5) That the venous pressure – stroke volume relationship was non-linear and reached a plateau consistent with the finite pumping capacity of the heart (Fig. 6 ). 6) For convenience, two additional parameters were introduced (S1 and S2) representing the state of the sympathetic nervous system. These gave the capacity to adjust the proportionality term between blood pressure and heart rate (HRgain) and between CVP and stroke volume (kc). This allowed these scaling constants to be separated into a constant term that is solely used to convert measurement units (HRgain or kc) and another term (S1 or S2) that represents changes in underlying physiology for use when fitting data. Their normal values were 1 in each case (giving no effect for baseline conditions) and their function is summarised in the following equations: Figure 6 Effect of introducing constraints on the model. Top: Venous pressure – volume curves. Venous volume starts at V v 0 when the heart is not pumping, at which point the venous pressure is the mean circulatory pressure (MCP). With increased pumping, the venous volume and venous pressure is reduced. In the simple (Frank-Starling-Baroreceptor) model, the CVP – volume relationship was linear, with an intercept of zero. In the constrained model, a lower intercept was used which was necessary to produce realistic venous volumes under baseline conditions. The multiple curves in the plot show the effect of changing venous compliance (CPL v ). Middle: Arterial pressure – volume curves. Arterial volume starts at Va0 when the heart is not pumping, at which point the arterial pressure is the mean circulatory pressure (MCP). With increased pumping, the arterial volume and arterial pressure are increased. In the simple model, the MAP – volume relationship was linear, with an intercept of zero. In the constrained model, a lower intercept was the used, which was necessary to produce realistic arterial volumes under baseline conditions. The multiple curves in the plot show the effect of changing compliance (CPL a ). Bottom: "Cardiac output curves" In this case cardiac output is given by stroke volume, which is plotted against central venous pressure (CVP). In the simple model, this relationship was linear. In the constrained model the relationship was given by a logistic equation which rose to a limit. The left-hand side of the curves are pseudo-linear, and the slope of the lines increase with increasing contractility. This behaviour mimics the "Cardiac output curves" found in many cardiovascular textbooks (e.g. Guyton [19]). HR = MAPset + MAPdelta*(HRgain*S1)    ...(7) The resultant cardiac function curves for this model are shown in Fig. 7 , and the equations for the model are shown in Additional File 3 . Figure 7 Cardiac function curves for the Constained-Frank-Starling-Baroreceptor model. A summary of the behaviour of the final cardiovascular dynamic model. Baseline values for the cardiovascular model For convenience, the target cardiovascular variables of the final constrained model discussed above are summarised in Table 1 with references to their origins. The parameter set that produced cardiovascular variables similar to the target values is summarised in Table 2 . This was derived semi-empirically by inspection of cardiovascular function curves (Fig. 7 ) and pressure-volume relationships (Fig. 6 ) with incremental adjustment of parameter values. Note that some variables are also listed as parameters – this is purely for convenience. The distinction between variables (time-dependent) and parameters (time-independent) is semantic and depends on the proposed use of the model. Table 2 Baseline model parameters The parameters chosen as those producing representative baseline (pre-drug) cardiovascular variables (Table 1). The co-efficient of variation (CV (%)) of these parameter values as determined by the Monte-Carlo sensitivity analysis is also shown. Parameter Name Value Units CV (%) CPL v Venous compliance 0.45 L mmHg -1 12.7 CPL ratio Ratio of venous over arterial compliance 20 dimensionless 17.7 V blood Blood volume 3.5 L 17.9 SVR Systemic vascular resistance 17 RU 5.5 MAPset Mean arterial pressure set point 100 mmHg 4.2 HRgain Gain for heart rate control 1.8 bpm a mmHg -1 23.6 CNT Contractility 3000 mmHg sec -1 4.9 a bpm = beats per minute The sensitivity of the baseline cardio-vascular model to changes in parameter values was determined via Monte-Carlo simulation [ 14 ]. Multi-variate normally distributed noise was added to the parameter values for a series of 10,000 simulations of the resulting cardiovascular variables. Those parameter sets that produced a set of cardiovascular variables within 10% of the target set were selected and analysed for with respect to parameter variability and correlation. Fitting the cardiovascular model to the magnesium data Changes in cardiovascular variables with the administration of magnesium were analysed as percentage change from baseline. This removed the contribution of inter-animal variability in baseline cardiovascular variables (which was nevertheless minor [ 8 , 9 ]) to variability in the cardiovascular effects of magnesium. The analysis involved fitting cardiovascular radar plots to the measured magnesium data (Data set 1) for key time-points (1, 2, 4, 10 and 25 min) during and after magnesium administration. The cardiovascular model was parameterised in terms of primary cardiovascular variables that could be directly influenced by magnesium. These were SVR, CPL v , CPL ratio , CNT, S1 and S2. V blood could also be considered a primary variable, but it was considered unlikely that magnesium could change the blood volume. The remaining cardiovascular variables were considered secondary in that they would change in response to changes in the primary variables as given by Eqns 2 to 4. Initially, the only primary parameter fitted to the data for each time point was SVR while the other parameters were held constant. This was based on the prior knowledge that this was the primary mechanism of action of magnesium. If the MSC was low and the cardiovascular radar plot showed a poor fit between model predictions and the data, an additional parameter was fitted one at a time from the remaining parameters listed above. A parameter was removed from the fit if it produced an undefined estimate. The parameter was kept in the fit if it improved the MSC and the fidelity of observed vs. predicted plots on the cardiovascular radar. By this process, the values of the primary cardiovascular parameter at each key time point required to describe the observed data were determined. Recirculatory pharmacokinetic model of magnesium disposition Conventional mamillary pharmacokinetic models are essentially empirical and do not include parameters (other than clearance) that represent defined physiological processes. This is problematic when drugs affect the cardiovascular system, or it is necessary to predict the kinetics of the drug when the underlying physiology has changed. This was the case for magnesium, which affected cardiac output significantly (Figs. 1 & 10 ). Full physiological pharmacokinetic (PBPK) models are an alternative, but often require extensive data sets for their parameterisation. Recirculatory models have been used [ 7 , 15 ] as an alternative that retain the key physiological descriptions of important organs, but have lumped descriptions of the less important organs. Often, they can be parameterised by fitting blood concentrations alone. Figure 10 Best fits for the recirculatory pharmacokinetic model for magnesium. Top: The observed changes in cardiac output for Data set 1 (symbols). Also shown is the line of best fit for the empirical forcing function used for development of the kinetic model. The large and consistent increase in cardiac output illustrates why it was necessary to use a kinetic model that could account for the significant flow changes caused by magnesium. Middle: The observed arterial concentrations of magnesium for Data set 1 (symbols). Also shown is the line of best fit for the final kinetic model (not linked to the cardiovascular model) based on the parameter values given in Table 4. Bottom: A sensitivity analysis of the final kinetic model with respect to cardiac output when used to simulate the dose regimen used for Data set 1. Cardiac output was given values of 2, 4, 6, 8 or 10 L min -1 while the other parameters were fixed at the values given in Table 4. This illustrates how the cardiac output changes caused by magnesium can influence its own kinetics. This feedback process was inherent in the structure of the final kinetic-dynamic model. The magnesium concentration data from Data set 1 were used to develop a recirculatory model of magnesium kinetics that could account for the observed cardiac output changes. The processed used was similar to that described by the authors for other drugs [ 15 ]. The final form of the model is shown in Fig. 9 . Figure 9 Final recirculatory pharmacokinetic model for magnesium. A pictorial representation of the model. Parameter names are given in Table 4. Key points during the model development process were: 1) The representation of the lungs as a single compartment. 2) The representation of the cardiac output change as an empirical forcing function (see Fig. 10 , this would later be replaced by the predictions of the final cardiovascular model). 3) The representation of the body as extracellular and intracellular spaces connected by a permeability term, in keeping with the known slow cellular uptake of magnesium. 4) The clearance of magnesium is renal, but it can be reabsorbed or excreted in the tubules, as dictated by homeostatic requirements [ 16 ]. Thus, renal clearance may be variable. To confirm that the kinetics of magnesium were cardiac output dependent, the final kinetic model was subjected to a sensitivity analysis for this parameter. Cardiac output was assigned values of 2, 4, 6, 8 or 10 L min -1 while the other parameters were fixed at their best fit value. The time-course of the arterial magnesium concentration was recorded in each case. Linking the pharmacokinetic and pharmacodynamic models The relationship between the key cardiovascular parameters (effects) and the concentrations of magnesium in arterial and coronary sinus blood were examined using hysteresis plots (effect vs. concentration). A concentration-effect relationship was considered plausible if produced a predictable relationship with minimal hysteresis that was consistent with the known mechanisms of action of the drug. By these criteria, it was found that the arterial concentrations were the better predictor of the fitted cardiovascular parameters shown in Table 3 . The concentration – effect relationships are summarised in Fig. 11 . The major effect of magnesium was to drop systemic vascular resistance (SVR). SVR was related to the arterial magnesium concentration by a link model based on a linear relationship with a threshold (Fig. 11A ): Table 3 The fitted primary cardiovascular parameters for Magnesium data set 1 Units are as for Table 2. The parameter estimates are given with the standard deviation returned by the curve-fitting program. S1 could not be reliably fitted to the data. 0 min 1 min 2 min 4 min 10 min 25 min Fitted parameter (baseline) estimate (sd) estimate (sd) estimate (sd) estimate (sd) estimate (sd) MSC n/a 3.88 2.96 4.75 1.67 4.74 CPL v 0.45 0.490 (0.0028) 0.495 (0.0075) 0.497 (0.0019) 0.505 (0.0049) 0.492 (0.0007) SVR 17 11.99 (0.12) 9.84 (0.26) 11.84 (0.084) 14.64 (0.27) 16.74 (0.077) CNT 3000 2988 (41) 3031 (104) 3388 (32) 3108 (80) 3180 (23) S2 1 0.978 (0.023) 0.972 (0.055) 0.877 (0.014) 1.22 (0.06) 1.25 (0.017) Figure 11 Link models for concentration-effect relationships. A: The systemic vascular resistance (SVR) parameter (symbols, obtained by the fitting process that gave the radar plots shown in Fig. 8 and summarised in Table 3) plotted against the concurrent exogenous arterial magnesium concentrations. The final link model (Eqn. 9; line) based on a linear relationship with a threshold is also shown. B: The venous compliance (CPL v ) parameter (symbols, via Fig. 8) plotted against the concurrent exogenous arterial magnesium concentrations. The final link model (Eqn 10; line) based on a simple threshold that switches between two states of venous compliance is also shown. This is plausible if it is considered that magnesium, even at relatively low concentrations, causes maximal dilation of the venous capacitance vessels. C: The contractility (CNT) parameter (symbols, via Fig. 8) plotted against the concurrent exogenous arterial magnesium concentrations. The final link model (line) was based on the assumption that contractility was unaffected by magnesium (i.e parameter value was fixed). D: The sympathetic tone coefficient for contractility (S2) parameter (symbols, via Fig. 8) plotted against the concurrent exogenous arterial magnesium concentrations. The final link model (line) was based on the assumption that S2 was unaffected by magnesium (i.e parameter value was fixed). if C art < 2.66 then SVR = 17 else SVR = -1.759*C art + 21.68    ...(9) Magnesium also raised venous compliance (CPL v ). This was related to the arterial concentration using a simple threshold (Fig. 11B ): if C art < 2 then CPL v = 0.45 else CPL v = 0.50    ...(10) Magnesium had little effect on myocardial contractility (Fig. 11C ), and the linking function assumed that CNT remained at baseline values. Magnesium appeared to increase the sympathetic tone coefficient for contractility (S2) by approximately 25% at between concentrations of 2 and 4 mmol L -1 (Fig. 11D ). However, this rise in S2 only occurred late in the study (Table 3 ). It indicates subtle changes in the relationship between the filling pressure index (LVEDP) and the contractility index (dp/dt). This may reflect measurement error in these variables, non-stationarity in the experimental preparation or subtle delayed changes in myocardial compliance caused by magnesium. However, it was found that a link function assuming S2 remained at baseline values (Fig. 11D ) was an adequate account of the data and did not compromise the predictive power of the model in the validation stage. The final kinetic-dynamic model therefore consisted of the kinetic model shown in Fig. 9 linked to the Constrained-Baroreceptor-Frank-Starling cardiovascular dynamic model (Figs. 4 & 7 ) via the link Equations 9 and 10. This is summarised in Fig. 12 . The equations for the model are shown in Additional file 4 . Figure 12 Overview of the kinetic-dynamic model linking process. A schematic representation of how the final model was derived from Data set 1. The pharmacokinetic (PK) component of the model was developed by fitting the observed arterial magnesium concentrations (Fig. 10, middle). As cardiac output was a parameter of the recirculatory model, the magnesium induced changes in cardiac output were represented as a forcing function during fitting (Fig. 10, top). In the final model, this forcing function was replaced by the cardiac output predicted by the cardiovascular (CV) model. For the CV model, target baseline cardiovascular variables were derived from previous measurements and the literature (Table 1). A unique parameter set for the CV model was found that reproduced these values (Table 2). To account for the changes in cardiovascular variables from baseline following magnesium, four parameters (SVR, CPL v , CNT and S2) were fitted to the observed magnesium CV data (expressed as change from baseline) at selected time-points (Fig. 8; Table 3). Of these, two parameters (SVR, CPLv) showed concentration dependent changes that could be related via link functions to the time-course of magnesium concentrations (Fig. 11). The other parameters of the CV model were fixed at their baseline values. The final model was able to predict the concentrations and CV effects of magnesium for a different dose regimen (Data set 2, Fig. 13). Validation of the final model The final kinetic-dynamic model developed using Data set 1 was used to predict the arterial magnesium concentrations, cardiac output and mean arterial blood pressure for Data set 2. Data set 2 differed from Data set 1 in that the dose of magnesium was given over 5 min instead of 2 min. Consequently, although the dose was the same, the cardiovascular effects were less pronounced. For example, the lowest blood mean arterial pressure for Data set 1 was 76% of baseline, while for Data set 2 this was 86% of baseline. The only change made to the parameters of the final model was to alter the duration of infusion of the magnesium. Results Parameter sensitivity of cardiovascular model (baseline conditions) The baseline cardiovascular variables and the parameters that produced them are summarised in Tables 1 and 2 , respectively. Of the 10,000 random parameter sets examined in the Monte-Carlo sensitivity analysis, only 37 produced a set of cardiovascular variables that was within 10% of the target cardiovascular variables. The variability of these successful parameter values was low (Table 2 ), and the spread of each parameter showed a unimodal, approximately normal distribution. This suggests that there was a unique set of parameter values for the model that was consistent with normal baseline physiology. Visual inspection showed no obvious correlation between parameter values, except for CPL v and CPL ratio (correlation coefficient = 0.83). This suggests that specifying the value for one of these parameters significantly constrains the value that can be taken for the other, as would be expected on physiological grounds. It can be concluded that each parameter had an important role to play in the model, and that each could only take a limited range of values to be consistent with the required baseline physiology. By extension, the assumptions regarding the values of these parameters are likely to be appropriate. Furthermore, the changes in these parameters observed following magnesium administration therefore reflect the effects of this drug rather than uncertainty in the parameter space of the model. Parameter estimates – cardiovascular data The method of estimating cardiovascular model parameters from cardiovascular data for individual time points was effective. Thus, it was possible to find a parameter set at each time point (Table 3 ) that produced a fitted cardiovascular radar plot that closely matched the observed plot (Fig. 8 ). In general, the parameter estimates were precise. The most obvious effect of magnesium was a drop in systemic vascular resistance and a rapid and sustained increase in venous compliance. The changes in the other cardiovascular variables (e.g. HR and MAP) simply reflected reflex changes in response to these primary drug effects. Figure 8 Best fit cardiovascular radar plots for each key time point. Cardiovascular radar plots of the observed data (blue) and the best fit of the final cardiovascular model (red). Note that the shape of the radar plot changes with time, indicating the evolving effects of magnesium on the circulation. For each time-point, the fit was an adequate account of the data (Table 3). Parameter estimates – pharmacokinetic data The recirculatory pharmacokinetic model was able to fit the observed concentrations with adequate fidelity (Fig. 10 , middle) and produce precise parameter estimates (Table 4 ). As the clearance of magnesium was low, it would be expected that the permeability term into the deep compartment governed the rate of decline of the magnesium concentration rather than its clearance from the body. Table 4 The fitted pharmacokinetic parameters for the Magnesium data set 1 The parameter estimates are given with the standard deviation returned by the curve-fitting program. Fitted variable Value Units MSC 3.13 V lung 0.887 (0.221) L CL 0.0021 (0.1286) L min -1 V body 4.023 (0.486) L PS 0.589 (0.227) L min -1 V deep 8.63 (5.39) L A feature of recirculatory pharmacokinetic models is that their initial kinetics are governed by first-pass passage of drug through the lungs, and the dilution of the injected drug with the cardiac output [ 7 ]. The cardiac output sensitivity analysis for the model confirmed this behaviour for magnesium (Fig. 10 , bottom). This reinforces the need for a common cardiac output term for the cardiovascular and recirculatory kinetic model (Fig. 12 ). The resultant final model therefore accounts for the fact that by altering cardiac output, magnesium alters its own kinetics. Link functions Relating the estimated cardiovascular parameters in Table 3 to the concurrent arterial concentrations produced the concentration-effect curves shown in Fig. 11 . Link functions were established for SVR and CPLv, but not CNT or S2. The overall role of the link functions is summarised in Fig. 12 . Model validation – pharmacokinetic component The recirculatory model of magnesium disposition was able to accurately predict the time-course of the arterial magnesium concentrations observed for the validation Data set 2, despite the large change in cardiac output produced by magnesium (Fig. 13 ). The mean prediction error was 0.02% Figure 13 Observed and predicted results for Data set 2. The final kinetic-dynamic model developed using Data set 1 was used to predict the exogenous arterial magnesium concentrations (C art,x ), cardiac output (CO) and mean arterial pressure (MAP) for Data Set 2. The only change in the model between the two data sets was to increase the duration of the infusion from 2 to 5 min. The observed data are shown as symbols, together with the upper and lower 95% confidence intervals of the data (dotted lines). The predictions of the model are shown by the solid lines. Model validation – pharmacodynamic component The final pharmacodynamic model was able to accurately predict the time-course of the cardiac output changes observed for the validation Data set 2 (Fig. 13 ). The mean prediction error was 3.0%. The dynamic model captured the general trend of the mean arterial blood pressure for the validation data (Fig. 13 ), but some systematic deviations were evident. The model was accurate until the end of the infusion, but thereafter slightly over-estimated the rate of recovery of blood pressure. However, the model did predict that the drop in blood pressure would be considerably less for a 5 min versus 2 min infusion, and the overall magnitude of the changes in blood pressure for the 5 min infusion were small (less than 10% change). The mean prediction error was 6.1%. Discussion Concentration-effect relationships and recirculatory models In this paper, all cardiovascular effects were related to the arterial concentration of magnesium. As covered in the introduction, there may be other sites in the body that have a theoretical claim to being the most appropriate link concentration for certain cardiovascular dynamic effects. For example, the reductions in myocardial contractility caused by thiopental have been shown to have a better temporal relationship to the thiopental concentrations in the myocardium itself rather than in arterial blood [ 17 ]. This consistent with a direct thiopental effect on the myocardium. In recirculatory models, it is possible to add a "target organ" to represent organs such as the heart [ 18 ]. The fact that this was not necessary for magnesium may be the exception rather than the rule. As magnesium has small volumes of distribution, there is little difference in the time-course of the arterial and regional venous concentrations. Furthermore, the predominant effects of magnesium were directly on blood vessels (arterioles for SVR and large veins for capacitance) in direct equilibrium with blood rather than organs such as the heart or brain. Thus, a "systemic" recirculatory model was sufficient for magnesium. As other drugs are studied using this method, data on target organ kinetics and their incorporation into the kinetic model may be necessary. Limitations There are a number of limitations of this modelling approach, many of which are inherent in the assumptions made in the construction of the model. Other limitations may become apparent if the model is used outside of the range of the data used to develop the model. For example, the CL term in the kinetic model was very low (Table 4 ). This may reflect extensive tubular re-absorption, but may also reflect the fact that the concentrations were followed for only 25 min in the original paper (the time by which most cardiovascular variables had returned to baseline). Studies of a longer duration would help to define this clearance term better. The cardiovascular model also assumes an instantaneous baroreceptor response. While it is relatively easy (in modelling terms) to add a delay to this response, this was not supported by the data. However, if the model is extended to situations with very rapid blood pressure changes (e.g. orthostatic hypotension) this deficiency may become significant. Constructing physiologically based models, even of the simplicity presented here, requires crossing many decisions points where a choice must be made from multiple options – sometimes the choices are data driven, sometimes theory driven, sometimes the subjective experience of the model maker must be called upon. While a "wrong" model is evident because it does no match the data, there is clearly no "right" model of the cardiovascular system. It is anticipated that more limitations of the cardiovascular dynamic model will become apparent when model is rigorously compared to data for other drugs, and for other cardiovascular scenarios. It is should be expected that the model will continue to evolve as these data are collected and analysed. Conclusion The combination of the recirculatory kinetic model and the simple cardiovascular dynamic model was able to describe and predict the concentrations and cardiovascular effects of magnesium in sheep. It is proposed that the general methods used here could be applied to other drugs with cardiovascular effects. The authors are currently applying the method to intravenous anaesthetics. Abbreviations Cardiovascular term Description Frank-Starling model V blood Blood volume CVP = P v Central venous pressure MAP = P a Mean arterial pressure MCP Mean circulatory pressure V a Volume of blood in arterial compartment V v Volume of blood in venous compartment V a 0 Volume of blood in arterial compartment at MCP V v 0 Volume of blood in venous compartment at MCP CPL a Arterial compliance CPL v Venous compliance CPL ratio Ratio of venous over arterial compliance SVR Systemic vascular resistance CO Cardiac output CO L Cardiac output (left side) CO R Cardiac output (right side) HR Heart rate SV Stroke volume kc unit conversion factor – contractility CNT Contractility additional for Frank-Starling-Baroreceptor model MAPset Mean arterial pressure set point HRgain Gain for heart rate control additional for Constrained-Frank-Starling-Baroreceptor model P a S Pressure in arterial compartment when stressed P v S Pressure in venous compartment when stressed V a S Volume in arterial compartment when stressed V v S Volume in venous compartment when stressed slopeMAP slope for arterial pressure-volume relationship intMAP intercept for arterial pressure-volume relationship slopeCVP slope for venous pressure-volume relationship intCVP intercept for venous pressure-volume relationship S1 Sympathetic tone coefficient – Chronotropy S2 Sympathetic tone coefficient – Contractility SV max maximum for stroke volume-CVP relationship SV 50 half-volume for stroke volume-CVP relationship nSV "Hill factor" for stroke volume-CVP relationship Pharmacokinetic term Description Description Recirculatory model R 0 doserate of zero order infusion tau duration of zero order infusion C art Arterial magnesium concentration (total) C ven Venous magnesium concentration (total) C art,e Arterial magnesium concentration (endogenous) C ven,e Venous magnesium concentration (endogenous) C art,x Arterial magnesium concentration (exogenous) C ven,x Venous magnesium concentration (exogenous) V lung Apparent distribution volume of the lung CL Clearance V body Apparent distribution volume of the body compartment PS Permeability-surface area product of deep compartment V deep Apparent distribution volume of the deep compartment Authors' contributions RNU participated in the original magnesium studies and performed the modelling. GLL participated in the original magnesium studies and acted as a resource for cardiovascular theory. Both authors contributed to the manuscript. Supplementary Material Additional File 1 The simple Frank-Starling model. The simple Frank-Starling model written in pseudo-code to generate cardiac function curves. The code is intended to run in the "Scientist" differential equation solving program. Click here for file Additional File 2 The Frank-Starling-Baroreceptor model. The equations for the Frank-Starling-Baroreceptor model with baroreceptor control written in pseudo-code to generate cardiac function curves. Click here for file Additional File 3 The Constrained Frank-Starling-Baroreceptor model. The equations used for the final cardiovascular model written in pseudo-code to generate cardiac function curves. Click here for file Additional File 4 The final pharmacokinetic-pharmacodynamic model. The equations used for the final model written in pseudo-code. Click here for file

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pRb Inactivation in Mammary Cells Reveals Common Mechanisms for Tumor Initiation and Progression in Divergent Epithelia

Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb f , collectively), play a pivotal role in regulating eukaryotic cell cycle progression, apoptosis, and terminal differentiation. While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of pRb f loss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that mouse mammary epithelium is similar in its requirements for pRb f function; Rb f inactivation by T 121 , a fragment of SV40 T antigen that binds to and inactivates pRb f proteins, increases proliferation and apoptosis. Mammary adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on aberrations commonly observed in the human disease.

Introduction Aberrant retinoblastoma 1 (pRb) pathway activity, resulting from defects in pRb itself, cyclin-dependent kinase inhibitor 2A (p16 INK4a ), cyclin D1 (CCND1), or cyclin-dependent kinase 4 (CDK4), is observed in the majority of human sporadic cancers ( Marshall 1991 ; Weinberg 1995 ; Sherr 1996 ; Ortega et al. 2002 ). This pathway is commonly altered early in cancer development, indicating an ability to predispose cells to tumorigenesis. However, whether the mechanism(s) is similar among cell types is not known. Examination of pRb inactivation in specific cell types in vivo has been technically challenging due to the apparent functional compensation or redundancy among pRb, retinoblastoma-like 1 (p107), and retinoblastoma-like 2 (p130) in many cell types of the mouse ( Luo et al. 1998 ; Robanus-Maandag et al. 1998 ; Dannenberg et al. 2000 ; Sage et al. 2000 ). Thus, genetic inactivation of the Rb gene alone, either by conditional deletion ( Marino et al. 2000 ) or by the generation of chimeric mice harboring pRb-deficient cells ( Maandag et al. 1994 ; Williams et al. 1994 ) yields only medulloblastomas, pituitary, and thyroid tumors. We have begun to systematically examine the role of retinoblastoma protein family (pRb f ) inactivation in multiple cell types of the mouse by dominant expression of T 121 , a truncation mutant of simian virus 40 (SV40) T antigen that inactivates all three pRb-related proteins ( DeCaprio et al. 1989 ; Dyson et al. 1989 ; Ewen et al. 1989 ; Stubdal et al. 1997 ; Sullivan et al. 2000 ). In this report we determine the role of pRb inactivation in mammary adenocarcinoma predisposition, establish a role for p53 inactivation in subsequent mammary adenocarcinoma progression, and, together with our previous studies, provide a comprehensive comparison of these mechanisms in distinct epithelial lineages. pRb plays a critical role in eukaryotic cell cycle progression, when cells exit G0 or G1 and enter S phase, thereby acting as a crucial negative regulator of cellular proliferation and neoplasia ( Sherr and McCormick 2002 ). In quiescent or early G1-phase cells, pRb is hypophosphorylated and associates with specific members of the E2F transcription factor family, converting them to active transcriptional repressors ( Hamel et al. 1992 ; Weintraub et al. 1992 ). Gene repression is also mediated by pRb and p130 recruitment of histone deacetylase to promote formation of inhibitory nucleosomes ( Brehm et al. 1998 ; Luo et al. 1998 ; Magnaghi-Jaulin et al. 1998 ). The many proteins found in association with pRb suggest other regulatory mechanisms may also be involved ( Morris and Dyson 2001 ), although the biological potential for most of these interactions remains yet unproven. Cell cycle progression from G to S phase occurs when complexes of D-type cyclins/CDK4/CDK6 phosphorylate pRb, thereby derepressing E2Fs to direct transcription of DNA-replication machinery and nucleotide biosynthesis genes ( Dyson 1998 ). Like most human solid tumors, breast cancers harbor frequent alterations in the pRb pathway, including CCND1 overexpression in 45% ( Buckley et al. 1993 ), p16 INK4A loss in 49% ( Geradts and Wilson 1996 ), and pRb loss in 6% of breast tumors ( Geradts and Wilson 1996 ). In the Rb -deficient mouse mammary gland, p107 and/or p130 may play overlapping or compensatory roles, as they do during embryonic development, given that pRb is dispensable for normal mammary development and mammary tumor suppression. pRb-deficient embryonic stem cells participate in normal mammary gland formation in chimeric mice ( Maandag et al. 1994 ), and donor pRb −/− mammary precursor cells transplanted into wild-type mice can populate a normal mammary gland without evidence of neoplasia, even after multiple pregnancies ( Robinson et al. 2001 ). The interplay between pRb signaling and the tumor protein p53 pathway is also critical to the understanding of breast cancer biology. Since the pRb pathway is defective in a majority of human tumors and the p53 gene is mutated in about half of them, including approximately a fifth of sporadic breast cancers ( Nigro et al. 1989 ; Greenblatt et al. 1994 ), these aberrations often coexist. Whether loss of these tumor suppressor pathways collaborate in tumorigenesis is also cell type-specific. In a brain epithelial tumor model, we previously demonstrated that, in the absence of pRb f function, inactivation of p53 significantly decreases apoptosis and accelerates tumor growth in vivo ( Symonds et al. 1994 ). However, in astrocytic brain tumors induced by pRb f inactivation, tumor progression is not accelerated by reduced p53 activity; rather, the phosphatase and tensin homolog ( PTEN ) regulates the apoptosis, and reduction in its function accelerates tumor growth ( Xiao et al. 2002 ). In this report, we extend our analysis of pRb function in vivo and examine the consequence of pRb f loss specifically in mammary epithelium. These studies serve not only to provide insight into the cell specificity of tumor suppression mechanisms, but also to model the stepwise evolution of breast adenocarcinomas that harbor defects in this pathway. Results Generation of Mice with Inducible pRb f Deficiency in Mammary Cells Seven founder mice were generated in which the T 121 gene was regulated by the whey acidic protein (WAP) transcriptional signals ( Figure 1 ; see Materials and Methods ). Of these, two founder animals died spontaneously of unknown causes, while the transgenic progeny of the third line died prematurely, also of unknown cause ( Figure 2 A). The extent to which the transgene contributed to these deaths was not investigated further; however, ectopic transgene expression was detected in several tissues (data not shown). Characterization of female mice of the four remaining lines is the focus of this report. Figure 1 Diagram of the WAP-T 121 Transgene and Protein The fragment consists of the 2.4 kb WAP promoter (hatched) and the mutant SV40 T-antigen coding region (white box) containing two deletions, the 196-bp amino-terminal deletion, which abolishes small t antigen production, and the dl 1137 deletion, which truncates T antigen. Both the J domain and the LXCXE domain are required for pRb family inactivation (see Materials and Methods ). Figure 2 Expression of T 121 Protein in WAP-T 121 Mice and a Summary of Gross Phenotypes As expected, each line showed mammary-specific expression following lactation induction, while line 4 showed more widespread expression, with protein detected in brain and kidney. Mice from the higher-expressing lines 3 and 4 failed to nurse because of lactation defects. Mammary glands of adult female mice from all four lines showed elevated proliferation and apoptosis. Glands from line 1 and 2 mice were hyperplastic, while glands from lines 3 and 4 were atrophic. Lines 3 and 4 later developed carcinomas and other neoplasms. T 121 protein was detected by Western blot analysis in lactating mammary glands of animals from all four lines (B), although the lower-expressing lines 1 and 2 required immunoprecipitation with anti-T-antigen antibody prior to Western blot analysis (right panel in [B]). Brain tumor extract (see Materials and Methods ) was used for a positive control, and nontransgenic mammary tissue extract was used for a negative control. A timecourse analysis of T 121 expression (C) shows lactation-induced expression peaking at 5 d postpartum. Abbreviations: Adeno-Ca, adenocarcinoma; AP, elevated apoptosis in mammary gland; At, atrophy; dpc, postcoital; FTN, failure to nurse; Hyp, hyperplastic acini; MG, mammary gland; MIN, mammary epithelia neoplasia; ND, not determined; nt, nontransgenic; pp, postpartum; Pr, elevated proliferation in mammary gland; pw, post-weaning. Footnotes: a Mosaic founder animal. b At earlier stages, development defects attributed to atrophy, while MIN and adenocarcinoma were observed at terminal stages. c Approximately half of progeny died of unknown cause. T 121 Is Expressed in Lactating Mammary Western immunoblotting analyses of mammary gland extracts demonstrated that this tissue expresses T 121 protein at the expected size in all four lines ( Figure 2 B). T 121 expression in lines 1 and 2 was only revealed following immunoprecipitation using an anti-T-antigen antibody prior to Western blot analysis, indicating lower levels of T 121 (right panel in Figure 2 B). A survey of select tissues showed that detectable expression was restricted to the mammary gland in lines 1–3, while expression was more widespread in the higher expressing line 4 (data not shown) and included brain and kidney expression. As expected, T 121 expression was induced by lactation with highest levels observed 5 d postpartum ( Figure 2 C). Southern blot analyses indicate that mice in line 3, which was used as a representative line for extensive characterization, harbor approximately ten copies of the transgene at a single insertion site (data not shown). Impact of Rb f Inactivation in Mammary Epithelium Representative histological analysis of lactating mammary glands (day 1) from single-pregnancy females of the line 2 founder (F 0 ) and a line 3 F 1 mouse shows that the impact of Rb perturbation is severalfold. Compared to an age- and parity-matched control tissue, the normal architecture of the lactating mammary tissue is disturbed. In contrast to normal tissue where acini consist of a single layer of secretory epithelia with milk-filled lumen ( Figure 3 A), transgenic animals have a lower density of acini ( Figure 3 K), consistent with atrophy, and are often atypical ( Figure 3 I). T 121 -positive mammary epithelial cells were associated with abnormalities ( Figure 3 B, 3 F, and 3 J). The line 2 F 0 animal was mosaic for T 121 protein expression with distinct regions of expressing and nonexpressing cells ( Figure 3 F), whereas T 121 expression in the line 3 animal was in secretory epithelium distributed throughout the gland ( Figure 3 J). Increased proliferation, indicated by proliferating cell nuclear antigen (PCNA) staining, was also observed in transgenic mammary glands ( Figure 3 C, 3 G, and 3 K), concomitant with increased levels of apoptosis assayed by TUNEL staining ( Figure 3 D, 3 H, and 3 L). Quantification of T 121 expression and apoptosis revealed higher protein expression levels (see Figure 2 B) correlate with higher percentages of apoptotic cells ( Figure 4 A). Consistent with a model for cell-autonomous functioning of T 121 , the pattern of abnormalities of morphology, proliferation, and apoptosis in the mosaic animal mimicked the regionalized T 121 expression pattern, and conversely, where T 121 protein was absent, the tissue appeared normal. Figure 3 Mammary-Specific Inactivation of the pRb Pathway Induces Extensive Abnormalities Histologic comparisons of nontransgenic (A–D), mosaic (F 0 line 2 [E–H]), and transgenic (F 1 , line 3 [I–L]) lactating mammary glands reveals that T 121 expression results in increased proliferation and apoptosis. Hemotoxylin and eosin staining shows acini of the normal lactating gland are composed of a single layer of secretory epithelial cells (A) with milk-filled lumen. Consistent with atrophy, transgenic animals have a lower density of acini demonstrated by the presence of lipid-filled adipocytes (asterisk in [K]). Acini composed of T 121 -expressing cells are atypical. Many are collapsed and composed of tall columnar epithelia of large hyperchromatic cells with papillary tufting (arrows in [I]). Transgene-expressing cells have large pleomorphic nuclei (open arrows in [G]) as compared to nuclei of nonexpressing cells (arrows in [G]). Staining for T 121 expression (blue in [B]–[J]) indicates the line 2 F 0 animal is mosaic, showing localized expression (F), whereas the transgene expresses throughout the gland of an F 1 line 3 animal (J). Increased proliferation assayed by PCNA staining (red) is also localized in the mosaic founder (G), but found throughout the F 1 transgenic gland (K). Similarly, TUNEL staining (brown) demonstrates increased apoptosis in transgenic animals (H and L); moreover, the regionalized apoptosis in the mosaic gland (H) strongly suggests that transgene expression and not precocious involution is the cause. All samples are from primiparous females on lactation day 1. Figure 4 Reduced p53 Activity Decreases Apoptosis but Does Not Increase Proliferation Representative apoptosis levels of each mouse line correlate with T 121 expression as indicated by the percentage of TUNEL positive cells (A). Decreasing levels of p53 activity correlate with lower levels of apoptosis in transgenic mammary glands (B). The mean percentage of apoptotic cells in p53 wild-type transgenic glands was 21%; in p53 heterozygous animals, 9%; and in p53 null animals, 5% (B), indicating that 75% of the apoptosis is p53-dependent. Apoptosis levels are further reduced to 2% in terminal stage tumors (B, Tumors). The percentage of PCNA staining cells remains unchanged in p53 heterozygous or nullizygous animals (C), indicating that reduction of p53 activity levels had no significant impact on cell proliferation. Samples were derived from primiparous animals on lactation day 1, except as indicated as tumor samples (B). Transgenic animals in (B) and (C) were from line 3. Role of p53 in Apoptosis To investigate the impact of germline loss of p53 on apoptosis levels in Rb f -deficient mammary glands, we mated line 3 animals to p53 null mice to generate transgenic and nontransgenic females of distinct p53 genotypes (+/+, +/−, −/−). Transgene expression was induced by a single pregnancy, and mammary glands were examined on lactation day 1. As expected, nontransgenic mammary glands showed no appreciable apoptosis regardless of p53 status ( Figure 4 B). However, in transgenic animals, decreased levels of p53 activity were correlated with lower levels of apoptosis. The mean percentage of apoptotic cells in p53 wild-type transgenic glands was 21%; in p53 heterozygous animals, 9%;and in p53 null animals, 5% ( Figure 4 B), indicating that 75% of the apoptosis is p53-dependent. That we could detect haploinsufficiency of p53 for apoptosis is remarkable, since in the previously characterized T 121 -expressing choroid plexus epithelium, apoptosis levels were the same in p53 heterozygous and wild-type backgrounds ( Lu et al. 2001 ). This observation indicates that there is a threshold for p53 levels in eliciting apoptosis and that either the threshold is different between cell types or that the absolute functional p53 level is distinct. Such differences could have significant impact on the requirements for tumorigenesis. Role of p53 in Proliferation In two other transgenic mouse models of breast cancer, where tumors were initiated by activated Harvey rat sarcoma viral oncogene homolog (v -Ha-ras ) ( Hundley et al. 1997 ) or wingless -related murine mammary tumor virus (MMTV) integration site 1 ( Wnt-1 ) ( Donehower et al. 1995 ), inactivation of p53 did not result in a reduction of apoptosis; rather, loss of p53 was associated with increased proliferation of the mammary epithelium. To determine whether p53 inactivation also impacted mammary cell proliferation induced by Rb f inactivation, glands from primiparous lactating (day 1) mice were assessed for the expression of nuclear PCNA. Unlike the tumors initiated by activated Ras or Wnt-1 , p53 heterozygosity or nullizygosity had no significant impact on the level of cell proliferation ( Figure 4 C). This experiment indicates that p53 can have distinct mechanisms of action depending on the nature of the initiating lesion. pRb Inactivation Predisposes to Tumorigenesis All females from higher-expressing lines (lines 3 and 4) failed to nurse pups because of lactation defects and developed mammary tumors after multiple pregnancies. Because line 4 mice expressed T 121 in nonmammary tissues, further characterization focused on line 3. For this line, the median time following initial transgene induction until a palpable tumor appeared was 10 mo, and within 16 mo, all mice developed palpable tumors ( Figure 5 A). Interestingly, latency in this line on a BALB/cJ background (see Materials and Methods ) was reduced to a median time of 8.5 mo ( p = 0.0077; Figure 5 A) indicating the presence of modifier alleles. The condensed timeframe for tumor development in this strain will also be valuable for future preclinical studies using this model. However, all further studies in the current report were carried out on the original B6D2F1 background. Figure 5 Mammary Tumor Onset and Growth Are Accelerated by p53 Reduction Among line 3 animals, the median time following initial transgene induction until a palpable tumor appeared was 10 mo, and within 16 mo, all mice developed palpable tumors (red line in [A]). In p53 +/− transgenic animals (blue line in [A]), mammary tumors were detected significantly earlier ( p < 0.0003) with a median onset of 6 mo. Among mice with BALB/cJ background (black line in [A]), median mammary tumor latency (8.5 mo) was significantly shorter ( p = 0.0077) compared to mice of the hybrid BDF1 background strain and indistinguishable ( p = 0.2466) from WAP-T 121 ;p53 +/− mice. Once palpable, WAP-T 121 ;p53 +/− tumors grew faster than the p53 wild-type counterparts (B). The average growth rates for p53 +/+ (black solid) and p53 +/− (dashed) are indicated. The median onset for mammary tumors in line 4 was 14 mo ( n = 3; data not shown), which indicates that the transgene and not its insertion caused tumorigenesis. With two exceptions, line 3 WAP-T 121 mice, regardless of p53 status, developed a single palpable tumor (87% of p53 +/+ , n = 15; 78% of p53 +/− , n = 9). A single mouse with either two or three palpable tumors was also observed in both p53 +/+ and +/− backgrounds. At least one additional nonpalpable tumor was visible during necropsy in approximately one-third of all tumor-bearing mice. While the two lower-expressing lines, lines 1 and 2, were able to nurse pups and appeared grossly normal, both had hyperplastic lobular alveoli associated with increased levels of proliferation and apoptosis. However, females from low-expressing lines did not develop adenocarcinomas after at least four pregnancies and 20 mo of age (line 1, n = 2; line 2, n = 6) (data not shown). Most terminal stage tumors in either wild-type or p53 +/− backgrounds were adenocarcinomas ( Figure 6 A, 6 B, and 6 E); however, we also observed four pilar tumors ( Figure 6 C and 6 E) and one spindle cell carcinoma ( Figure 6 D and 6 E). Terminal-stage mammary adenocarcinomas resembled poorly to moderately differentiated invasive ductal adenocarcinoma in humans. Morphologically, we designate these tumors as mixed solid and glandular carcinomas with necrosis and fibrosis. Poorly differentiated solid tumors ( Figure 6 A) are composed of nests of epithelial cells with large pleomorphic nuclei and delicate chromatin patterns with inverted nuclear:cytoplasmic ratios, while glandular tumors ( Figure 6 B) are composed of irregular glands with varying degrees of differentiation. While most animals had a single tumor mass, the adenocarcinomas were multifocal, with solid tumors consisting of subclones of distinct expansile masses, and with only two exceptions, glandular tumors were coincident with solid tumors. The adenocarcinomas were malignant, infiltrating dense, fibrous connective tissue, and were accompanied by strong peripheral immune response ( Figure 6 A). Figure 6 Tumor Morphologies Hemotoxylin and eosin staining of WAP-T 121 (C and D) and WAP-T 121 p53 +/− (A and B) (also representative of WAP-T 121 ) tumor sections shows that terminal stage adenocarcinomas have varied morphologies. Poorly differentiated solid tumors were comprised of nests (A) or cords of epithelial cells (Tu) that infiltrate a fibrous stroma and were accompanied by necrosis (arrow) and strong immune response (arrowheads). Moderately differentiated glandular tumors (B) consisted of irregular, disorganized glands. In animals of wild-type p53 background, four pilar tumors (C), distinguished by swirls of laminar acellular keratin (arrow), and a single spindle cell carcinoma (D) were also observed. For comparison, a lactating gland from a wild-type animal is shown in Figure 3 A. The percentage of animals displaying each of the phenotypes is summarized in (G). Since many tumors shared multiple morphologies, the sum exceeds 100%. Mammary Tumor Onset and Growth Are Accelerated by p53 Reduction Since 75% of the apoptosis induced by Rb f inactivation was mediated by p53 and was indeed reduced even in p53 +/− mice, we investigated the impact of p53 loss on tumor onset and growth kinetics. Animals harboring either one or two p53 null alleles were monitored for mammary tumors. As expected, a subset of p53 +/− and p53 −/− mice developed nonmammary tumors (either thymic lymphomas or sarcomas), consistent with published reports ( Jacks et al. 1994 ; Sandgren et al. 1995 ; Dannenberg et al. 2000 ). All p53 −/− mice ( n = 4) succumbed to these tumors by 4 mo of age, prior to developing palpable mammary tumors, so acceleration of this phenotype could not be assessed. In p53 +/− animals, mammary tumors were detected significantly earlier (see Figure 5 A; p = 0.0003) compared with p53 +/+ mice. Furthermore, once palpable, WAP-T 121 ;p53 +/− tumors grew significantly faster than the p53 wild-type counterparts (see Figure 5 B). The observation of four pilar tumors in p53 +/+ animals and none in p53 +/− animals is a statistically significant difference (Fisher–Freeman–Halton's exact test, p = 0.0177) and suggests that the reduction of p53 activity drives tumors to the adenocarcinoma phenotype. Taken together, these studies indicate that p53 heterozygosity leads to increased tumor growth rates and/or progression and may alter the spectrum of tumor morphologies. Selective Pressure for p53 Inactivation during Adenocarcinoma Development Since apoptosis was significantly reduced in WAP-T 121 ;p53 +/− mammary tissue compared with that of WAP-T 121 ;p53 +/+ mice, it was possible that p53 heterozygosity was sufficient for tumor acceleration. To assess whether this was the case or whether there was selective pressure for p53 inactivation during tumor progression, real-time PCR analysis was employed to determine the status of the wild-type p53 allele in WAP-T 121 ;p53 +/− tumors. Of ten tumors, eight showed loss of the wild-type p53 allele ( Table 1 ), indicating that the apoptosis reduction observed in WAP-T 121 ;p53 +/− mammary epithelium was not sufficient for tumor progression. Significant selective pressure favored cells that had completely inactivated p53, indicating that tumor progression requires further reduction of apoptotic activity and/or that p53 loss contributes to tumor progression through additional mechanisms that confer selective advantage. Assessment of apoptosis levels in terminal tumors showed apoptosis levels were indeed reduced in comparison to preneoplastic tissue (see Figure 4 B). Table 1 p53 LOH among the Majority of p53 +/− Tumors Real-time PCR was performed in duplicate to determine the status of the wild-type p53 alleles in the mammary tumors or tissues as indicated. Analysis of standard samples indicates that copy numbers of 2, 1, and 0 are indicated by 2 -ΔΔCt values of greater than or equal to 0.7, 0.2–0.7, and less than 0.2, respectively ( Lu et al. 2001 ). Of ten WAP-T 121 ;p53 +/− tumors, eight show LOH of p53 gene, while all three WAP-T 121 ;p53 +/+ tumors retained both p53 alleles. Abbreviation: Tg, transgenic a Tumor samples were derived from line 3 animals, except tumor 1, which was derived from a line 4 animal b ΔΔC t = [sample C t ( p53 ) − sample C t ( β-actin ) ] − [ p53 +/+ control Ct ( p53 ) − p53 +/+ control C t ( β-actin ) ]. C t = the number of cycles required to reach a threshold value, which is set within the exponential phase of the logarithmic scale amplification plot Comparative Genomic Hybridization Reveals Recurrent Chromosomal Imbalances in Tumors, but Limited Chromosomal Instability Among the multiple mechanisms of tumor suppression attributed to p53, a common hypothesis is that p53 prevents genetic instability. Indeed, studies using other mouse models indicate loss of p53 function in tumors often correlates with chromosomal instability. These include other breast cancer models such as Wnt-1p53 +/− ( Donehower et al. 1995 ) and MMTV-ras p53 +/− ( Hundley et al. 1997 ) and p53 +/− thymic lymphomas and sarcomas ( Venkatachalam et al. 1998 ). In marked contrast, our study of p53 deficiency in an evolving brain epithelial tumor showed that tumorigenesis progresses without chromosomal instability, indicating p53 loss contributes via alternative mechanisms ( Lu et al. 2001 ). To determine whether this difference was due to cell-type specificity, differences in initiating mechanisms, or differences in experimental approaches, we analyzed the genome of mammary WAP-T 121 ;p53 +/− tumors. We employed two methods of comparative genomic hybridization (CGH): chromosome-based CGH (cCGH) (Panel I in Figure 7 ) ( Kallioniemi et al. 1992 ) and microarray CGH (aCGH) (Panel II in Figure 7 ) ( Solinas-Toldo et al. 1997 ; Pinkel et al. 1998 ). Figure 7 CGH Analysis Shows Limited Genomic Instability Twelve tumors were analyzed by CGH: ten by cCGH (Panel I, A–J), eight by aCGH (Panel II, B, C, E, and H–L), and six by both procedures (Panels I and II, B, C, E, and H–J). In Panel I, green and red lines adjacent to the ideograms indicate relative gain or loss, respectively. Tumor sample identities are indicated by letters above gain and loss lines. Only a single sample (Panel I, D) shows loss of Chromosome 11. Telomeric sequences of many chromosomes are increased, most frequently Chromosomes 5 and 15. Recurrent losses are seen on Chromosomes 10 and X. For aCGH (Panel II), the genomic map is depicted with chromosomes horizontally aligned centromere to telomere. The relative fluorescence intensities (tumor:normal) are indicated along the vertical axis. Individual BACs are plotted according to their physical map position versus relative fluorescence, with sample identities indicated by a unique symbol for each tumor. To simplify visualization, only BACs with relative intensities greater than 1.25 (gains) or less than 0.75 (losses) are shown. X Chromosome values were halved to account for sex-mismatched samples. Changes spanning the entire length of the chromosome are readily detected on Chromosomes 6, 8, 10, 15, 18, and X. None of the clones showing loss on Chromosome 11 spans the p53 locus. The original p53 background of the animal and the p53 LOH status of each tumor are also indicated in the legend. Twelve mammary tumors were assayed by CGH: ten by cCGH, eight by aCGH, and six by both procedures. Both assays revealed limited genomic imbalances ( Figure 7 ), yet only a single tumor showed loss of Chromosome 11 (which harbors p53 ). Among samples tested by both methods, there is strong concordance among large chromosomal changes, encompassing multiple cytological bands to whole chromosome lengths. For example, there is apparent whole chromosomal duplication of Chromosomes 6 and 15 in tumor C and of Chromosomes 8 and 18 in tumor H, monosomy of Chromosome 10 in tumor J, and loss of X Chromosome in tumors E and H (all or partial, respectively). Making comparisons among imbalances spanning shorter chromosome lengths was more difficult, mainly due to the challenge of reconciling cytological and physical maps. Furthermore, technical limitations may account for real differences between the two assays: small imbalances detected by one to several bacterial artificial chromosome (BAC) clones are irresolvable by cCGH; on the other hand, the relatively low density of BAC clones may not adequately sample smaller regions detected by cCGH. Nevertheless, on average, about five imbalances per tumor were detected by cCGH. This number is comparable to the number of changes observed in myelocytomatosis oncogene (c -myc )-induced mouse mammary tumors ( Weaver et al. 1999 ) and human tumors ( Ried et al. 1995 ), yet less than the number of changes seen in breast cancer 1 ( Brca1 )-deficient mouse tumors (8.0) and more than v- erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER2/ neu )-induced tumors (2.8) ( Montagna et al. 2002 ; Weaver et al. 2002 ). Discussion Common Mechanisms for Tumor Progression in Epithelial Cells of Distinct Origin Here we report that loss of pRb family function in mammary epithelium predisposes to malignant adenocarcinoma. Using a single transgenic allele, we have thus far inactivated the pRb pathway in several cell types in the mouse: brain choroid plexus epithelium, astrocytes, and mammary epithelium. In each case, despite the marked differences among these divergent cell types, pRb inactivation causes a similar response, initially evoking increased proliferation and apoptosis and, ultimately, predisposing to tumorigenesis ( Chen et al. 1992 ; Saenz-Robles et al. 1994 ; Symonds et al. 1994 ; Xiao et al. 2002 ). Not surprisingly, the long latency of mammary adenocarcinomas indicates that additional events are required for tumor progression. We show that mammary epithelium is similar to brain epithelium ( Symonds et al. 1994 ; Lu et al. 2001 ) in its requirement for p53 activity in the apoptotic response to aberrant proliferation caused by pRb f inactivation. Previous models using wild-type large T antigen ( Li et al. 1996b ; Husler et al. 1998 ; Green et al. 2000 ; Schulze-Garg et al. 2000 ) are unable to address the relative contribution of pRb and p53, since T antigen also binds and inactivates p53. As in brain epithelium, we show here that when the mammary tumor phenotype is initiated by pRb f inactivation, most of the apoptosis is mediated through p53. Furthermore, as in brain epithelium, heterozygosity for a null p53 allele significantly shortens tumor latency (discussed further below). Importantly, the Rb f deficiency-induced apoptotic response and inhibition of tumor progression are not universally dependent on p53. In astrocytes, we recently showed that PTEN, and not p53, modulates these same responses to Rb f inactivation. In contrast to the p53-dependent apoptosis of mammary epithelial cells in response to pRb f deficiency, apoptosis associated with normal mammary involution subsequent to lactation does not require p53 ( Li et al. 1996a ). Thus, the “wiring” of the apoptotic response within this cell type is not global, but rather depends on the signal. Although loss of p53-dependent apoptosis accounts for the acceleration of mammary tumorigenesis in WAP-T 121 ;p53 +/− mice, in models expressing either activated v-Ha-ras ( Hundley et al. 1997 ) or Wnt-1 ( Jones et al. 1997 ), earlier tumor formation in p53 heterozygous and homozygous null mice is accounted for by increased proliferation rather than attenuated apoptosis. An important caveat to this comparison is that the latter studies compared apoptosis in terminal tumors in which loss of apoptosis might have been selected regardless of initial p53 status, leaving open the possibility that tumor growth rates in these models reflect the combined effects of increased proliferation as well as reduced apoptosis. Nevertheless, there is a clear difference in WAP-T 121 mammary gland in that, unlike the Ras and Wnt-1 models, proliferation levels do not depend on p53 status. Taken together, these observations indicate that the specific cellular response to an oncogenic stimulus depends on the nature of the initial insult. Given that the pRb pathway is directly disrupted in T 121 -expressing cells, this could be explained if these other initiating events evoke p53-dependent growth arrest which, in part, functions upstream of pRb. High Selective Pressure for p53 Inactivation in the Transition to Aggressive Mammary Adenocarcinoma Most of the apoptosis induced by pRb f deficiency in both mammary (75%) and brain (85%) epithelia is p53 -dependent as determined by comparing p53 +/+ and p53 −/− tissue. However, while p53 heterozygosity had no impact on the level of apoptosis in the brain epithelium, in the mammary gland the level was reduced by half in p53 +/− tissue. Given that apoptosis is the basis for selective inactivation of p53 in the brain tumor model ( Lu et al. 2001 ; X. Lu and T. Van Dyke, unpublished data), it was possible that the pressure was relieved or reduced in WAP-T 121 ;p53 +/− mice. However, aggressive adenocarcinoma growth was accelerated with 100% penetrance, and 80% of these tumors underwent selective loss of the wild-type p53 allele, just as in the brain tumor model ( Lu et al. 2001 ). This result indicates that tumor progression requires more than a simple reduction in the level of apoptosis; it follows that p53 may contribute to tumor suppression by multiple mechanisms. While both mammary and brain carcinomas show high rates of p53 loss of heterozygosity (LOH), the mechanism of loss may be distinct. Chromosome loss clearly explains p53 LOH in the brain carcinoma model ( Lu et al. 2001 ) where nearly all tumors (greater than 90%) are monosomic for Chromosome 11, whereas only a single mammary tumor analyzed by CGH showed Chromosome 11 loss. Alternative mechanisms that may explain p53 LOH in the mammary tumors include somatic recombination or chromosomal reduplication following mitotic nondisjunction. Whether these alternative routes of LOH represent bona fide tissue-specific phenomena or are due to relatively small sample sizes will require further analyses. Interestingly, most mammary tumors derived from Brca1 -deficient mice lost p53 ; however, regions distal to p53 were amplified ( Weaver et al. 2002 ). Thus, it is possible that mammary tumor promoting factor(s) is located on distal Chromosome 11, selecting against loss. Limited Chromosomal Instability in the Absence of p53 Genomic instability is a hallmark of most human solid tumors, and a widely held view is that p53 represses instability to suppress tumorigenesis, although evidence for this activity has been mostly correlative. Contrary to this model, we demonstrated previously that in the absence of p53 activity in brain epithelia, tumors progress without chromosomal instability; except for Chromosome 11 loss, in a p53 +/− background these carcinomas are diploid ( Lu et al. 2001 ). Here we show that mammary tumors similarly harbor limited genome-wide alterations. While the number of aberrations within the mammary tumors is small, it is intriguing that some changes are recurrent, suggesting that their accrual is causal in tumorigenesis. T 121 -induced mammary carcinomas harbor more genomic imbalances than brain tumors (approximately five versus approximately one). One explanation for this observation is that, because the brain is a vital organ, animals succumb to their illness when the brain tumor is at a relatively earlier stage at which fewer changes have accumulated. However, chromosome content of choroid plexus tumors passaged further in xenografts remained stable (X. Lu and T. Van Dyke, unpublished data). The converse experiment, analyses of early mammary tumors subsequent to p53 loss, will be required to determine the kinetics of chromosomal changes in this tissue. Pocket Protein Redundancy Chimera and tissue-grafting experiments with pRb-deficient cells indicate the absence of pRb alone is not sufficient for abnormal mammary development or tumor formation ( Maandag et al. 1994 ; Robinson et al. 2001 ). Yet mammary-directed overexpression of CCND1, an upstream regulator of pRb f , leads to mammary adenocarcinoma ( Wang et al. 1994 ). Given other recent studies indicating the possibility for compensation of pRb function by p107 and/or p130 ( Dannenberg et al. 2000 ; Sage et al. 2000 ) and the clear redundancy of function in some murine cell types ( Robanus-Maandag et al. 1998 ; Xiao et al. 2002 ), it is likely that the discrepancy among our results can be explained by overlapping functions of other family members, p107 and/or p130. In our studies, T 121 abrogates the activities of all Rb family members by a dominant interfering mechanism. A subtly distinct alternative explanation is that the acute loss of pRb signaling, rather than a chronic loss as of pRb during mammary development, as in the chimera and grafting models, accounts for the difference. Cell culture experiments that support this hypothesis were recently reported ( Sage et al. 2003 ). In this model, p107 and p130 may be more responsive to pRb regulatory signals during development than in the terminally differentiated tissue; therefore, the developing tissue more easily accommodates for the absence of pRb in the pool of available pocket proteins. In the WAP-T 121 model, the gland undergoes normal development and then is subsequently subjected to acute pRb pathway loss. We presume that this scenario more closely mimics the situation of spontaneous somatic loss in adult human breast. The test of this alternative hypothesis awaits analyses of tissue-specific inactivation of pRb and the paralogous pocket proteins using conditional alleles. A Model for Mammary Tumorigenesis Initiated by Targeting the pRb Pathway The WAP-T 121 model is a significant addition to the current repertoire of preclinical mammary tumor models exploring the role of pRb pathway in tumorigenesis. Despite the prevalence of pRb pathway defects in human sporadic cancers, mice harboring germline mutations of p16 INK4a do not develop mammary cancer ( Krimpenfort et al. 2001 ; Sharpless et al. 2001 ). In addition, mammary-directed expression of CCND1 is only mildly oncogenic ( Wang et al. 1994 ), and as mentioned above, inactivation of pRb alone is not sufficient for tumorigenesis. Although the WAP promoter was a convenient means of directing mammary-specific expression for an initial assessment this model, it also presents the major shortcoming to this model in that expression of T 121 is linked to lactogenic hormone activity, as in most existing murine mammary tumor models. Future improvements aim to direct expression of T 121 through hormone-independent methods. Finally, the advantage over wild-type T antigen models is that WAP-T 121 uncouples the simultaneous inactivation of pRb and p53 and permits an assessment of the relative contributions of the individual oncogenic pathways. Testing the combinatorial effects of Rb loss and other breast cancer mutations (e.g., BRCA1 and BRCA2 ), along with the further characterization of WAP-T 121 tumors, should help provide additional insights into human breast cancer biology. Materials and Methods Derivation and characterization of transgenic mice. The 2.4 kb WAP promoter region was isolated from a WAP-TGFα construct (a gift from David Lee, University of North Carolina at Chapel Hill, United States [ Sandgren et al. 1995 ]) and was cloned upstream of a 2.4 kb KpnI–SalI fragment of the dl 1137′t plasmid ( Chen et al. 1992 ). We targeted T 121 expression to mammary gland using the WAP promoter, which is induced late in pregnancy and expressed during lactation ( Pittius et al. 1988 ) (see Figure 1 ). T 121 contains the first 121 amino acids of the SV40 T antigen (see Figure 1 ) that encodes a J domain and a pRb-binding domain, which together are sufficient to cause transformation by inactivating the pRb f proteins ( DeCaprio et al. 1989 ; Dyson et al. 1989 ; Ewen et al. 1989 ). Importantly, in contrast to other wild-type T antigen constructs encoding the entire SV40 early region ( Husler et al. 1998 ; Green et al. 2000 ; Schulze-Garg et al. 2000 ), small t antigen expression is absent due to a deletion that removes the splice acceptor site. The importance of this is demonstrated by the recent observation that small t antigen alone is sufficient for tumorigenesis in the mammary gland ( Goetz et al. 2001 ). Furthermore, p53 and EP300 (E1A-binding protein p300), which map to the carboxyl half of T antigen, are also abolished, thus permitting assessment of pRb f inactivation without the confounding effects of altering additional suppressor pathways. An EcoRI fragment containing the full transgene (see Figure 1 ) at a concentration of 4 ng/μl was injected in to fertilized eggs harvested from B6D2F1 (Jackson Laboratory, Bar Harbor, Maine, United States) mice as described previously ( Yan et al. 1990 ). Transgenic mice were identified by PCR amplification of a 160 bp fragment using primers 5′-GAATCTTTGCAGCTAATGGACC-3′ and 5′-GCATCCCAGAAGCTCCAAAG-3′ with toe-derived genomic DNA as template. Cycling profile was as follows: 94°C, 2 min; 35 cycles of 94°C, 20 s; 62°C, 45 s; 72°C, 45 s; and final incubation of 72°C, 2 min. TgWAP-T 121 mouse lines were maintained by crossing to nontransgenic B6D2F1 mice (Jackson Laboratory) and therefore are designated as B6;D2-Tg(WAP-T 121 ) Tvd. To study the effect of background differences, WAP-T 121 males were backcrossed to BALB/cJ (Jackson Laboratory) female mice. To increase sample size, tumor onset analysis for BALB/c background mice combined data for N6 ( n = 6), N7 ( n = 1), and N9 ( n = 4) generation mice. For tumor induction, female mice, unless noted as virgin, were housed with male mice to maximize the number of pregnancies, because WAP promoter activity is lactation-dependent ( Pittius et al. 1988 ). To study the effect of p53 mutation on mammary tumorigenesis in WAP-T 121 mice, male WAP-T 121 + mice were mated to p53 +/− females ( p53 tm1Tyj ; Jackson Laboratory). p53 genotypes were determined by PCR using two reactions ( Lowe et al. 1993 ), one that amplifies the neomycin insertion site (neomycin primer: 5′- TCCTCGTGCTTTACGGTATC-3′, p53 primer: 5′-TATACTCAGAGCCGGCCT-3′; 525 bp product) and a second that amplifies the endogenous p53 allele (substituting 5′-ACAGCGTGGTGGTACCTTAT-3′ for the neo primer, 475 bp product). Cycling parameters were the same as the above WAP-T 121 reaction. We performed the cross WAP-T 121 − ;p53 +/− × WAP-T 121 + ;p53 +/− , and transgenic female mice that were p53 +/+ , p53 +/− , or p53 −/− were used for analyses while nontransgenic littermates served as controls. Western immunoblotting analysis. Protein expression levels were assayed as previously described ( Symonds et al. 1993 ). Fresh or flash-frozen tissue samples were homogenized in lysis buffer (50 mM Tris [pH 8.0], 5 mM EDTA, 150 mM NaCl , and 1% NP-40) using a Polytron ® homogenizer (Kinematica, Littau-Lucerne, Switzerland). Total protein (10 μg) was electrophoresed through a 15% polyacrylamide denaturing gel and then transferred to nitrocellulose membrane (15 V, 30 min). Alternatively, for low-expressing lines, immunoprecipitation was performed prior to electrophoresis as previously described ( Symonds et al. 1991 ). The filter was preincubated in 3% bovine serum albumin, followed by incubation with primary antibody against SV40 T antigen (PAb419 at a dilution of 1:5,000; Harlow et al. 1981 ). The filter was then washed, followed by incubation at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham Biosciences, Little Chalfont, United Kingdom). The enhanced chemiluminescence method (Amersham Biosciences) was used for autoradiography. Histopathology and immunohistochemistry. Mammary tissue and tumor samples were dissected from WAP-T 121 transgenic or age- and parity-matched B6D2F1 animals. A portion of each tumor was flash-frozen in liquid nitrogen and the remaining tissue was fixed in 10% phosphate buffered formalin, embedded in paraffin, cut to a 5-μm thickness, and stained with hemotoxylin and eosin or immunostained using the Vector ABC system (Vector Laboratories, Burlingame, California, United States) for histopathological examination. Apoptosis levels were evaluated by TUNEL assay ( Gavrieli et al. 1992 ) essentially as described in Symonds et al. (1994 ). Real-time PCR. Quantitative real-time PCR analysis was performed using a TaqMan approach on DNA derived from terminal tumors to determine the status of the wild-type p53 allele as previously described ( Lu et al. 2001 ). The primers for the p53 allele were 5′-ATGGCCATCTACAAGAAGTCACAG-3′ and 5′-ATCGGAGCAGCGCTCATG-3′. The sequence of the p53 probe was 5′-ACATGACGGAGGTCGTGAGACGCTG-3′. The primers for the internal control β-actin gene were 5′-AAGAGCTATGAGCTGCCTGA-3′ and 5′-ACGGATGTCAACGTCACACT-3′. The sequence of the β-actin probe was 5′-CACTATTGGCAACGAGCGGTTCCG-3′. Each 25-μl reaction mixture contained 50 ng of DNA template, 18 nM p53 primers, 80 nM β-actin primers, 8 nM probe, and 12.5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, California, United States) containing AmpliTaq Gold polymerase, deoxynucleoside triphosphates, and PCR buffer. The cycling conditions were 50°C for 2 min and 95°C for 10 min for 1 cycle, and 95°C for 15 s and 60°C for 1 min for 40 cycles. The reactions were performed using an ABI 7700 Sequence Detection system (Applied Biosystems), and the data analyzed using Sequence Detector 1.7 (Applied Biosystems) and standard protocols ( http://www.appliedbiosystems.com ). The copy number of each sample was determined by calculating ΔΔCt based on the formula ΔΔC t = [sample C t(p53) − sample C t( β-actin ) ] − [p53 +/+ control Ct (p53) − p53 +/+ control Ct ( β-actin ) ], where C t is the number of cycles required to reach a threshold based on linear amplification. Analyses of standard samples (L. Chin, Harvard University, Cambridge, Massachusetts, United States, personal communication) indicate copy numbers of 2, 1, and 0 are indicated by 2 -ΔCtn values of greater than 0.6, 0.15–0.6, and less than 0.15, respectively. Standard samples analyzed along with experimental samples confirmed the accuracy of these assignments. Statistical analyses. Kaplan–Meier survival analysis was used to determine median tumor latencies (StatsDirect, Camcode, Sale, United Kingdom), and the Log-Rank (Peto, StatsDirect) test was performed to evaluate significance. The equivalence of tumor morphology distributions was tested using the Fisher–Freeman–Halton's exact test. CGH. Genomic DNA was extracted from end-stage tumors (1 cm in diameter) or tails using a DNeasy genomic tip (Qiagen, Valencia, California, United States) and further purified by proteinase K digestion followed by phenol/chloroform extraction, ethanol precipitation, and resuspension in sterile H 2 O. cCGH was performed as described in Kallioniemi et al. (1992 ), Donehower et al. (1995 ), and Lu et al. (2001 ). aCGH was performed as described in Snijders et al. (2001 ). For both methods, genomic DNA from tumor and normal tissue was labeled with different fluorochromes and then cohybridized together with Cot-1 DNA to either normal metaphase chromosomes from cultured cells (cCGH) or microarrayed BAC clones containing mouse genomic DNA (aCGH). Nonequivalent fluorescence intensities indicate relative imbalances of genomic DNA. Aneuploidy and partial chromosome gains and losses are detectable by cCGH with approximately 10 Mb resolution. Graphical output of cCGH data was generated using the National Cancer Institute and National Center for Biotechnology Information Spectral Karyotyping SKY and Comparative Genomic Hybridization CGH Database ( http://www.ncbi.nlm.nih.gov/sky/skyweb.cgi ). For aCGH, approximately 1,500 BAC clones span the entire mouse genome with 2–20 Mb spacing. Tumor DNA and normal DNA were sex-mismatched; thus, the X Chromosome served as an internal control, while normal tail DNA was used as a negative control. Gains or losses were scored based on tumor:normal fluorescence ratios that were greater than 1.25 or less than 0.75, respectively. Supporting Information Accession Numbers The accession numbers for the genes and gene products discussed in this paper are Brca1 (LocusLink ID 12189), CDK2 (LocusLink ID 1017), CDK4 (LocusLink ID 1019), CDK6 (LocusLink ID 1021), c -myc (LocusLink ID 17869), cyclin D1 (LocusLink ID 595), cyclin E (LocusLink ID 898), E2F (InterPro ID IPR003316), HER2/ neu (LocusLink ID 13866), histone deacetylase (LocusLink ID 3065), p16 INK4a (LocusLink ID 1029), p53 (LocusLink ID 7157), p107 (LocusLink ID 5933), p130 (LocusLink ID 5934), p300 (LocusLink ID 2033), PCNA (LocusLink ID 18538), pRb (LocusLink ID 5925), PTEN (LocusLink ID 5728), v -Ha-ras (LocusLink ID 3265), WAP (LocusLink ID 22373), and Wnt-1 (LocusLink ID 22408). These databases may be found at www.ncbi.nlm.nih.gov/LocusLink/ (LocusLink), and www.ebi.ac.uk/InterPro/ (InterPro).

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Ecology Drives the Global Distribution of Human Diseases

It's no surprise that the Amazonian rainforest contains far more species than, say, the Siberian tundra. Over 50% of the world's species live in tropical rainforests, which cover just 6% to 7% of the earth's terrestrial surface. That the number of marine and terrestrial species declines with distance from the equator is a well-documented phenomenon called the latitudinal species diversity gradient. What's proven challenging, however, is figuring out what drives this pattern. Over 30 hypotheses have been proposed in the past two decades, but only four have garnered serious attention. These four focus on variables relating to area and energy factors, geographic constraints, and habitat diversity. Understanding the factors—both contemporary and ancient—responsible for the diversity gradient could help answer one of the fundamental questions in evolutionary ecology: what regulates species diversity? But teasing out the likely mechanisms behind this diversity has practical implications as well: mounting evidence suggests that ecological and climatic conditions influence the emergence, spread, and recurrence of infectious diseases. Global climate change is likely to aggravate climate-sensitive diseases in unpredictable ways. The number of pathogen species increases towards the equator Increasingly, public health programs aimed at preventing and controlling disease outbreaks are considering aspects of the ecology of infectious diseases—how hosts, vectors, and parasites interact with each other and their environment. The hope is that by understanding how ecological factors impact the global distribution of parasitic and infectious diseases, public health officials can predict and contain future outbreaks. Even though parasitic and infectious organisms account for a major fraction of the biological diversity on the planet, few studies have analyzed the factors affecting the spatial distribution of these organisms or attempted to quantify their contribution to biodiversity. In this issue, Vanina Guernier, Michael Hochberg, and Jean-François Guégan address the influence of ecological factors on the biological diversity and distribution of parasitic and infectious diseases and find that climatic factors are the most important determinant of the global distribution of human pathogens. The current understanding of human disease and availability of complete datasets on many parasitic and infectious diseases, the researchers explain, present a unique opportunity to explore the relationship between parasitic and infectious disease species richness (defined in their study as total number of pathogens within a given country's borders) and latitude. This information, in turn, can help identify potential factors that affect diversity gradients. After compiling epidemiological data on 332 different human pathogens across 224 countries, Guernier et al. used sophisticated statistical modeling methods to identify and characterize the influence of a number of potential contributing factors on species richness. After adjusting the model to control for cofactors that might influence the relationship between latitude and species richness indirectly rather than directly (cofactors such as the size of countries and demographic, economic, and environmental variables), the researchers confirmed that, on average (seven times out of ten), tropical areas harbor a larger number of pathogen species than more temperate areas. In other words, the species richness of human pathogens follows the same pattern seen in other species. These results, Guernier et al. argue, suggest that the latitudinal species diversity gradient “might be generated in large part by biotic interactions.” This in turn indicates that current estimates of species diversity, which ignore parasites and infectious organisms, are “substantially underestimated.” The authors went on to explore groupings of individual pathogen species within larger parasitic and infectious disease communities along the gradient and found that species present at northern latitudes are a subset of those present in equatorial areas, rather than a different set of species (a phenomenon called “nestedness”). Since nestedness is strongly associated with latitude, which is typically used as a proxy for a range of climatic factors, the researchers investigated the relationship between various climatic variables and pathogen diversity. The climatic variable most strongly correlated with diversity was the maximum range of precipitation of a region. The finding that climatic factors are largely responsible for the spatial distribution of human pathogens has important implications for predicting and managing future infectious disease outbreaks. These results counter the conventional assumption that socioeconomic conditions are the most important factor in controlling disease, indicating that global climate change could have far more significant effects on global patterns of disease, with diseases once relegated to the tropics migrating to temperate zones, for example. Identifying the links between ecology and disease, however, could lay the foundation for effective preventive strategies.

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549602

Why Blood Glucose Control Matters for the Kidney

One of the most common and most serious complications of both type 1 and type 2 diabetes is diabetic nephropathy. It occurs in around 30% of patients with type 1 diabetes and 10% to 40% of patients with type 2 diabetes. Diabetic nephropathy is the leading cause of renal failure in the developed world. The main effect of diabetic nephropathy is proteinuria, initially in very small amounts but which increases, leading to nephrotic syndrome and end-stage renal disease in most cases. Apoptotic renal tubular cells in diabetic nephropathy Various risk factors in individuals with diabetes are known to increase the chance of developing diabetic nephropathy, including South Asian or African background, male sex, long history of diabetes, poor blood sugar control, high blood pressure, and smoking. One early change associated with diabetic nephropathy is degeneration of the renal tubular epithelium, but the exact cause of this at the cellular level is unclear. Erwin Böttinger and colleagues have dissected out one key point in the progression to diabetic nephropathy. They looked at cell lines of renal tubular cells from humans and mice and kidney biopsies from patients with diabetic nephropathy, patients with non-diabetic renal disease, and mice with genetic and induced diabetes. In the human cell lines they showed that glucose induced the expression of CD36, a receptor known to have a role in adhesion and signal transduction (in addition to being the receptor for malaria-infected erythrocytes). They then went on to show that apoptosis of these cells occurred in the presence of glycated (glucose-modified) albumins or free fatty acids, which are present in increased amounts in patients with diabetes, and that CD36 was essential for the apoptosis to occur. They then examined how CD36 triggered apoptosis and found that it involved src kinase, p38 MAP kinase, and caspase 3. Comparing mice and humans, the researchers found that the two species are not alike: diabetic mice did not show an increase in tubular expression of CD36—even though the gene is present in mice—and had normal tubular epithelium and no tubular apoptosis. They confirmed this difference between humans and mice by showing that normal mouse epithelial cell lines were resistant to apoptosis caused by the glycated albumins; however, artificially expressing CD36 in these lines made them susceptible to apoptosis by these modified albumins. These results provide insight into one of the crucial steps in diabetic nephropathy and, in humans at least, might help to explain why high blood glucose is so damaging to the kidney, hence providing a good reason—if another is needed—for encouraging patients to control blood glucose as tightly as possible.

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526762

Transmembrane carbonic anhydrase isozymes IX and XII in the female mouse reproductive organs

Background Carbonic anhydrase (CA) classically catalyses the reversible hydration of dissolved CO 2 to form bicarbonate ions and protons. The twelve active CA isozymes are thought to regulate a variety of cellular functions including several processes in the reproductive systems. Methods The present study was designed to investigate the expression of transmembrane CAs, CA IX and XII, in the mouse uterus, ovary and placenta. The expression of CA IX and XII was examined by immunoperoxidase staining method and western blotting. CA II and XIII served as positive controls since they are known to be present in the mouse reproductive tract. Results The data of our study indicated that CA XII is expressed in the mouse endometrium. Only very faint signal was observed in the corpus luteum of the ovary and the placenta remained mainly negative. CA IX showed weak reaction in the endometrial epithelium, while it was completely absent in the ovary and placenta. Conclusion The conservation of CA XII expression in both mouse and human endometrium suggests a role for this isozyme in reproductive physiology.

Background Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that are responsible for the reversible hydration of carbon dioxide in a reaction CO 2 + H 2 O ↔ H + + HCO 3 - . CAs are produced in a variety of tissues where they participate in several important biological processes such as acid-base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis, gluconeogenesis, lipogenesis and body fluid generation [ 1 , 2 ]. The mammalian α-CA gene family includes at least twelve enzymatically active isoforms with different structural and catalytic properties. CA I, II, III, VII and XIII are cytosolic enzymes [ 1 , 3 , 4 ]. CA VA and VB are mitochondrial proteins encoded by nuclear DNA [ 5 , 6 ]. CA VI is the only secretory form being present in saliva and milk [ 7 ]. The cluster of membrane-bound CAs includes four isozymes: CA IV, IX, XII, and XIV [ 8 - 11 ]. The other members of the CA gene family (CA VIII, X and XI) are inactive isoforms whose functions have not yet been described [ 3 , 12 , 13 ]. It has been previously suggested that CAs may play important roles in the uterine endometrium by maintaining the appropriate pH balance through the catalysis of the production of bicarbonate ions [ 14 ]. Indeed, the role of bicarbonate in fertilization has been demonstrated in a number of previous studies. It is functionally involved in some key processes such as sperm cell capacitation and regulation of sperm motility [ 15 - 17 ]. Similarly, CAs may have several functions also in the placenta. They can be active in intermediary metabolism and provide ions for exchange in transepithelial movement of ions and fluid [ 18 ]. CA activity has been studied in pig, horse, cow, mink, rat and human placentas, and the results show considerable heterogeneity among different species [ 18 ]. Previous immunochemical studies have shown evidence for expression of CA II but not CA I or III in the bovine placenta [ 19 ]. Both CA I and II are expressed in the human syncytiotrophoblasts [ 20 - 22 ] and, especially CA II, in the fetal villous endothelium of mature placenta [ 22 ]. CA IV-positive staining has been reported in the mouse placenta by Rosen and coauthors [ 23 ]. Their data showed strong CA IV immunoreactivity in the mouse trophoblasts and endodermal layer of the yolk sac. In the mouse genital tract, CA I, II and III have been reported by Ge and Spicer [ 24 ]. These isozymes were reported to be present in the theca interna cells in the mouse ovary, and CA I was found in the zona pellucida and cytoplasmic foci in follicular granulosa cells. In the mouse oviductal epithelium, CA II expression showed distinct variation. The reaction was absent in the infundibulum, whereas the ampulla and isthmus showed positive staining. CA XIII is the newest member of the CA enzyme family, which has been described in the mouse and human endometrium along with several other positive tissues [ 4 ]. As a cytosolic isozyme it may be one of the major proteins regulating the pH and bicarbonate homeostasis not only in the endometrial cells but also in the lumen of the uterus. These mechanisms are complex due to the presence of several isozymes, however, and may greatly differ between species. For example, the human endometrium contains CA II only in the capillaries, whereas this high activity isozyme is abundantly expressed in the epithelial cells of the mouse endometrium [ 4 , 24 ]. CA IX is expressed at the basolateral plasma membrane of the human, rat and mouse epithelial cells [ 25 , 26 ]. In a recent extensive study, Ivanov et al [ 27 ] analyzed a number of normal human tissues for the expression of CA IX. Among reproductive organs, they reported positive signal for CA IX mRNA and protein in the efferent ducts, rete testis, and rete ovarii. Human CA XII is expressed in several organs including colon, kidney, and pancreas [ 28 - 30 ]. In the human female reproductive tract, CA XII has been shown both in the glandular and surface epithelium of the endometrium, while it was only occasionally present in the cervix [ 14 ]. Ivanov et al [ 27 ] further confirmed CA XII expression in the glandular epithelium during the proliferative phase. In this report we studied the expression of CA II, IX, XII and XIII in mouse female genital organs including uterus, ovary and placenta. The studies were specially focused on CA IX and XII, which have been designated as tumor-associated isozymes [ 9 , 10 ]. In addition to some normal tissues, both isozymes are overexpressed in several carcinomas such as renal and colorectal cancers [ 9 , 27 , 31 , 32 ]. A previous study has also demonstrated CA IX and XII expression in a number of neoplasias derived from the female reproductive tract [ 27 ]. However, there have been no previous studies on these isozymes in the female murine reproductive organs. The conservation of CA XII expression in both mouse and human endometrium shown in the present paper suggests a role for this isozyme in reproductive physiology. Materials and methods Antibodies In the present study, we used the following antibodies which have been produced and characterized earlier: rabbit anti-mouse CA II [ 4 ], rabbit anti-mouse CA IX [ 26 ], rabbit anti-mouse CA XII [ 33 ], and rabbit anti-mouse CA XIII [ 4 ]. Collection of tissue samples Two adult Balb/c mice were sacrificed by CO 2 asphyxiation followed by decapitation. Uterus, ovary and placenta samples were collected from both animals. The samples were immersion-fixed overnight in Carnoy's fluid (ethanol, chloroform and acetic acid (6:3:1)). Then the specimens were treated with absolute ethanol for 30 min, with 1:1 mixture of ethanol and chloroform for 15 min, and finally with chloroform for 30 min. Paraffin embedding was performed in a vacuum oven for 2 h at +58°C. Paraffin wax was purchased from Fluka Chemie GmbH (Buchs, Schwitzerland). To collect a placenta sample, a mouse was sacrificed at 9 days of pregnancy. The ninth day was chosen since it represents the middle gestational phase. It is also the time when the most critical steps of organogenesis occur in mouse. For western blotting, uterus, kidney and colon were removed and rapidly frozen in liquid nitrogen. The tissue samples for western blot were homogenized with HEPES buffer. Total protein concentration was determined after homogenization using BCA Protein Assay Kit (Pierce, Rockford, IL) according to manufacturer's instructions. The study protocols were approved by the Animal Care Committee of Tampere University. Immunohistochemistry In the mouse tissues, the localization of CA IX and XII was examined by immunoperoxidase method. Antibodies against CA II and XIII were used as positive controls for the immunostaining. All experiments were performed in duplicate and included control staining with non-immune normal rabbit serum (NRS). NRS was obtained from a rabbit that was later immunized against mouse CA XIII. The tissue samples fixed in Carnoy's fluid and embedded in paraffin were cut at 5 μm sections and placed on microscope slides. The peroxidase-anti-peroxidase complex method included the following steps: a) pretreatment of the sections with undiluted cow colostral whey (Biotop, Oulu, Finland) for 40 min and rinsing in phosphate-buffered saline (PBS); b) incubation for 1 h with the primary antiserum (anti-mouse CA II, CA IX, CA XII or CA XIII) or NRS diluted 1:100 in PBS containing 1% bovine serum albumin (BSA) (BSA-PBS solution); c) treatment with undiluted cow colostral whey (40 min); d) incubation for 1 h with swine anti-rabbit IgG (Dakopatts, Copenhagen, Denmark) diluted 1:100 in 1% BSA-PBS; e) incubation for 30 min with peroxidase-anti-peroxidase rabbit conjugate (Dakopatts) diluted 1:500 in PBS; f) incubation for 2 min with 3,3'diaminobenzidine tetrahydrochloride (DAB) solution (6 mg DAB in 10 ml PBS plus 3.3 μl H 2 O 2 ) as chromogen. The sections were washed three times for 10 min in PBS after incubation steps b and d and four times for 5 min after incubation step e. All of the incubations and washings were carried out at room temperature. The sections were finally mounted in Neo-Mount (Merck, Darmstadt, Germany). The stained sections were examined and photographed with a Zeiss Axioskop 40 microscope (Carl Zeiss, Göttingen, Germany). Western blot The samples containing 50 μg of protein from mouse uterus, kidney and colon were analyzed by SDS-PAGE under reducing conditions [ 34 ]. All of the reagents and the protein standard (BenchMark™ Prestained Protein Ladder) for SDS-PAGE were purchased from Invitrogen (Carlsbad, CA) except Laemmli sample buffer that was obtained from Sigma (St. Louis, MO). Electrophoresis (200 V for 40 min) was performed in a Novex Xcell II mini cell electrophoresis unit (Invitrogen) with a 10% Bis-Tris gel (Invitrogen). The separated proteins were transferred electrophoretically from the gel to a polyvinylidene fluoride (PVDF) membrane (Invitrogen) in a Novex Xcell II blot module (Invitrogen). The transfer buffer (NuPAGE Transfer Buffer™) was purchased from Invitrogen. The blot module was filled with the transfer buffer until the gel/membrane assembly was covered. The outer buffer chamber was filled with 650 ml deionized water. The protein transfer was performed using a constant voltage of 36 V for 1 h 20 min. After the transblotting, the sample lines were detected by ECL western blotting detection reagents and analysis system (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions. First, the sample lines were incubated with TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0,3 % Tween 20) containing 10 % cow colostral whey for 25 min and then the first antibodies diluted 1:2000 (anti-CA II, anti-CA IX, anti-CA XII, NRS) or 1:1000 (anti-CA XIII) in TBST buffer for 1 h. The PVDF membranes were washed five times for 5 min with TBST buffer and incubated for 1 h with peroxidase-linked ECL Anti-Rabbit IgG (Amersham Biosciences) diluted 1:25 000 in TBST buffer. After washing four times 5 min in TBST buffer, the polypeptides were visualized by a chemiluminescence substrate (ECL detection reagents 1 + 2, Amersham Biosciences). Kodak™ Biomax™ MS-1 films (Amersham Biosciences) were exposed to the chemiluminescence for 5 min (CA IX and XII) or 1 min (CA II and CA XIII). All the steps were carried out at room temperature. The western blotting experiments were performed in triplicate to confirm the reproducibility of the results. Results Immunohistochemistry All the studied CA isozymes showed positive immunostaining in the epithelial cells of the mouse endometrium (Fig. 1 ). CA II and XII showed a somewhat reciprocal distribution pattern in that CA II was confined to the surface epithelial cells (Fig. 1C ), while CA XII was more intensely stained in the deeper endometrial glands (Fig. 1A ). It is noteworthy, however, that CA XII was clearly expressed also in the surface epithelial cells, but the staining intensity was weaker compared to the glands. As expected, the strongest reaction for CA XII was associated with the basolateral plasma membrane, and unexpectedly, also CA II immunoreaction was most intense at the plasma membrane. CA IX and XIII showed weak reactions in both surface and glandular epithelia (Fig. 1B,1D ). The control immunostaining with NRS was negative (Fig. 4A ). Figure 1 Immunohistochemical staining of CA XII (A), CA IX (B), CA II (C), and CA XIII (D) in the mouse endometrium. All the studied CA isozymes show positive immuostaining, although the staining intensity varies between different isozymes. CA XII shows stronger reaction in the deep endometrial glands compared to the surface epithelium. This pattern is inversed with CA II showing high reaction in the surface epithelium. Insert in panel A shows that the CA XII immunostaining is most abundant in the basolateral plasma membrane of the epithelial cells. Insert in panel C demonstrates that CA II immunoreactivity is also closely associated with the plasma membrane. CA IX and XIII show faint immunoreactions in both the surface and glandular epithelia. Arrows = endometrial glands, arrowheads = surface epithelium. Original magnifications: × 400. Figure 4 Control immunostaining of mouse uterus, ovary and placenta with normal rabbit serum. No immunoreaction is seen. Oringinal magnifications: × 400. In the ovary, immunoreactions for different CA isozymes were negligible (Fig. 2 ). In fact, only CA XII showed occasional positive cells in the corpus luteum (Fig. 2A ). No staining for these isozymes was observed in the developing follicles. No immunoreaction was obtained with NRS (Fig. 4B ) Figure 2 Immunolocalization of CA XII (A,B), CA IX (C,D), CA II (E,F), and CA XIII (G,H) in the mouse corpus luteum (A,C,E,G) and follicle (B,D,F,H). Only faint positive reaction for CA XII can be seen in occasional cells of the corpus luteum that is indicated in the insert of the panel A (arrows). Original magnifications: × 200 (A,C,E,G), × 400 (B,D,F,H). In the 9-days-old mouse placenta, the immunostaining reactions for CA isozymes remained quite weak or absent (Fig. 3 ). CA II was located to the endothelium of the placenta blood vessels and erythrocytes (Fig. 3E ), and it was also present in the amnionic epithelium (Fig. 3F ). The amnionic epithelium showed no or very weak staining for CA XII, whereas the decidual glands were strongly labeled (Fig. 3B ). The control staining again showed no positive signal (Fig. 4C ). Figure 3 Immunohistochemical staining of CA XII (A,B), CA IX (C,D), CA II (E,F), and CA XIII (G,H) in the mouse placenta (A,C,E,G) and amnionic epithelium (B,D,F,H). CA II is located in the endothelium of the blood vessels and erythrocytes (arrows in the panel E). It is also expressed in the amnionic epithelium (arrows in the panel F). Insert of the panel B shows that CA XII is highly expressed in the decidual glands, while the amnionic epithelium is negative. DE = Decidua, P = placenta. Original magnifications: × 400. Western blot Western blotting was performed for the mouse uterine protein to evaluate the specificity of the immunoreactions. Mouse kidney and colon samples were used as positive control tissues, since they are known to express CA II, XII and XIII [ 4 , 33 , 35 ], and the colon contains CA IX [ 36 ]. CA II and XIII were positive in all tissue specimens (Fig. 5 ). Both CA IX and XII showed weak positive reactions in the mouse uterus. The molecular weights for these isozymes were 51 and 46 kDa, respectively. Based on the western blotting the expression of CA XII was weaker in the uterus than in the colon or kidney. On the other hand, CA IX showed the strongest signal in the colon. It is notable that anti-mouse CA XII serum cross-reacted with 30 kDa polypeptide in the western blotting. Previous immunostaining of gastric mucosa with the same anti-CA XII and anti-CA II antibodies has clearly indicated that anti-CA XII serum does not recognize CA II which has a molecular mass of 30 kDa in western blot [ 35 ]. Even though gastric epithelial cells contain high levels of CA II, no immunoreaction was obtained with anti-CA XII serum in those cells. Furthermore, no staining has been obtained by anti-CA XII antibody in the red cells which contain high levels of CA I and II, nor in the brain which expresses high levels of CA II and XIII (data not shown). Figure 5 Western blotting of total homogenate from mouse uterus, kidney and colon for CA II, IX, XII and XIII. Normal non-immune rabbit serum (NRS) was used instead of the first antibodies as a negative control. Both CA IX and XII show weak positive reactions for the uterine proteins (arrowheads). The molecular weights for these isozymes are 51 and 46 kDa, respectively. The signal for CA XII is weaker in the uterus than in the colon or kidney. Note that anti-CA XII serum cross-reacts with a 30-kDa polypeptide. This cross-reaction is evident only in western blotting conditions as pointed out in the Results section. CA IX shows the strongest signal in the colon. CA II and XIII are positive in all tissue specimens. Discussion This study describes the expression of CA II, IX, XII and XIII in mouse female genital organs including uterus, ovary and placenta. CA II showed a very limited distribution pattern in the mouse placenta, being present only in the erythrocytes, endothelium of the blood vessels and amnionic epithelium. In previous studies, CA II has been detected by immunohistochemistry in the human villous syncytiotrophoblasts and in varying amounts in fetal villous endothelium [ 21 , 22 ]. Using a histochemical staining method, Ridderstråle et al [ 18 ] showed in several species that the highest CA activity located in the maternal capillaries, and the membrane-bound CA activity varied among different species. To date, CA IV is the only membrane-bound CA isozyme which has been detected in the mouse placenta [ 23 ]. In our study, CA IX and XII were not found in the mouse placental tissue except that CA XII showed a very weak reaction in the amnionic epithelium. Concluding from the results of the present and previous studies, CA I and II appear to represent the enzyme forms that are most relevant for the placental function [ 22 ], while CA IX and XII may play a role in other reproductive organs such as the male excurrent duct and female uterus [ 14 , 37 ]. It is known that CA activity facilitates transport of CO 2 across biological membranes by converting it to bicarbonate and hydrogen ions. These ions are then translocated across the plasma membrane through specific carrier proteins in a coordinated manner. It is of considerable interest that CA isozymes II and IV have been recently described to form active metabolon systems with ion exchanger proteins such as anion exhanger isoform 1 (AE1) and Na + /H + -exhanger isoform 1 (NHE1) [ 38 - 40 ]. Even though these associations have not yet been described in the placental tissue, it is possible that such metabolons play a role in facilitating placental ion transport processes. Previous studies have shown CA activity in the endometrium of several mammalian species [ 41 , 42 ]. Until now the only established isozymes in the human endometrium are CA XII [ 14 ] and CA XIII [ 4 ]. Interestingly, the high activity isoenzyme, CA II, is not expressed in the human endometrial epithelium [ 4 ]. In the present study, all the examined CA isozymes – including CA II – showed positive immunostaining in the epithelial cells of the mouse endometrium. To our knowledge, there are only a few examples of clear species-specific difference in CA expression. These include e.g. CA XII expression in the kidney (human principal cells versus mouse intercalated cells) [ 33 , 43 ] and CA XIII in the human and mouse testis [ 4 ]. What would be the physiological consequence of such variation between different species? Of course, there are marked differences in human and rodent reproductive physiology. Mouse is characterized by tremendous reproductive potential. Females generally have 5–10 litters per year if conditions are suitable. Gestation period is 19–21 days. Litters consist of 3–12 (generally 5 or 6) offspring, and the mice reach sexual maturity at 5–7 weeks. Even though our observations do not provide any clues whether CA expression could contribute to some of the described characteristics, these differences may have fundamental physiological effects that should be addressed in future investigations. In the present study, CA XII and II showed more intense staining in the surface endometrial epithelia than CA IX and XIII. CA XII was more intensely stained in the deeper endometrial glands, while CA II was confined to the surface epithelial cells. Interestingly, CA II showed positive immunoreaction not only in the cytoplasmic compartment but also at the plasma membrane of the cells that is quite surprising for a cytosolic enzyme. The same phenomenon is detectable in some other tissues including the human gallbladder [ 25 ] and gut [ 44 ]. The cell membrane reactivity may reflect a possible physical association between CA II and ion transport proteins, which has been demonstrated in cell cultures [ 38 - 40 ]. When CA XII was first discovered in the normal human endometrium, it was suggested to play a role in reproductive functions [ 14 ]. In the endometrium, pH and ion balance has to be tightly regulated to ensure normal fertilization. For example, the bicarbonate concentration has been implicated in the regulation of sperm motility, capacitation, and acrosome reaction [ 15 , 17 , 45 ]. One major regulatory pathway includes a bicarbonate-sensitive adenylate cyclase that is present in the plasma membrane of the sperm cell [ 46 ]. In the female genital tract, the endometrial and oviductal epithelium may produce an alkaline – bicarbonate rich – environment for maintaining the sperm motility. This suggestion is in agreement with the observations by Guerin et al. [ 47 ], that the sperm motility is improved by co-culture of human spermatozoa with either endometrial or oviductal epithelial cells. In the future studies, it will be important to investigate whether the hormonal status regulates the expression of different CA isozymes – particularly CA XII – in the endometrium. Another interesting line of investigations would be to analyze the fertilization capacity of CA XII knockout mice as soon as they become available. One could hypothesize that endometrial CA isozymes are important factors, contributing to the appropriate bicarbonate concentration and pH balance in the cervical and endometrial mucus needed for normal fertilization process. Based on our recent studies, CA IX-deficient mice showed no apparent phenotypic changes linked to fertility [ 26 ]. Even more interesting from this point of view is that CA XII may be an important isozyme present in the endometrium, and therefore, CA XII knockout mice will be very attractive targets for reproductive physiological studies. Conclusions The present paper demonstrates for the first time the expression of transmembrane carbonic anhydrase isozymes IX and XII in the female murine reproductive tract. The data indicates that the endometrial epithelium is a prominent site for CA XII expression. The conservation of CA XII expression in the endometrium of different species (mouse and human) suggests a role for this isozyme in reproductive physiology. Authors' contributions All authors participated in the design of the study. PH, JL and SP collected the tissue samples. PH, JL, ET, PK and SP drafted the manuscript. PH, JL and SP performed the western blotting. SPas, JP, AW and WSS provided the antibodies. PH, JL and SP participated in the immunohistochemical staining. All authors read and approved the final manuscript.

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544859

The involvement of survival signaling pathways in rubella-virus induced apoptosis

Rubella virus (RV) causes severe congenital defects when acquired during the first trimester of pregnancy. RV cytopathic effect has been shown to be due to caspase-dependent apoptosis in a number of susceptible cell lines, and it has been suggested that this apoptotic induction could be a causal factor in the development of such defects. Often the outcome of apoptotic stimuli is dependent on apoptotic, proliferative and survival signaling mechanisms in the cell. Therefore we investigated the role of phosphoinositide 3-kinase (PI3K)-Akt survival signaling and Ras-Raf-MEK-ERK proliferative signaling during RV-induced apoptosis in RK13 cells. Increasing levels of phosphorylated ERK, Akt and GSK3β were detected from 24–96 hours post-infection, concomitant with RV-induced apoptotic signals. Inhibition of PI3K-Akt signaling reduced cell viability, and increased the speed and magnitude of RV-induced apoptosis, suggesting that this pathway contributes to cell survival during RV infection. In contrast, inhibition of the Ras-Raf-MEK-ERK pathway impaired RV replication and growth and reduced RV-induced apoptosis, suggesting that the normal cellular growth is required for efficient virus production.

Introduction Rubella virus (RV) is the sole member of the Rubivirus genus of the Togaviridae . It has a positive-sense single stranded RNA genome that is 9762 nucleotides (nt) in length and contains two non-overlapping open-reading frames (ORFs). The 5' proximal ORF encodes the p200 polyprotein precursor for the nonstructural proteins (NSPs) p150 and p90 [ 1 , 2 ]. The 3' proximal ORF encodes the structural proteins: capsid (C), and glycoproteins E1 and E2 [ 3 , 4 ]. RV infection usually causes mild disease with few complications. However, infection during the first trimester of pregnancy results in fetal infection, and in more than 75% of cases this leads to the development of congenital abnormalities. These abnormalities include sensorineural deafness, mental retardation, and congenital heart defects, and are collectively termed congenital rubella syndrome (CRS) [ 5 ]. The cellular mechanisms activated by RV, which lead to the disruption of organogenesis, are not fully understood. However, in permissive cell cultures, the cytopathic effect (CPE) of RV has been shown to be due to caspase-dependent apoptosis [ 6 - 12 ]. Apoptosis is a key component of developmental processes in mammals, which functions to delete vestigial structures, control cell number and remodel tissues and organs [ 13 ]. Thus, it has been proposed that RV-induced apoptosis may cause irreparable damage to fetal tissues, resulting in the abnormalities observed in CRS [ 12 ]. However, the outcome of RV infection is likely to depend on multiple signaling events that control the balance between cell death and cell survival. Eukaryotic cells contain a large number of mitogen activated protein kinase (MAPK) signaling cascades that are activated in response to growth factors, cytokines and stress stimuli such as viral infection and UV irradiation. In common with apoptotic proteins, MAPKs are highly conserved and ubiquitously expressed [ 14 , 15 ]. These cascades integrate external stimuli and transmit signals to the nucleus resulting in the activation of transcription factors, which regulate expression of genes required for proliferation, differentiation, survival and apoptosis. Two well-studied mitogenic pathways are the phosphoinositide 3-kinase (PI3K)-Akt pathway and the Ras-Raf-MEK-ERK pathway, which are central to cell survival and proliferative signals respectively. PI3Ks phosphorylate plasma membrane inositol lipids at the 3' position of the inositol ring. These 3'phosphoinsoitides recruit proteins such as Akt and phosphoinositide dependent kinases 1 and 2 (PDK1/2) to the plasma membrane via their pleckstrin homology (PH) domains [ 16 , 17 ]. At the plasma membrane PDK1/2 activate Akt through phosphorylation at Ser 473 and Thr 308 . Activated Akt promotes cell survival by phosphorylating and inhibiting a number of pro-apoptotic proteins including BAD, caspase-9, GSK-3β and Forkhead transcription factors [ 18 , 19 ]. The Ras-Raf-MEK-ERK is a classical MAPK pathway where growth factor-receptor interactions trigger intracellular activation of the small G-protein Ras. Ras recruits and directly activates the MAPK kinase kinase (MAPKK) Raf, which phosphorylates and activates the MAPK kinase (MAPKK) MEK1/2, which in turn activate the MAPK ERK1/2. Activated ERK1/2 translocates to the nucleus where it can activate a number of transcription factors including c- myc , c- jun , and Elk-1 , which regulate cell cycle progression responses [ 20 ]. Activation of PI3K-Akt and Ras-Raf-MEK-ERK signaling cascades during virus infection is thought to play an important role not only in cellular growth and survival, but also in virus replication and growth during both acute and chronic virus infections [ 21 - 25 ]. This study was carried out to examine the role of PI3K-Akt and Ras-Raf-MEK-ERK signaling during RV infection in RK13 cells. The PI3K inhibitor LY294002 and the MEK inhibitor U0126 were used to investigate PI3K-Akt and Ras-Raf-MEK-ERK signaling respectively during RV replication, growth and induction of apoptosis. Apoptosis was measured in RV-infected cells by caspase activity and cell viability assays, DNA fragmentation analysis, and trypan blue exclusion staining. Involvement of PI3K-Akt and Raf-Raf-MEK-ERK signaling in RV-induced apoptosis was also examined by expression of constitutively active Akt and MEK in RV-infected cells. Results Phosphorylation of Akt, ERK1/2 and their downstream targets during RV infection The effect of RV infection on PI3K-Akt and Ras-Raf-MEK-ERK pathways was investigated by examining the expression and phosphorylation profiles of Akt, ERK1/2 and their downstream targets. Cell lysates from RV and mock infected RK13 cells were collected 12–96 hours post-infection (p.i.), separated by SDS-PAGE, and analyzed for total and phosphorylated Akt and ERK1/2 by Western blotting. Phosphorylated Akt and ERK1/2 could be detected in RV-infected cells from 48 hours p.i., and band intensity increased from 48–96 hours p.i. compared to total levels (Fig. 1A ). Phosphorylated Akt and ERK2 (but not ERK1) were detected in the mock-infected cells at 96 hours p.i. but not before, whereas total levels of Akt and ERK 1/2 were detectable at all time points (Fig. 1A ). Treatment of RV-infected cells with PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126 completely inhibited activation of Akt and ERK1/2 respectively (data not shown). Figure 1 Kinase phosphorylation during RV infection. Serum-starved RK13 cells were mock infected or infected with RV at an m.o.i. of 4 PFU/cell. At indicated time points cell lysates were collected and proteins (30 μg/lane) were separated by SDS-PAGE, and analysed by Western blotting using phospho-specific antibodies. Blots were also probed with anti-tubulin antibody to demonstrate equal loading. A – Total and phosphorylated Akt and ERK (24–96 hours p.i.). B – Total and phosphorylated Akt, ERK, and p70S6K, and phosphorylated GSK-3β and c-myc. The data were consistently repeated in two independent experiments. The phosphorylation of Akt and ERK and their downstream targets p70S6K, GSK-3β, c- myc and BAD were also examined by Western blotting between 12–96 hours p.i. (Fig. 1B ). Phosphorylated Akt and ERK1/2 were detectable in RV-infected cells at 48 and 36 hours p.i. respectively. p70S6K is phosphorylated by FRAP/mTOR downstream of Akt at Thr 389 and at Thr 421 /Ser 42 , downstream of the Ras-Raf-MEK-ERK pathway. Phosphorylation at Thr 389 was observed at 12, 24, 60, 84 and 96 hours p.i. (Fig. 1B ). Phosphorylation of the Thr 421 /Ser 42 site was observed at all time points, although increases in band intensity could be seen at 12, 24, 60, 84 and 96 hours p.i., mirroring the phosphorylation at Thr 389 . Phosphorylation of Thr 421 /Ser 424 but not Thr 389 was observed in the mock-infected cells, albeit at a lower level than in RV-infected cells. The phosphorylation of GSK-3β, downstream of Akt, increased from 12 and 96 hours p.i. and was similar to that of Akt. Phosphorylation of BAD, another substrate for Akt, however, could not be detected in RV-infected or mock-infected cells. The phosphorylation of c- myc , a transcription factor activated by ERK1/2 phosphorylation, decreased between 12 and 96 hours p.i., in contrast to the phosphorylation profile of ERK1/2. GSK-3β and c- myc were also detectable in the mock-infected cells at 96 hours p.i. The effects of LY294002 and U0126 on cell viability in RV-infected cells RV induces apoptosis in RK13 cells with characteristic morphological and biochemical features [ 6 , 8 , 9 ]. The XTT assay was used to examine the effect of RV infection and LY29002 and U0126 treatment on cellular metabolism over time. XTT is a tetrazolium salt, which is cleaved by the succinate dehydrogenase system of mitochondria in metabolically active cells, to yield a soluble orange formazan product. A decrease in the intensity of formazan was used to monitor changes in cellular metabolism and cell viability in RV-infected cells by spectroscopy. Cellular viability during RV infection did not appear to be disrupted, supporting previous observations which reported that a large number of monolayer cells remain in tact and do not rapidly undergo apoptosis in RV infected cells [ 9 , 12 ] (Fig. 2 ). LY294002 treatment of RK13 cells reduced cell viability by 20%, which remained constant throughout the 12–96 hour period. Cell viability was reduced to 60% in the presence of both RV and LY294002. Thus the combined effect of PI3K inhibition and RV-infection caused a significant reduction in cell viability. Figure 2 The effect of PI3K and MEK1/2 inhibition on cell viability during RV infection. Serum-starved RK13 cells were mock infected or infected with RV at an m.o.i of 4 PFU/cell with or without LY294002 (30 μM) or U0126 (15 μM). At indicated time points cell viability was determined by XTT assay. Tetrazolium salt (XTT) and electron coupling reagent were added directly to cells, and after 24 hours the absorbance at 405–690 nm was determined. Data represent mean ± S.E. from three independent experiments. As Ras-Raf-MEK-ERK signaling is crucial to the regulation of cell growth in many cell lines, inhibition of this pathway often has detrimental effects. A typical dose-response curve can be seen with MEK inhibitor U0126 in RK13 cells, with cell viability completely abolished by 60–72 hours p.i. (Fig. 2 ). With the addition of RV, the U0126 curve moved to the right, the effect of the drug was delayed by approximately 12 hours. Inhibition of PI3K results in an increase in the speed and magnitude of RV-induced apoptosis To evaluate the role of PI3K-dependent signaling during RV infection, the effects of PI3K inhibitor LY294002 on the development of RV-induced apoptosis were examined, 12–96 hours p.i., by caspase activity assay, trypan blue exclusion staining, DNA fragmentation and light microscopy. (Fig. 3A–D ). RV-induced apoptotic signaling has been reported to occur between 12–24 hours p.i., with peak caspase activity occurring around 72 hours p.i. at a multiplicity of infection (MOI) of 3 PFU/cell [ 6 ]. Fig. 3A shows that with a MOI of 4 PFU/cell the peak of RV-induced caspase activity occurs earlier at 60 hours p.i. When RV infection was carried out in the presence of LY294002, the maximum caspase activity increased by 53.9 % (P < 0.05) and occurred 12 hours earlier than with RV alone (Fig. 3A ). Figure 3 The effect of PI3K and MEK1/2 inhibition on RV-induced apoptosis. Serum-starved RK13 cells were mock infected or infected with RV at an m.o.i of 4 PFU/cell with or without LY294002 (30 μM) or U0126 (15 μM). Cells were harvested and analyzed for markers of apoptosis. A – At indicated time points, cell lysates were collected and incubated with artificial caspase substrate Ac-DEVD-pNA. Free pNA due to caspase cleavage was measured at an absorbance of 405 nm. Data represent mean ± S.E. from three experiments, *P < 0.05. B – The number of measurable dead floating cells in the cell culture supernatant was determined by trypan blue exclusion staining at indicated time points. Data represent mean ± S.E. from three experiments, *P < 0.05. C – Total DNA was extracted from detached and monolayer cells at 72 hours p.i. and apoptotic DNA fragments were resolved on a 1.5% agarose gel, stained with ethidium bromide, and visualized using UV transillumination. Molecular size markers were run in the left hand lane . D – Light microscopy photographs of cell monolayers at 72 hours p.i., at a magnification of 20X. This increase in speed and magnitude of RV-induced apoptosis is more strikingly observed in Fig. 3B , which shows the number of dead floating cells by trypan exclusion staining in the culture supernatant fluid of RV infected and LY294002 treated cells. LY294002 treatment doubles (and at 84 hours p.i. triples) the number of floating cells produced in RV-infected cells. Increases in the number of apoptotic floating cells are statistically significant at 84 and 96 hours p.i. (P < 0.05). Fragmented DNA patterns can be seen at 72 hours p.i. with both RV and RV in the presence of LY294002 (Fig. 3C ). However, the interesting feature of these apoptotic ladders is that in RV-infected cells, a significant proportion of genomic DNA is still intact, whereas when RV-infected cells are also exposed to LY294002, the majority of the genomic DNA is fragmented. The morphological changes caused by RV-infection and LY294002 were examined by light microscopy (Fig. 3D ). At 72 hours p.i. CPE and induction of apoptosis by RV can be clearly seen. RV-induced CPE is characterized in the earlier stages by clumps of apoptotic cells, surrounded by healthy cells. In the later stages the cell sheet is completely destroyed and the majority of cells have become apoptotic floaters [ 6 ]. In the presence of LY294002, RV-infected cells are almost all dead by 72 hours p.i., resembling the later stages of RV-induced CPE. LY294002-only treatment of RK13 cells did not induce apoptosis as evidenced by the lack of caspase activity (Fig. 3A ), DNA fragmentation (Fig. 3C ), and measurable floating cells (data not shown). Morphological examination of LY294002 treated RK13 cells show the cell monolayers were in tact with no visible cytotoxicity (Fig. 3D ). Inhibition of MEK1/2 reduces RV-induced apoptosis The role of Ras-Raf-MEK-ERK signaling in RV-induced apoptosis was investigated using MEK inhibitor U0126 as described above for LY294002 (Fig. 3A–D ). U0126 treatment reduced caspase activity in RV-infected cells by 51.9% (P < 0.05), with a low peak occurring at 48 hours p.i. (Fig. 3A ). The number of dead floating cells in RV and U0126-treated cells was slightly lower than in RV-infected cells at all time points (Fig. 3B ). DNA fragmentation was observed in both RV-infected cells and RV in the presence of U0126 (Fig. 3C ), although the presence of the drug also appeared to result in smearing of high molecular weight DNA, characteristic of necrosis [ 26 , 27 ]. The detrimental effect of U0126 on RK13 cell morphology is shown in Fig. 3D ; this correlates with the rapid decline in cell viability. Inhibition of MEK1/2 inhibits RV replication and growth To examine the effect of LY294002 and U0126 on RV replication and growth, RV-infected and drug-treated cell culture supernatants were tested for RV capsid gene expression by RT-PCR, and virus growth by TCID 50 assay 24–96 hours p.i.. The capsid gene is the first gene to be transcribed from the second ORF encoding the structural proteins. Therefore detection of capsid RNA by RT-PCR is a good measure of RV replication [ 1 , 28 ]. In RV-infected cells simultaneously treated with LY294002, levels of RV capsid RNA increased over time, as in RV-infected cells (Fig. 4A ). In the presence of U0126, however, levels of capsid RNA were reduced, and remained lower than that seen at 24 hours p.i. in RV-infected cells. Figure 4 The effect of PI3K and MEK1/2 inhibition on RV growth and replication. Serum-starved RK13 cells were infected with RV at an m.o.i of 4 PFU/cell with or without LY294002 (30 μM) or U0126 (15 μM). Cell culture supernatants were extracted from cells at indicated time points. A – RV RNA was extracted from virus-infected cell culture supernatants and the capsid gene was amplified by RT-PCR as described under 'Experimental Procedures'. B – Monolayers of RK13 cells in 96-well plates were infected with RV-infected cell culture supernatants, and virus titers were determined using the TCID 50 assay. Results are representative of a least two independent experiments. Both LY294002 and U0126 affected virus growth (Fig. 4B ). During RV-infection of RK13 cells with 4 PFU/cell of virus, virus titers reached 10 8 TCID 50 /ml by 96 hours p.i. However, in the presence of U0126 the titer was approximately 10 2 lower at 24 hours p.i., 10 3 lower at 48 hours p.i., and 10 4 lower at 72–96 hours p.i. LY294002 reduced virus growth to a similar extent, but unlike with U0126, by 96 hours p.i. the virus titer recovered slightly. Constitutively active Akt and MEK1/2 enhance RV-induced apoptosis To determine the importance of PI3K-Akt and Ras-Raf-MEK-ERK in the transduction of cell survival and proliferative mechanisms during RV-infection, RK13 cells were transiently transfected with constitutively active forms Akt and MEK. Significant expression of both proteins was seen after 24 hours (Fig. 5A ). Over-expression of both activated Akt and MEK enhanced RV-induced caspase activity (Fig. 5B ). RV infection in the presence of the empty pUSEamp(+) control vector slightly decreased caspase activity. Caspase activity following Lipofectamine treatment alone or pUSEamp(+) transfection was below that of the mock-infected cells (data not shown). Figure 5 Over-expression of Akt and MEK enhances RV-induced apoptosis. RK13 cells were transfected with eukaryotic expression vector pUSEamp(+) containing constitutively active HA-tagged MEK1 or myristoylated myc-tagged Akt1 under the control of a CMV promoter, or with an empty pUSEamp(+) control. A – Expression of MEK1 and Akt1 was determined by Western blotting. Cell lysates were collected 24 hours post-transfection and 30 μg protein separated by SDS-PAGE and transferred to nitrocellulose membranes. MEK1 and Akt1 were detected by anti-HA and anti-myc antibodies respectively. B – RK13 cells in 6-well plates were transfected with Akt, MEK or pUSEamp(+) control constructs for 24 hours and subsequently infected with RV or mock-infected. 24 hours later cell lysates were collected and tested for caspase activity using artificial caspase substrate Ac-DEVD-pNA. Discussion We have previously shown that RV induces caspase activation during the early stages of infection in vitro , prior to the appearance of morphological apoptotic changes [ 6 ]. In this study we demonstrated that, in common with other viruses such as Coxsackievirus B3 virus, human cytomegalovirus, influenza virus A, and respiratory syncitial virus (RSV) (Cooray, 2004; Johnson et al., 2001; Opavsky et al., 2001; Pleschka et al., 2001), signaling downstream of PI3K stimulates a survival response in the cell following RV infection and that signaling downstream of MEK1/2 is required for RV replication, growth and induction of apoptosis. Analysis of phosphorylation profiles during RV infection demonstrated that the presence of the virus stimulated an increase in the phosphorylation of ERK1/2, Akt, and Akt target GSK-3β over time. The presence of phosphorylated Akt (and occasionally ERK2) at 96 hours p.i. in the mock-infected cells, suggests that cell survival mechanisms may be activated in older uninfected cell cultures. The phosphorylation pattern of downstream target p70S6K did not follow that of Akt and ERK1/2. Apart from being phosphorylated by ERK1/2 and mTOR/FRAP downstream of Akt, p70S6K can be phosphorylated by an array of different proline-directed kinases, including PDK1, PKCζ, JNK and cdc2 which may explain this difference [ 29 - 33 ]. The phosphorylation of c- myc , a downstream target of ERK1/2, did not follow the same pattern. Levels of phosphorylated c- myc decreased as infection progressed, which was probably due to its targeted degradation or the action of cellular phosphatases. RV infection has been observed to slow cell cycle progression both in vivo and in vitro [ 12 , 34 ]. As c- myc is a transcription factor that stimulates cell cycle progression, its de-phosphorylation or degradation as RV infection progresses supports these observations. The expression and activity of c- myc and other downstream transcription factors in relation to the cell cycle during RV-infection requires further investigation. Phosphorylation of BAD, downstream of Akt, could not be detected in RV-infected cells (data not shown). However, BAD is not ubiquitously expressed and therefore may not be produced in the rabbit kidney epithelial cells (RK13) used [ 16 ]. Inhibition of PI3K signaling with LY294006 significantly increased the speed and magnitude of RV-induced apoptosis as shown by increased caspase activity, dead floating cells, apoptotic laddering of genomic DNA and decreased cell viability. Thus, RV-induced apoptotic signaling appears to be held in check by host cell survival signals downstream of PI3K. Although inhibition of PI3K did not affect RV replication, virus growth was affected. The speed of apoptotic monolayer death may have prevented production of optimal virus titers. The importance of PI3K survival signaling has been observed with other viruses. Recently phosphorylation of Akt, GKS3β and PKCζ (another downstream target of PI3K signaling), has been demonstrated in Vero E6 cells early during infection with severe acute respiratory syndrome (SARS)-associated corona virus (CoV) [ 35 ]. However, unlike in this study the survival response due to PI3K-Akt signaling was deemed to be weak, as LY294002 treatment did not result in an increase in apoptotic DNA laddering. PI3K, Akt and NFκB have also been shown to be activated prior to epithelial cell apoptosis in RSV-infected cells [ 36 ]. As with RV, inhibition of PI3K increased the speed and magnitude of RSV-induced apoptosis, although host-cell survival was suggested to occur prior to apoptotic signaling, as opposed to RV where caspase activation and Akt phosphorylation occur concomitantly [ 6 ]. PI3K-Akt signaling has also been shown to reduce coxsackievirus B3 (CVB3)-induced apoptosis. However, in contrast to RSV, the replication of CVB3 was also reduced, suggesting that PI3K-Akt survival signals may also serve as a defense mechanism against virus infection [ 37 ]. Inhibition of the MEK1/2 in RK13 cells by U0126 resulted in necrotic monolayer destruction and a significant decrease in cell viability. XTT assay and light microscopy demonstrated that RV infection appeared to delay the effect of U0126. As discussed above, RV infection stimulates ERK activity, downstream of MEK, and may therefore counteract the effect of the inhibitor. Despite this, U0126 impaired RV replication, growth, and induction of apoptosis. Therefore it appears that although RV infection slows the cell cycle progression, cells must be cycling and metabolizing normally for RV replication to occur. ERK1/2 phosphorylation has also been observed during infection with a number of other viruses, and inhibition of ERK1/2 signaling by U0126 has consistently been shown to be detrimental to virus growth. Infection of Jurkat cells with CVB3, for example, leads to up-regulation of ERK1/2 phosphorylation, and elevated levels of phosphorylated ERK1/2 have been observed in the myocardium of mice susceptible to CVB3-induced myocarditis [ 38 ]. Treatment of cultured cells with U0126 reduced CVB3 titers and inhibited the release of virus progeny [ 38 , 39 ]. Similarly, HCMV infection in human embryonic lung fibroblasts (HELs) has been shown to stimulate biphasic activation of MEK1/2 and ERK1/2, and treatment of infected cells with U0126 reduced viral DNA replication, protein production and virus titer [ 40 ]. Influenza A virus infection in vitro has also been shown to stimulate biphasic activation of MEK1/2 and ERK1/2, and U0126 treatment prevented export of ribonucleoprotein complexes from the nucleus and inhibited virus production [ 24 ]. Inhibition of MEK1/2 during HIV infection has been demonstrated to reduce infectivity, but unlike the other viruses mentioned herein, did not affect protein levels or virus production [ 25 ]. These findings, along with the results of this study, suggest that signaling downstream of MEK1/2 and ERK1/2 is important for viral infectivity and efficient virus replication and growth in vitro . Over-expression of Akt and MEK1/2 increased RV-induced caspase activity in RK13 cells. This response was not due to the transfection procedure, as the increase in caspase activity was not observed in the pUSEamp(+) or lipofectamine controls. Such a response is also seen in malignant cells, which are more readily killed by apoptotic stimuli. Thus, the over-expression of these mitogenic pathways may have resulted in a cell survival response whereby a negative feedback loop occurred that sensitized cells to RV-induced apoptosis. In order to study this further, it would be necessary to construct stable cell lines over-expressing active Akt and ERK1/2 as well as their dominant negative mutants and other signaling proteins. It is clear from the results of this and previous studies that the outcome of RV infection in vitro depends on numerous signaling events. It has been suggested that RV capsid protein, when anchored to the ER can independently induce apoptosis in culture (Duncan et. al, 2000). However this has not been confirmed by other groups and there is conflicting evidence that virus replication and the presence of the RV NSPs (which are necessary for replication) is required [ 10 , 12 , 41 ]. Interestingly the NSP p90 has been shown to interact with the retinoblastoma (pRB) cell cycle-regulatory protein and the cytokinesis regulatory protein citron-K kinase (CK), and it has been suggested that this may perturb the cell cycle [ 42 , 43 ]. How these interactions interfere with signaling pathways and modulate cellular responses, however, remains to be determined. In relation to CRS, study of the expression and localization of apoptotic and mitogen activated signaling proteins in RV-infected fetal tissues would be necessary to confirm the theory that the pathogenesis of the disease is related to perturbation of the cell cycle. However as CRS is now rare in the UK and work with fetal tissues is tightly regulated, such a study would be hard to carry out. In vivo studies are difficult, as a reliable animal model does not exist for CRS. However, it may be possible to extrapolate findings from cell culture systems. We used RK13 cells because they are the best cells in which to detect rubella-induced apoptosis; further studies are required to confirm our findings in primary human embryonic cells. Materials and methods Chemical Compounds Stock concentrations of PI3K inhibitor LY294002 [2-(4-Morpholinyl)-8-phenyl-1-4H-1-benzopyran-4-one] and MAPK/MEK inhibitor U0126 [1, 4-Diamino-2, 3-dicyano-1, 4-bis (2-aminophenylthio) butadiene] (Calbiochem, UK) were made up in dimethyl sulfoxide (DMSO). In all experiments LY294002 and U0126 were used at concentrations of 30 μM and 15 μM respectively. Cell Culture & Viral Infection Mycoplasma-free rabbit kidney epithelial (RK13) cells were obtained from the European Collection of Cell Cultures and cultured as previously described (3). RV (wild type strain RN) was propagated as previously described (3). For infection, cells were grown to confluence in minimal essential medium (MEM) supplemented with 15 mM L-glutamine and 5% FCS (v/v) (Invitrogen, UK) at 37°C in 5% CO 2 in air, and serum starved overnight. Cells were infected with RV at a MOI of 4 plaque forming units (PFU) per cell and maintained in MEM with 1% FCS until harvested at indicated time points. Where appropriate kinase inhibitors (LY294002 and U0126) were added to the media at the same time as the virus, and were present during subsequent incubation periods. Mock-infected cells were treated and harvested in the same manner as those infected, except that MEM without virus was used during the infection. RV titers, in the presence of inhibitors, were determined by TCID 50 assay in RK13 cells as the sample number was too large to perform plaque assays. Inhibitor, virus and serum concentrations were optimized to ensure that the effect of both the virus and the inhibitors could be monitored. Transfection Control and expression plasmids [pUSEamp(+), and constitutively active HA-Akt1 and Myc-MEK1 in pUSEamp(+)] were purchased from Upstate Biotechnology Inc. (UK). RK13 cells were grown to confluence in 25 cm 2 tissue culture flasks and transiently transfected with 0.25 μg of control or expression plasmids. Tranfections were carried out in serum-free MEM using Lipofectamine Plus (Invitrogen, UK), according to the manufacturer's instructions. For optimal transfection, cell monolayers were incubated with the DNA-liposome mixture for 5 hours at 37°C. Following transfection, the DNA liposome complexes were removed and replaced with fresh medium. After 24 hours, RV was added to cells, which were maintained on MEM with 1% serum (as above). After an additional 24 hours, cells were analyzed for protein expression by Western blot analysis, and for apoptosis by caspase activity assay. Western Blot Analysis Polyclonal anti-PI3K p85, anti-HA Tag, anti-myc Tag, and monoclonal anti-β-tubulin antibodies were from Upstate Biotechnology inc. (UK). Polyclonal anti-caspase-3 antibody was from Sigma (UK). All other primary antibodies were purchased from Cell Signaling Technology (UK). Cells were treated as described above and at indicated times post-infection (p.i.), washed in PBS and harvested in cell lysis buffer [50 mM Tris, 150 mM NaCl, 1% Triton-X-100, 2 mM EDTA, 2 mM EGTA, 100 μM protease inhibitor cocktail, and 100 μM each of phosphatase inhibitor cocktails 1 and 2 (Sigma, UK)]. Protein concentrations were determined using the BioRad assay (BioRad, Hemel Hemstead, UK), and equal protein loading was determined by Coomassie staining (Invitrogen, Paisley, Scotland). Lysates were electrophoresed on 12% Bis-Tris polyacrylamide gels (Invitrogen, UK) and transferred onto Hybond™ ECL nitrocellulose or PVDF membranes (Amersham Biosciences, UK). Membranes were blocked with 5% non-fat dried milk in PBS containing 0.1% Tween-20, and subsequently incubated with primary antibody (1:1000) overnight at 4°C. Specific antibody binding was detected using horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG (1:2000) (Dako, UK), and immunoreactive bands were visualized using the ECL detection system according the manufacturer's instructions (Amersham Biosciences, UK). XTT Assay RK13 cells were grown to confluence in 96-well tissue culture plates at 37°C in 5% CO 2 in air. Cells were treated, in a final volume of 100 μl, with RV and kinase inhibitors as described above. At indicated times p.i., 50 μl of labeling mixture containing XTT (sodium 3'- [1-(phenylaminocarbonyl)-3, 4-tetrazolium]-bis (4-methoxy-6-nitro) and coupling reagent PMS (N-methyl dibenzopyrazine methyl sulphate) (Roche Applied Science, Mannheim, Germany) was added directly to the wells to give final concentrations of 0.3 mg/ml and 2.5 μg/ml respectively. Plates were incubated in a humidified atmosphere (37°C, 5% CO 2 ) for 24 hours. The absorbance of the formazan product was measured at a test wavelength of 450 nm and a reference wavelength of 690 nm. Caspase Activity Assay DEVD specific caspase activity assay (Promega, UK) was carried out as previously described (3). Briefly, RK13 cells were grown to confluence, and treated with RV, LY294002, and U0126 (as above). Cell lysates were collected at indicated times p.i. and stored at -70°C until required. For the assay, lysates were incubated with colorimetric substrate DEVD-p-NA for 4 hours at 37°C, and absorbance of free pNA cleaved by endogenous caspases-3 and -7 was measured at 405 nm. DNA Fragmentation Analysis Analysis of apoptotic DNA fragmentation was carried out as previously described (3). Briefly, RK13 cells in 6-well plates were treated with RV, LY294002 and U0126 as above, and harvested 72 hours p.i. Total cellular DNA was extracted from 2 × 10 6 cells according to the manufacturer's instructions (Calbiochem, Nottingham, UK). Nucleic acids were precipitated using 3 M sodium acetate, 2-propanol, and ethanol. DNA pellets were dried and re-suspended in 10 mM Tris pH 7.5, 1 mM EDTA. Ladder fragments were electrophoretically separated on 1.5% Tris-Acetate EDTA (TAE) agarose gels. Gels were stained in ethidium bromide solution (5 mg/ml) and fragmented DNA was visualized under UV light. Examination of floating cells Floating dead cells in the supernatant following infection with RV or drug treatment (as described above) were quantified by trypan blue exclusion staining. The morphological changes to the cells were examined by light microscopy using a Nikon Eclipse TS100 light microscope. Pictures of cells were taken at a magnification of 20X using a Nikon COOLPIX 4500 digital camera and processed with Adobe Photoshop 7.0 software. RV Capsid RT-PCR Total RNA was extracted from 100 μl tissue culture supernatants, collected at indicated times p.i., using a silica-guanidinium isothiocyanate method [44]. Prior to reverse transcription, RV RNA was heated to 95°C for 1 minute and kept on ice. RNA was transcribed to cDNA using Superscript III RNase H - reverse transcriptase (Invitrogen, UK). Reverse transcription was performed in 20 μl reaction volumes containing 200 U enzyme, 10 μl sample RNA, 0.5 mM of each dNTP, and 5 pmoles external reverse primer (5'-CCTGTACGTGGGGCCTTTAA-3'). RNA bound to cDNA in RNA-DNA hybrids was removed by incubation of the cDNA with RNase H (Roche Diagnostics, UK) for 20 minutes at 37°C. PCR amplification was carried out using a GC-Rich PCR System (Roche Diagnostics, UK). In the PCR reaction 10 μl cDNA was added to 40 μl of PCR reaction mix to give final concentrations of 1X GC-Rich PCR buffer, 1.5 mM MgCl 2 , 0.2 mM each dNTP, 0.5 M GC-rich resolution solution™, 0.5 pmole of forward and reverse primers (5'-TAGGAGGTGCCGCCATATCA-3' and 5'-CCTGTACGTGGGGCCTTTAA-3' respectively), and 2U Taq polymerase and a mixture of proof-reading polymerases. The cycling conditions, as recommended by the manufacturer were: 95°C for 3 minutes followed by 10 cycles of 95°C for 30s, 57°C for 30s, 72°C for 1 minute; and 25 cycles of 95°C for 30s, 57°C for 30s, 72°C for 1 minute (plus an additional 5 seconds per cycle), and a final extension of 72°C for 7 minutes. Amplified capsid product (1053 b.p.) was electrophoretically separated on 1% Tris-Borate (TBE) agarose gels, stained with ethidium bromide solution (5 mg/ml) and visualized under UV light. Authors' Contributions SC conceived of the study, carried out the virological and biochemical assays and drafted the manuscript. JL participated in the design of the study. JMB participated in design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.

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423140

A Gene Responsible for Hybrid Incompatibility in Drosophila

Nearly 150 years after Darwin published On the Origin of Species , biologists are still debating how new species emerge from old—and even the definition of species itself. Darwin demurred from offering a hard and fast definition, suggesting that such a thing was “undiscoverable.” One of the more enduring definitions characterizes organisms as distinct reproductive units and species as groups of individuals that can interbreed and produce viable, fertile offspring. The lack of genetic exchange between species, called reproductive isolation, lies at the heart of this definition. Environmental changes can create physical barriers between populations that preclude mating between the populations. Reproductive isolation can also involve changes at the genetic level, when molecular barriers prevent two recently diverged populations from producing viable or fertile offspring. Such factors limit gene flow between diverging species and allow the emergence of genetically novel yet sound populations—that is, new species. Multiple regions of Hmr show evidence for divergence driven by positive selection At the heart of reproductive isolation is a phenomenon called hybrid incompatibility, in which closely related species are capable of mating but produce inviable or sterile offspring. The classic example of hybrid incompatibility is the male donkey–female horse cross, which yields a sterile mule, but many other cases have been documented among mammals, and thousands of plant crosses produce infertile offspring. Much has been learned about the genetic architecture of hybrid incompatibility by studying the offspring of closely related, or “sibling,” fruitfly species in the lab. Sibling species are morphologically very similar, or even indistinguishable, but typically do not interbreed in nature. In the lab, their offspring are either sterile or inviable, a fate that varies depending on the gender of the offspring and species of the parents. To elucidate the molecular mechanisms of reproductive isolation, biologists must first identify candidate hybrid incompatibility genes. Species- or lineage-specific functional divergence is an essential trait of these genes. (That is, the genes evolve different functions after the species diverge from their common ancestor.) While several such candidate genes have been identified in the fruitfly Drosophila melanogaster , none has been shown to display this functional divergence. Now, working with D. melanogaster and its sibling species D. simulans and D. mauritiana , Daniel Barbash, Philip Awadalla, and Aaron Tarone establish the functional divergence of a candidate hybrid compatibility gene and confirm its status as a true speciation gene. Since the 1930s, investigations of reproductive isolation have been guided by the Dobzhansky-Muller model, which attributes hybrid incompatibility to the interactions between two or more genes that have evolved independently in two isolated populations. These independently evolving genes diverge functionally, and the interactions of these functionally divergent genes in a hybrid individual are responsible for the defective phenotypes observed (either inviability or sterility). If this is the case, the alleles, or versions, of the gene causing hybrid incompatibility should have distinct phenotypes in the two species. A corollary of the model says that the diverged allele (A) and not the ancestral allele (a) causes the incompatibility phenotype, which means that experimental manipulations of A but not a should affect the hybrid incompatibility phenotype. Barbash et al. tested the model's predictions by genetically manipulating the alleles of the Hybrid male rescue (Hmr) gene from each sibling species and observing the mutations' effects on the flies' hybrid offspring. In previous experiments the researchers had shown that loss-of-function mutations in the D. melanogaster Hmr gene “rescue” hybrid individuals from the hybrid incompatibility phenotype (male inviability) typically observed in the offspring of crosses between D. melanogaster and its sibling species, and that increased Hmr activity suppresses rescue and kills hybrids. If D. melanogaster Hmr has functionally diverged between the species, then transgenes containing Hmr from sibling species should not cause the hybrid incompatibility phenotype caused by the D. melanogaster Hmr . The researchers tested this hypothesis by introducing transgenic Hmr genes from sibling species into D. melanogaster . In all cases, the hybrid male offspring of D. melanogaster / D. mauritiana and D. melanogaster / D. simulans crosses “were at least as viable as their brothers without the transgene.” To examine this divergence at the genomic level, Barbash et al. compared the divergence of 250 genes in D. melanogaster and D. simulans and found that the Hmr gene was among the most rapidly evolving genes. By examining the frequency of mutations that have accumulated between D. melanogaster and sibling species relative to the number of mutations accumulated within species, the authors show that the mutations between species were by and large not neutral and that they occurred after D. melanogaster diverged from its sibling species, indicating that the gene has been under positive natural selection. Barbash et al. have not only identified a bona fide speciation gene by demonstrating its functional divergence, they've also created a platform for investigating the mechanisms through which such genes cause hybrid incompatibility and lay the groundwork for speciation.

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Pudendal nerve decompression in perineology : a case series

Background Perineodynia (vulvodynia, perineal pain, proctalgia), anal and urinary incontinence are the main symptoms of the pudendal canal syndrome (PCS) or entrapment of the pudendal nerve. The first aim of this study was to evaluate the effect of bilateral pudendal nerve decompression (PND) on the symptoms of the PCS, on three clinical signs (abnormal sensibility, painful Alcock's canal, painful "skin rolling test") and on two neurophysiological tests: electromyography (EMG) and pudendal nerve terminal motor latencies (PNTML). The second aim was to study the clinical value of the aforementioned clinical signs in the diagnosis of PCS. Methods In this retrospective analysis, the studied sample comprised 74 female patients who underwent a bilateral PND between 1995 and 2002. To accomplish the first aim, the patients sample was compared before and at least one year after surgery by means of descriptive statistics and hypothesis testing. The second aim was achieved by means of a statistical comparison between the patient's group before the operation and a control group of 82 women without any of the following signs: prolapse, anal incontinence, perineodynia, dyschesia and history of pelvi-perineal surgery. Results When bilateral PND was the only procedure done to treat the symptoms, the cure rates of perineodynia, anal incontinence and urinary incontinence were 8/14, 4/5 and 3/5, respectively. The frequency of the three clinical signs was significantly reduced. There was a significant reduction of anal and perineal PNTML and a significant increase of anal richness on EMG. The Odd Ratio of the three clinical signs in the diagnosis of PCS was 16,97 (95% CI = 4,68 – 61,51). Conclusion This study suggests that bilateral PND can treat perineodynia, anal and urinary incontinence. The three clinical signs of PCS seem to be efficient to suspect this diagnosis. There is a need for further studies to confirm these preliminary results.

Background The objective of perineology is to treat each defect of the perineum with the right procedure [ 1 - 3 ]. Pudendal nerve decompression (PND) is theoretically a basic procedure in perineology thanks to its ability to treat the defect "pudendal neuropathy". Before going into details of this procedure, it is necessary to remember the anatomy of the pudendal nerve. This anatomy is still controversial. While summarizing the data of the literature and the results of our dissections, the likeliest anatomy of the pudendal nerve presents itself as follows. The pudendal nerve is a mixed nerve carrying motor and sensory fibers. Its fibers are derived from the sacral roots S2, S3 and S4 [ 4 , 5 ]. Once the roots traverse the sacral foramen, they divide into autonomic branches forming the pelvic plexus (parasympathetic supply of the pelvic organs) and somatic branches merging to form the pudendal nerve travelling under the piriformis muscle. Near its formation point, it gives a levator branch running on the inner (upper) surface of the levator plate and providing the innervation of this muscle [ 4 ]. For Barber et al [ 6 ], this levator nerve originates directly from the S3, S4 or S5 roots. Some somatic fibers coming from S2 and S3 run close to the pelvic plexus to innervate the levator ani and the urethral sphincter [ 4 ]. Caudally, the pudendal nerve enters a small space ("clamp") between the sacro-spinal and sacro-tuberous ligaments very near the ischial spine. Just inferior to the ischial spine, the nerve gives its first branch, the dorsal nerve of the penis [ 4 ] or the clitoridal nerve. These nerves are separated from the main trunk by the pudendal vein and artery. Then, it enters the Alcock's canal formed by a division of the obturator muscle aponeurosis. In the canal the nerve crosses the sharp edge of the sacro-tuberous ligament (falciform process) [ 7 , 8 ]. Caudally, at the level of the anus, the nerve gives medially the inferior rectal nerves (usually two branches) which innervate the anal sphincter (and probably the pubo-rectalis) and the skin of the posterior perineum and anterolaterally the transversus perinei branch (for this muscle, for the ischiocavernosus muscle and maybe for the urethral sphincter) [ 4 ]. The remaining part of the nerve is usually called the perineal nerve. This nerve gives a bulbocavernosus branch and finally divides into a sphincteric branch (innervation of the urethra) and a branch which innervates the skin of the anterior perineum [ 9 ]. The pudendal canal syndrome (PCS) and its surgical treatment have been described by Shafik in 1991 [ 5 ]. This syndrome is induced by the compression or the stretching of the pudendal nerve in the Alcock's canal. The complete syndrome presents with anal incontinence, pain, hypo or hyperesthesia and urinary incontinence (and impotence in males). Some important studies were done earlier by Amarenco [ 10 ] and Robert [ 7 ] but these authors focused mainly on pain which is only a part of the syndrome. The cause of the PCS is not always clear but it is often possible to find a compression (biking, long time sitting, haematoma...) or a stretching (descending perineum, surgery, delivery....) of the pudendal nerve in the Alcock's canal [ 10 - 19 ] in the history of the patient. A change in the shape or orientation of the ischial spine induced by some athletic activities during the youth could also explain some cases [ 20 ]. The clinical signs and investigation results proposed by Shafik [ 5 ] to confirm the diagnosis of PCS before surgery are: tenderness over the pudendal canal in the ischio-rectal fossa, diminished perineal sensation, weak or absent anal reflex, reduced EMG activity of the external anal sphincter and increased PNTML. The surgical procedure described by this author (trans-perineal approach) consists in opening the Alcock's canal to give the pudendal nerve a sufficient length to be unstretched and/or to suppress compression. The trans-gluteal approach proposed by Robert to treat pudendal neuralgia aimed to open also the "clamp" between the sacro-spinal and the sacro-tuberous ligament by cutting one or two of them [ 8 ]. Since Shafik's study in 1991, some questions about the effect of PND on the PCS remained open. No peer reviewed publications confirming the efficacy of this surgery on anal incontinence or on urinary incontinence could be found. Even the existence of a genuine PCS has never been validated. The aim of this study was to answer the following questions: Is there any effect of the bilateral PND according to Shafik on: - three main symptoms of PCS ? - perineodynia (vulvodynia, perineal pain, proctalgia) [ 21 ] - anal incontinence - urinary incontinence - three clinical signs of PCS ? - abnormal sensibility - painful Alcock's canal - painful "skin rolling test" [ 22 ] - two neurophysiological tests ? - electromyography (EMG) of the anal sphincter and of the bulbocavernosus muscles. - pudendal nerve terminal motor latencies (PNTML) of the anal and perineal branches. What is the clinical value of the aforementioned three clinical signs in the diagnosis of PCS ? Methods Studied sample A retrospective analysis to study the effects of a bilateral PND. The studied sample comprised 74 female patients who underwent a bilateral PND between 1995 and 2002 done by the same surgeon. The average age was 56.1 years (range: 28 – 77). All these patients underwent a complete history and clinical examination following the three perineal axis (gynaecological, urological and colo-proctological) according to the concept of perineology [ 1 - 3 ]. The frequency of the 3 main symptoms of the PCS (anal incontinence, perineodynia, urinary incontinence) in the 74 patients before surgery is presented in Figure 1 . Figure 1 Frequency of the 3 main symptoms of the pudendal canal syndrome (perineodynia, anal incontinence, urinary incontinence) before surgery. Associated surgical procedures were performed at the same time as the PND to treat all the defects revealed by the clinical examination, and are presented in Table 1 . Table 1 Procedures associated with the bilateral pudendal nerve decompression Associated procedures Operated (n = 74) Reviewed after one year or more (n = 56) None 17 10 MVT according to Mouchel [48-50] 46 38 Correction of rectocele 49 42 Correction of cystocele 20 17 Vaginal hysterectomy 16 13 Levatorplasty according to Shafik [26] 14 13 Urethral meatotomy 4 3 Prepubien section [45] 2 1 Anal sphincteroplasty 2 2 Urethrolysis 1 1 Diagnostic tools for PCS The following variables were used: Three main symptoms of the PCS Perineodynia For perineodynia, four situations were encountered: no pain, proctalgia, unilateral pain, bilateral pain. The effect of surgery was estimated by the patient using one of the following proposals: cured, improved, unchanged or worsened. Anal incontinence For anal incontinence, a four levels ordinal scale was used: no incontinence, gas incontinence, liquid incontinence, solid incontinence. "Cured" was defined as "no incontinence". The patient was considered "improved" if there was a change of at least one level in the scale going from "solid" to "gas" incontinence. The patient was defined as "worsened" if there was a change of at least one level in the opposite direction. Urinary incontinence For urinary incontinence, a four levels ordinal scale was used: no incontinence, mild incontinence, moderate incontinence and severe incontinence. The two types of urinary incontinence, stress and urge incontinence, were evaluated separately even if both were present in the same patient (mixed incontinence). "Cured" was defined as "no incontinence". The patient was considered "improved" if there was a change of at least one level in the scale going from "severe" to "mild" incontinence. If the change observed was in the opposite direction the patient was considered "worsened". Three clinical signs of the PCS (the examinations were done in gynaecological position) Abnormal anal or vulvar sensibility Sensibility was tested with a needle comparing the left and the right sides of the vulva and of the skin 2 cm lateral to the anus. The interpretations of the results were done using a four levels ordinal scale: 0 = total anaesthesia, 1 = reduced sensibility, 2 = normal sensibility, 3 = hypersensibility. 0, 1 and 3 were considered as "abnormal sensibility". Painful Alcock's canal on rectal examination The pain induced by the palpation of the pudendal canal by rectal examination was evaluated using a seven levels ordinal scale : 0 = no pain, 1 = mild pain, 2 = mild pain with Tinel sign (irradiation of the pain), 3 = moderate pain, 4 = moderate pain with Tinel sign, 5 = severe pain, 6 = severe pain with Tinel sign. The Alcock's canal was considered "painful" if the pain was 4 or more. Painful "skin rolling test" Beginning from 5 cm behind the level of anus the skin was pinched and then rolled to the front until the skin fold was at the level of the clitoris. The skin rolling test was considered "painful" if it induced a severe pain at least at one level (Figure 2 ). Figure 2 Skin rolling test : the skin of the perineum is pinched just beneath the level of the anus and then rolled to the front searching for a sharp pain at one level. This sign is well known in the diagnosis of neuralgia. Two neurophysiological tests Concentric needle EMG Concentric needle EMG was done at rest and during voluntary contraction on both sides of the external anal sphincter and on each bulbocavernosus muscle. The richness of the EMG was grossly evaluated using a six levels scale: 1 = simple, 2 = poor, 3 = intermediate poor, 4 = intermediate, 5 = intermediate rich and 6 = rich. Anal and perineal PNTML Anal and perineal PNTML were measured before and after the operation using the St Marks electrode to stimulate the pudendal nerve by the rectal route just under the ischial spine. For anal PNTML the electrical potentials induced in the striated anal sphincter were collected using the ring of this electrode. For the perineal PNTML the method described by Kiff [ 23 ] and Snooks [ 24 ] was modified according to Amarenco [ 25 ]. The electrical potentials were collected with a concentric needle in the two bulbocavernosus muscles. In our laboratory the normal values were: less than 2.5 msec for the anal PNTML and less than 5 msec for the perineal PNTML. EMG and PNTML were done by the same physician before and at least one year after the operation. Minimal criteria for surgery At least one of the 3 following symptoms resistant to conservative treatments (physiotherapy, drugs, infiltrations, modification of diet or behaviour): a. Anal incontinence b. Perineodynia c. Urinary incontinence Associated with at least two of the five following criteria: a. increased anal or perineal PNTML b. pathological EMG of the anal sphincter or bulbocavernosus muscles (neurogenic trace, reduced activity: richness "poor" or "simple"). c. painful Alcock's canal on rectal examination (at least on one side) d. abnormal perineal sensibility (at least at one level) e. painful "skin rolling test" (at least on one side). Surgical procedure Surgical procedure as described by Shafik in 1991 [ 5 ]. The operations were done under spinal or general anaesthesia. The patients were installed in the gynaecological position. The different steps of the procedure were: - Vertical incision of the skin between the anus and the ischial tuberosity. - Opening of the ischio-rectal fossa with scissors. - The inferior rectal nerve is hooked under the finger and followed to the entrance of the Alcock's canal (Figures 3 and 4 ). Figure 3 Left Alcock's canal (showed by the tip of the forceps) viewed from the mid side on a female cadaver: on the left the pudendal nerve, on the right the inferior rectal nerve on the finger. Figure 4 Alcock's canal viewed from below like in the operating room (right side of a female cadaver): inferior rectal nerve (horizontal) showing the entrance of the canal. - Opening of this canal (without opening the clamp between the sacro-spinal and sacro-tuberous ligament). - Control of the haemostasis. - Self draining closing of the skin with nylon. Evaluation of PND The efficacy on the symptoms, on the clinical signs and on the neurophysiological tests was evaluated during a follow up consultation one year or more after the surgical procedure because the nerve healing can be very slow [ 5 ]. Statistical methods Firstly, the efficiency of the PND on the symptoms and clinical signs was studied by means of descriptive statistics. Tests of hypothesis were done to compare the mean values of the neurophysiological tests before versus after PND. Secondly, the diagnostic value of the clinical signs was evaluated in a "case control" setting. A subject belongs to the "controls group" when PCS is considered to be absent, namely if the patient does not present any of the following symptoms, signs or risk factors for PCS: perineodynia, anal incontinence, prolapse, previous surgery in the area, dyschesia. The clinical signs are not used to decide if a subject belongs to the controls or cases group. The statistical comparison was done between the patient's group before ("cases group") and after the operation, and the "controls group" of 82 women (average age: 48.8 years, extremes 27–76). Statistical analysis of differences was performed using chi-square testing for categorical variables and t-tests for continuous variables. Results Effects of surgery Effect on the symptoms of the PCS The effect of PND on the symptoms of PCS is presented in Table 2 . Table 2 Effect of PND on the 3 main symptoms of the PCS Parameters Perineodynia (pain) Perineodynia (pain) Anal incontinence Anal incontinence Stress urinary incontinence Stress urinary incontinence Urge incontinence Urge incontinence All Without: levat All Without: sphincteroplasty, levat, recto All Without: levat, mvt, cysto, prepubien, meato, urethrolysis All Without: levat, mvt, cysto, prepubien, meato, urethrolysis Number of cases studied 74 59 74 22 74 22 74 22 Number of pathological results 26 22 46 9 47 4 33 4 Follow up less than 1 year or lost 8 8 10 4 10 3 6 0 Follow up 1 year or more 18 14 36 5 37 1 27 4 Mean follow up in months (range) 22,2 (12–48) 24,5 (12–48) 26,4(12–70) 17,2 (12–26) 32 (12–96) 12 26,7(12–72) 18,5 (12–26) Cured (%) 11 (61,1%) 8 (57,1%) 23 (63,9%) 4 (80%) 26 (70%) 0 (0%) 17 (62,9%) 3 (75%) Improved (%) 3 (16,6%) 2 (14,3 %) 7 (19,4%) 1 (20%) 7 (18,9%) 1 (100 %) 6 (22,2%) 0 (0%) No change (%) 4 (22,2%) 4 (28,6%) 4 (11,1%) 0 (0%) 4 (10,8 %) 0 (0 %) 3 (11,1%) 0 (0%) Worse (%) 0 (0 %) 0 (0 %) 2 (5,5%) 0 (0%) 0 (0%) 0 (0 %) 1 (3,7%) 1 (25%) levat = levatorplasty, recto = cure of rectocele, cysto = cure of cystocele, prepubien = prepubien section, meato = meatotomy. In order to treat completely the patient, PND was frequently associated with other procedures which might have an effect on the symptom studied. Therefore, the results for each symptom were presented in two columns: the first corresponds to the entire sample ("all") and the second to the small group of patients in which the symptom was treated by PND only ("without"). Effect on perineodynia On the 26 patients with pain before the operation, 18 were reviewed 12 months or more after the operation. The pain had disappeared in 11 and was reduced or had another origin (painful puborectalis) in 3. The cure rate with a mean follow-up of 22,2 months was 61,1 % (77,7% cured or improved). As none of the surgical associated procedures used in this study were known to improve or cure perineodynia, the only procedure removed was levatorplasty. Theoretically this operation can reduce the stretching on the pudendal nerves by reducing the sagging of the levator plate. The results were similar in the group without levatorplasty. Effect on anal incontinence On the 46 patients with anal incontinence before the operation, 36 were reviewed 12 months or more after the operation. 23 of them were cured, 7 improved, 2 were worse and 4 reported no change. The cure rate with a mean follow-up of 26.4 months was 63.9 % (83,3% cured or improved). The results according to the severity of incontinence are presented in Table 3 . Table 3 Effect of PND on anal incontinence according to the incontinence level. Cured Improved Unchanged Worsened Solid (n = 5) 3 2 0 0 Liquid (n = 20) 12 5 3 0 Gas (n = 11) 8 0 1 2 To study the real impact of PND on anal incontinence, we removed the following cases in which PND was associated with anal sphincteroplasty, levatorplasty or cure of rectocele by fascia and perineal body restoration: - 2 patients had anal sphincteroplasty together with the PND: one was cured and the other improved. - Levatorplasty according to Shafik [ 26 ] was used in 8 patients who had a severe levator plate sagging. This procedure could have a "post-anal repair effect" [ 27 ] and therefore improve anal incontinence. - 30 cases had a cure of rectocele by fascia and perineal body restoration (Ayabaca and coll. [ 28 ] found an improvement of anal incontinence in 25 of their 34 patients. Nevertheless, none of them gained full continence post-operatively). In the small group of patients with PND only, 5 were reviewed one year or more after the operation: 4 were cured (3 incontinences for liquid and 1 for gas) and 1 improved (liquid incontinence). Anal ultrasound was done before the operation in 13 of the 36 patients reviewed. Only 4 were normal, 3 showed a rupture of the internal and external sphincters, and 6 presented a disruption of the external sphincter alone. In the 7 patients who had a rupture of the anal sphincter (5 external only, 2 internal and external) without anal sphincteroplasty and a follow up of more than 12 months (mean 18.5 months), 4 were cured and 3 were a failure. 5 patients who were still incontinent after 1 year follow up (2 incontinences for flatus and 3 for liquid) became continent 2 years after the operation. Effect on urinary incontinence Five patients presenting urinary incontinence (4 urge and 1 stress incontinence) had PND without any other procedure around the urethra. The mean follow up was 18,5 months for the 4 patients with urge incontinence. In this small group, 3 patients were cured and one was a failure. The patient with stress urinary incontinence was improved (the number of pads used per day was reduced from 9 to 4) one year after surgery. Effect on the clinical signs The effect of PND on the three clinical signs is described in Table 4 . Table 4 Effect of PND on the three clinical signs Parameters Abnormal sensibility (at least at one level) Abnormal sensibility (at least at one level) Painful Alcock's canal (at least on one side) Painful Alcock's canal (at least on one side) Painful skin rolling test (at least on one side) Painful skin rolling test (at least on one side) All Without: levat All Without: levat All Without: levat Number of tests before surgery 42 27 46 32 39 26 Follow up less than 1 year or lost 11 9 19 16 22 16 Follow up 1 year or more 31 18 27 16 17 10 Normal test before (reviewed 1 year or more after) 15 8 9 6 8 2 Abnormal test before (reviewed 1 year or more after) 16 10 18 10 9 8 Mean follow up (range) 27,7 (12–68) 32,2 (12–68) 28,1 (12–68) 32,8 (12–68) 28,7 (12–60) 33,7 (12–60) Normal before => Normal after 14 8 9 6 7 2 Normal before => Abnormal after (%) 1 0 0 0 1 0 Abnormal before => Normal after (%) 11 (68%) 6 (60%) 11 (61%) 7 (70%) 6 (66,6%) 5 (62,5) Abnormal before => Abnormal after 5 4 7 3 3 3 Because levatorplasty can theoretically reduce the stretching of the pudendal nerve, the evaluation of the effect on the clinical signs has been done for the entire sample ("all") and for the same group without the patients who had a levatorplasty ("without: levat"). The cure rate of the 3 clinical signs was between 60 and 70 % depending of the sign and of the type of sample studied. Effect on EMG and PNTML 38 patients underwent a complete EMG evaluation before and after surgery. A relevant comparison was possible in 35 patients. 3 patients were excluded because one of the two EMG explorations was insufficient for technical reasons. The average follow up was 16,9 months (range: 12 – 35,4 months). Left and right values for one parameter correspond to two different nerves. Therefore, these values were considered independent (maximum number of analysed cases: 35 × 2 = 70). The effect of PND on EMG and PNTML is presented in Table 5 . Table 5 Effect of PND on EMG and PNTML Anal Richness (range 1 to 6) BC Richness (range 1 to 6) Anal PNTML (msec) Perineal PNTML (msec) All subjects 74 74 74 74 Follow up less than 12 months or lost 39 39 39 39 Analysed cases(left and right) 70 61 70 51 Mean Before 2,70 2,23 3,38 5,63 Mean After 3,11 2,44 2,63 5,21 t-test p-value(one-tail) 0,00007 0,06989 0,00004 0,00816 Left and right values for each level (anal and perineal) were included in the same group. P-values at the bottom line correspond to one-tailed significance tests of the mean differences "before" versus "after". The "Anal Richness" on EMG after surgery was significantly higher than before. The mean "Bulbocavernosus Richness" after surgery was slightly higher than before but this difference was not significant. Both anal and perineal PNTML after PND were significantly reduced compared to values before. The box-plots of the 4 studied parameters are presented in Figures 5 and 6 . Figure 5 Effect of PND on anal and bulbocavernosus (BC) richness on EMG. The box is defined by the sample mean plus or minus one standard error of the sample mean. The probability to obtain a value in the box is 67 %. The whiskers represent the 95% confidence intervals of the population means. Figure 6 Effect of PND on anal and perineal PNTML. The box-plots definitions are the same as in Figure 5. Evaluation of the clinical signs We present here the results concerning the evaluation of the three clinical tests: abnormal sensibility, painful Alcock's canal and painful "skin rollling test" as diagnostic tests for PCS. The statistical analysis is based on the following contingency tables presented in Tables 6 to 9 . Table 6 "Abnormal sensibility" in the diagnosis of pudendal canal syndrome Cases Controls Abnormal sensibility Before Surgery After Surgery Abnormal 24 7 19 Normal 18 36 63 Total 42 43 82 Chi-square versus Controls 12,691 0,449 P-value < 0,001 0,503 At the bottom lines, p-values and chi-square test statistics correspond to the homogeneity tests comparing the "Cases Before Surgery" to "Controls". The results obtained for the homogeneity test comparing "Cases After Surgery" versus "Controls" are also reported in the tables, although this does not contribute to the sensibility/specificity analysis. Table 7 "Painful Alcock's canal" in the diagnosis of pudendal canal syndrome Cases Controls Painful Alcock's canal Before Surgery After Surgery Abnormal 32 10 24 Normal 14 30 58 Total 46 40 82 Chi-square versus Controls 17,842 0,0776 P-value < 0,001 0,781 Table 8 "Painful skin rolling test" in the diagnosis of pudendal canal syndrome. Cases Controls Painful skin rolling test Before Surgery After Surgery Abnormal 21 6 13 Normal 17 28 69 Total 38 34 82 Chi-square versus Controls 17,968 0,001 P-value < 0,001 0,97 Table 9 The three clinical signs in the diagnosis of pudendal canal syndrome Cases Controls The three clinical signs Before Surgery After Surgery Abnormal – All positive 13 3 6 Abnormal – Two positive 8 3 9 Abnormal – One positive 5 3 20 Normal – All negative 6 24 47 Total 32 33 82 Chi-square versus Controls 26,528 3,834 P-value < 0,001 0,280 The proportions of observations in the "Cases Before Surgery" and "Controls" columns of the contingency table vary significantly from row to row (p-values <0,001), whereas no significant difference is observed between the proportions of observations between "Cases After Surgery" and "Controls" (p-values >0,05). Estimated sensibility, specificity, predictive values (positive and negative) and odd ratio (estimated values and 95% confidence intervals) corresponding to each of the three clinical tests and combinations of all of them are presented in Tables 10 and 11 , respectively. The predictive values were calculated for a PCS prevalence of 20 %. All the indicators in Tables 10 and 11 are estimated from data in Tables 6 to 9 , columns "Cases Before Surgery" and "Controls". Table 10 Evaluation of each of the three clinical signs of pudendal canal syndrome Tests SE SP PPV NPV OR 95%IC(OR) Nb of Cases Before Nb of Controls Abnormal sensibility 0,57 0,77 0,38 0,88 4,42 1,99 – 9,82 42 82 Painful Alcock's canal 0,70 0,71 0,37 0,90 5,52 2,51 – 12,15 46 82 Painful skin rolling test 0,55 0,84 0,47 0,89 6,56 2,74 – 15,68 38 82 SE = sensibility, SP = specificity, PPV = positive predictive value, NPV = negative predictive value, OR = odd ratio, 95%IC (OR) = 95 % confidence interval of the odd ratio. PPV and NPV for a prevalence of 20 %. Table 11 Evaluation of different combinations of the three clinical signs of pudendal canal syndrome Test: All Three Clinical Signs SE SP PPV NPV OR 95%IC(OR) Nb of Cases Before Nb of Controls All positive vs. All negative 0,68 0,89 0,60 0,92 16,97 4,68 – 61,51 19 53 At least 2 positive vs. At least 2 negative 0,66 0,82 0,47 0,90 8,53 3,40 – 21,39 32 82 At least 1 positive vs. All negative 0,81 0,57 0,32 0,92 5,82 2,16 – 15,66 32 82 SE = sensibility, SP = specificity, PPV = positive predictive value, NPV = negative predictive value, OR = odd ratio, 95%IC (OR) = 95 % confidence interval of the odd ratio. PPV and NPV for a prevalence of 20 %. The most sensible test is the "Painful Alcock's canal" and the most specific is the "Skin rolling test". Using the three signs altogether, the most sensible combination is "At least 1 positive versus All negative" and the most specific combination is the "All positive versus All negative". Side effects During one operation a heavy bleeding coming from the pudendal artery just near the pudendal nerve was very difficult to treat (selective ligature). This patient had a blood transfusion but no long term side effect. Since the operation, one patient has presented sometimes a short lasting clitoridal pain. This patient had also an increase in her anal incontinence (gas incontinence became a liquid incontinence). Three patients had wound healing problems which resolved with simple disinfection. Prevalence In the literature there is no data available about the prevalence of the PCS. Therefore, it seems to be a rare event. In this study, we evaluated the prevalence of the PCS in an outpatient perineology clinic. By using three different methods during the last 24 months (percentage of pudendal nerve decompressions in the treatment of prolapse or incontinence : 13/78; percentage of anal incontinence and/or perineodynia in our outpatient consultation : 78/316 ; percentage of positive skin rolling tests : 9/55) the estimated prevalence should be around 20%. Discussion Effects of surgery Before discussing the results of surgery, the first important issue is about the interest of a bilateral decompression. The benefit – risk ratio must be studied. The results of Shafik were obtained after bilateral decompression [ 5 , 29 , 30 ]. For urinary and anal incontinence, it seems logical to treat both nerves because the sphincters have a bilateral innervation and if one nerve is suffering maybe there is a problem on the other. The EMG exploration is not very sensitive and doesn't study the sensory pathway, which could be very important for continence. For the same reason, bilateral decompression seems logical in the treatment of proctalgia. For unilateral pain, the dilemma is more important. The risk is to induce pain at the "normal" side. Until now we have been performing bilateral operations without such a side effect but a controlled randomised study would be necessary to conclude. The treatment of pain starts with a holistic approach of the woman (drugs, psychotherapy, relaxation...) with exclusion of other causes of pain: piriformis syndrome, coccygodynia, interstitial cystitis, endometriosis... The other neurological causes must be excluded by a complete electrophysiological study of the perineum (sacral latencies, PNTML, detection EMG and sensory evoked potentials) and imaging of the spinal cord [ 31 ]. If the diagnosis is confirmed an infiltration of the Alcock's canal under scanner control can be tried. This infiltration is successful in 57% in the short term but only in 15 % of the cases after one year [ 32 ]. It can be repeated maximum 3 times to avoid a nerve irritation. In the treatment of pain the results of this study are similar or better than those obtained in previous studies [ 32 - 34 ]. Even with the transgluteal approach where the "clamp" between the sacro-spinal and the sacro-tuberous ligament is opened by sectioning the sacro-spinal ligament, the cure rate remains around 50%. In the 4 cases of proctalgia fugax the results were better (3 cured and 1 improved). Shafik [ 5 ] had also very good outcomes with this type of pain (100% cured). In this study, the results were worse if the pain was bilateral. For Robert early diagnosis appears to be the determining factor in improving results [ 35 ]. He used the infiltration of the Alcock's canal with lidocaïne-corticoïds as a test before operating. According to him a sufficient pain relieve, lasting during a short period, is a good indication for surgery. Mauillon et al also thinks that complete disappearance of pain for at least two weeks after a nerve block repeated twice before surgery may be the best criterion to predict success [ 34 ]. In this study, the patients presenting with perineodynia only (n = 10; Figure 1 ) had an infiltration before surgery but the number of cases was not sufficient to give a relevant impression about the infiltration test. For anal incontinence, our results were in the same range as Shafik [ 29 ]. In a previous study we also had similar results [ 36 , 37 ]. The exclusion of the patients who had sphincteroplasty, levatorplasty (possible post-anal repair effect [ 27 , 28 ]) and/or a cure of rectocele did not change the cure rate. More interesting was the cure rate in the group of patients with a clear rupture of one or the two anal sphincters. The traumatic rupture of the anal sphincter (delivery, sphincterotomy...) usually induces an immediate anal incontinence. In the patients who remained continent the power of the broken muscles remain sufficient to avoid flatus or faeces leakage. In the long term the continence is probably maintained with the help of the fibrous tissue located between the two edges of the ruptured muscle which acts like a bridge and therefore enables the sphincter to be efficient during many years. The aging process of the muscle and the pudendal neuropathy reduce the power of the muscle (and probably the sensibility in the anal canal) with time and explain the appearance of an anal incontinence. Therefore it is logical to restore continence by improving the conduction in the pudendal nerve. This fact can also explain why the results of sphincteroplasty decrease with time especially in the non diagnosed or treated pudendal neuropathies [ 38 ]. The fact that 5 patients were cured from their anal incontinence only 2 years after surgery emphasized the importance of a long follow up period to obtain relevant cure rates. Surprisingly, the cure rate seems to be not dependant of the degree of anal incontinence but the number of solid incontinences (5 cases; 3 cured and 2 improved) was too small to validate this impression. The results of the pudendal nerve decompression seem to be equivalent to these of neuromodulation [ 39 ] and the procedure is far less expensive because there is no need for a special material. If this study is confirmed by others, the treatment of the neuropathy should be done before any trial of neuromodulation. In fact it is logical to repair the electric cable before enabling the current to pass. For urinary incontinence the number of cases is too small to give a relevant cure rate but there were enough cases to suggest that this surgery can treat some patients with stress or urge incontinence. In a previous study, 3 of the 7 patients presenting a stress urinary incontinence were cured by bilateral pudendal nerve decompression alone [ 36 , 37 ]. In Shafik's study 6 patients were cured from their stress urinary incontinence, 5 improved and one was a failure [ 30 ]. For this author, the efficacy of the pudendal nerve decompression on stress urinary incontinence is due to an increase of the external urethral sphincter EMG activity and to a decrease in the straining urethral reflex latency (time between the expiration involved with the cough and the first deflection of the reflex muscle action potential complex) and PNTML. For Shafik [ 40 ] the increase of urethral pressure during abdominal hyperpressure is not only passive but is induced by an active contraction of the urethral sphincter. After an injection of lidocaïne in the sphincter the urethral hyperpressure was suppressed. Thind & al. clearly demonstrated the role of the pudendal nerve in urinary continence. These authors showed a clear reduction of the maximum urethral pressure and a decrease in the adjunctive urethral closure forces during stress after bilateral pudendal blockade [ 41 , 42 ]. This is also in agreement with the study of Constantinou which demonstrated that a fast-acting contraction occurs in the distal third of the urethra 240 plus or minus 30 msec before the bladder hyperpressure [ 43 ]. Furthermore, Ko and Kim demonstrated that pudendal nerve block with a 7% phenol solution is very effective in the treatment of external urethral sphincter hypertonicity in patients with spinal cord injury [ 44 ]. This study is the first one dealing with a possible effect of the pudendal nerve decompression on urge incontinence. It is probably due to a better control of the urethral sphincter which can reduce urethral instability [ 45 ] and improve the inhibition of the detrusor activity. One weakness of this study is the rough evaluation of the symptoms. We did not use any scoring system, pad test, quality of life questionnaires or "visual analog pain scale". Furthermore the number of anal and urethro-vesical manometries done before and after PND was too small to give relevant results. The objective evaluation of PND was done using two neurophysiological tests and the clinical examination. Like Shafik [ 29 , 30 ] we found a significant increase in anal richness on EMG, a significant reduction of anal and perineal PNTML after surgery and a significant reduction in the frequency of the clinical signs. The skin rolling test was improved as much as the perineal sensibility and the Alcock's canal pain, thus showing its relevant link with the PCS. Evaluation of the clinical signs and minimal criteria needed for the diagnosis Shafik described many clinical signs of the PCS [ 5 , 29 , 30 ]. In this study only three signs were studied carefully. Shafik described two of them: abnormal perineal sensibility and pain during the palpation of the Alcock's canal by a rectal examination. The third one is the "skin rolling test" which is well known in the diagnosis of neuralgia in other parts of the body [ 22 ]. This study is the first one in which this test was utilized as a clinical sign of PCS. Compared to patients with negative clinical signs, those having positive clinical signs have a 4,42 ; 5,52 and 6,56 higher likelihood of PCS for "Abnormal sensibility", "Painful Alcock's canal" and "Painful skin rolling test", respectively (Table 10 ). When patients with all three signs positive are compared to patients with all three signs negative, the odd ratio is 16,97 (Table 11 ). All the estimated 95% confidence intervals for the odds ratios are significantly higher than 1, indicating that the clinical signs can be considered as valuable signs in the diagnosis of PCS. The most specific sign was the "Painful skin rolling test" and the most sensitive was the "Painful Alcock's canal". The association of the three positive tests had a very high specificity in the diagnosis of a PCS (89 %). This high specificity was confirmed by the low frequency of this association after the operation (return to the same level as the control group). Therefore, in some cases the clinical examination should be sufficient to prove the existence of a PCS. For example, a patient presenting anal incontinence, an intact sphincter proven by ultrasound and the three clinical signs positive has almost certainly a PCS. Of course, from a scientific point of view it is still interesting to perform a complete electrophysiological study and a precise neurological examination to exclude a central problem (multiple sclerosis, tumor...) or a polyneuropathy. However, making the diagnosis of PCS is not usually an easy task. Many times, there is a high degree of suspicion but, as in many illness, all the symptoms or signs are not present. In this study, we decided to operate when at least two of the five clinical and neurophysiological signs described in the methods section were associated with one or more of the 3 classical symptoms (perineodynia, anal incontinence and urinary incontinence). At the beginning of this study, it was usually "increased PNTML" and "painful Alcock's canal". With the introduction of the "skin rolling test" and of the "sensibility test", clinical examination became more important in the decision. The more symptoms (especially anal incontinence and perineodynia) and signs were present, the more confident we were in the diagnosis of PCS. Further studies are necessary to validate this and to define more precisely the minimal criteria needed for the PCS diagnosis. Side effects The pudendal nerve decompression by the perineal route is a blind procedure. The search for the inferior rectal nerve and the opening of the Alcock's canal are done under finger control. In our experience it is not easier with retractors. Therefore it is necessary to have a clear anatomical vision of this area before performing the operation. Maybe the use of a laparoscope would help [ 46 ] but the procedure will become more expensive and time consuming. To suppress the blind character of the procedure the transgluteal approach proposed by Robert [ 8 ] or the more recent transvaginal approach from Bautrant [ 47 ] could be other ways to treat the PCS. Until now the results on pain are the same as those obtained by the Shafik's approach but with the concurrent sections of one or two ligaments of the pelvis (sacro-spinal and/or sacro-tuberous ligaments). However, we should be aware that the long term effects of these sections on the stability of the pelvic region are until yet unknown. Therefore, if the "clamp" must be open efforts should be done to open it without cutting a ligament. Up to now no data are available about a potential effect of the transgluteal or transvaginal procedures on urinary or anal incontinence. Despite the blind character of the procedure we only had one heavy bleeding probably coming from the pudendal artery. One patient still presents with a mild intermittent clitoridal pain and a worsening of anal incontinence. Because the nerve of the clitoris leaves the pudendal nerve just before the entrance into the Alcock's canal this problem is probably the result of a too large dissection in the upper part of this canal. The two cases of anal incontinence worsening (gas incontinence becoming liquid incontinence), including the aforementioned patient, are difficult to explain. Maybe the neuropathy increased with the stretching involved in the procedure, the scarring process or a too large dissection. It could also be the result of the changes in the posterior level anatomy induced by concomitant procedures (easier expulsion of gas or faeces). For the 2 patients the EMG data and the clinical examination after the operation did not improve therefore showing that the neuropathy was not healing. Prevalence Because the roughly estimate prevalence of PCS is around 20%, this "defect" seems to be a very frequent problem in Perineology. Therefore it should be logical to search for it in each clinical examination of a patient presenting with prolapse, perineodynia, urinary or anal incontinence. Conclusions Pudendal neuropathy is probably a frequent "defect" in perineology. Pudendal nerve decompression seems to be the defect specific procedure indicated in such a problem. In fact it can treat perineodynia, anal and probably urinary incontinence. Anal incontinence can be cured by pudendal nerve decompression alone even in the presence of a clear disruption of the anal sphincter on anal ultrasound. Anal richness on EMG increases and PNTML decrease significantly after surgery proving an objective effect on the nerve. The frequency of abnormal puncture sensibility, painful Alcock's canal and painful "skin rolling test" are significantly reduced by the operation. This study suggests that the three clinical tests could be used in practice to confirm or suspect the diagnosis of pudendal neuropathy in case of pain, urinary and/or anal incontinence. However, further studies are necessary to confirm these preliminary results. Abbreviations PCS = pudendal canal syndrome PND = pudendal nerve decompression EMG = electromyography PNTML = pudendal nerve terminal motor latency Competing interests The authors declare that they have no competing interests. Authors' contribution JB conceived the study, carried out surgery and clinical follow up and drafted the manuscript. DC participated in the design of the study and performed the statistical analysis. MB carried out the neurophysiological examinations. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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WINPEPI (PEPI-for-Windows): computer programs for epidemiologists

Background The WINPEPI (PEPI-for-Windows) computer programs for epidemiologists are designed for use in practice and research in the health field and as learning or teaching aids. They aim to complement other statistics packages. The programs are free, and can be downloaded from the Internet. Implementation There are at present four WINPEPI programs: DESCRIBE, for use in descriptive epidemiology, COMPARE2, for use in comparisons of two independent groups or samples, PAIRSetc, for use in comparisons of paired and other matched observations, and WHATIS, a "ready reckoner" utility program. The programs contain 75 modules, each of which provides a number, sometimes a large number, of statistical procedures. The manuals explain the uses, limitations and applicability of specific procedures, and furnish formulae and references. Conclusions WINPEPI provides a wide variety of statistical routines commonly used by epidemiologists, and is a handy resource for many procedures that are not very commonly used or easily found. The programs are in general user-friendly, although some users may be confused by the large numbers of options and results provided. The main limitations are the inability to read data files and the fact that only one of the programs presents graphic results. WINPEPI has a considerable potential as a learning and teaching aid.

Background This paper describes the WINPEPI (PEPI-for-Windows) programs recently added to the PEPI suite of computer programs for epidemiologists, and discusses some of their uses and limitations. The programs were developed for use in practice and research in the health field and as learning or teaching aids. PEPI (an acronym for P rograms for EPI demiologists) grew from a set of programs for programmable pocket calculators that was published in 1983 to "make life easier for investigators, extend the use of appropriate analytic methods, and enable researchers to concentrate on substantive issues rather than on procedural technicalities" [ 1 ]. The first version of PEPI appeared in 1993 [ 2 ], and was followed by version 2 (where the name "PEPI" was first used, in 1995) [ 3 ], version 3 (in 1999) [ 4 ], and version 4 (containing 43 programs, in 2001) [ 5 ]. The original programs were DOS-based. The first WINPEPI program, WHATIS, was included in version 4 of the PEPI package, a review of which stated: "WHATIS, the only Windows program, is our pick for the best program in PEPI. If all the programs could be converted into the WHATIS type of format, PEPI will be a truly outstanding package!" [ 6 ]. Four WINPEPI programs, containing 75 modules, have so far been issued. They provide many procedures not offered by the DOS-based programs, but do not include all those provided by the latter (which can be run in Windows as DOS applications, complementing the WINPEPI programs). Implementation There are at present four WINPEPI programs, DESCRIBE, COMPARE2, PAIRSetc, and WHATIS. The programs are free, and can be downloaded from the Internet. New versions have been issued at frequent intervals. Comprehensive manuals are provided. These furnish full information about each module, including explanations of the uses, limitations and applicability of specific procedures, and formulae or references. DESCRIBE DESCRIBE has 14 modules for use in descriptive epidemiology. It can appraise rates or proportions and categorical or numerical data (including survival data), examine a sequence of rates or other values (including the appraisal of seasonal variation), perform direct and indirect standardization, estimate prevalence from a cluster or stratified sample or by the capture-recapture method, determine required sample sizes, and appraise screening or diagnostic tests (with procedures for use in meta-analyses of studies of these tests). COMPARE2 COMPARE2 has 28 modules for use in comparisons of two independent groups or samples, and may be used to analyze cross-sectional, cohort and case-control studies, and trials. It can compare proportions or odds, risks, rates, and categorical and numerical data (including survival data), appraise the effect of misclassification, and determine power and sample size for a variety of tests. The program can deal with stratified data, analyzing the combined strata as well as each stratum; it permits the control of possible confounding by the stratifying variable or variables, and the assessment of heterogeneity as an indication of effect modification. It can be used in meta-analyses, to compare study results and, if warranted, combine them. PAIRSetc PAIRSetc has 29 modules for use in comparisons of paired and other matched observations, such as matched-control trials and cohort studies, matched case-control studies, before-after studies, and reliability studies that compare replicate observations or methods of measurement. The "etc" in its name indicates the program's ability to deal with matched sets larger than pairs. The program can compare dichotomous, categorical and numerical data (including paired survival data), appraise the effect of misclassification, and determine power and sample size for a variety of tests and for measuring kappa or intraclass correlation coefficients. Like COMPARE2, PAIRSetc can deal with stratified data. WHATIS WHATIS is a "ready reckoner" utility program with four modules. It provides a calculator (expression evaluator) that stores values and formulae, enabling them to be recalled when needed, and it computes confidence intervals for a variety of statistics, P-values corresponding to given values of z , t , chi-square, or F , or vice versa, and time-spans. The modules Some of the modules have very specific purposes; for example, to determine the sample size required to perform a specific test with a given power or precision, or to appraise the effect of misclassification in a given situation by computing the "true" findings that would give rise to the observed findings. Other modules provide many statistical procedures, as is illustrated by the following summaries of what two of the richer modules do. A module may not only provide numerous tests and measures, it may also use alternative methods of estimation. Comparison of proportions or odds (module A of COMPARE2) After entry of a 2 × 2 table, this module provides exact one-tailed and two-tailed tests (Fisher's, mid-P, and Overall's continuity-corrected tests and Tocher's test), chi-square tests (with and without Yates's, Upton's, and Haber's corrections), an optional equivalence test, the ratio of proportions (with its standard error and 90%, 95% and 99% confidence intervals and Jewell's low-bias estimator), the difference between proportions (with its standard error and 90%, 95% and 99% confidence intervals computed by Fleiss's procedure and by Wilson's score method, without and with a continuity correction), the odds ratio (with 90%, 95% and 99% confidence intervals – Cornfield's and exact Fisher's and mid-P intervals – and Jewell's low-bias estimator), Yule's Q, phi , and lambda . Separate results are shown for studies in which inverse sampling was used. For stratified data, the combined analysis provides Fisher and mid-P exact and Mantel-Haenszel tests, an optional equivalence test, three estimators of the overall ratio of proportions and of the overall difference between proportions (precision-based, Mantel-Haenszel, and DerSimonian-Laird estimators, with 90%, 95%, and 99% confidence intervals), and four estimators of the overall odds ratio (conditional and unconditional maximum-likelihood estimators, a Mantel-Haenszel estimator, and a DerSimonian-Laird estimator, with 90%, 95%, and 99% Fisher's, mid-P, Mantel-Haenszel, Cornfield-Gart, and Dersimonian-Laird intervals), heterogeneity tests and measures ( H and I -squared, with their 95% confidence intervals), and (for meta-analysis) estimates of the fail-safe N and two tests for a skewed funnel plot (regression asymmetry and adjusted rank correlation tests). Appraisal of numerical data (module D of DESCRIBE) This module appraises a frequency distribution, and also appraises a sequence of numbers. It describes the frequency distribution in terms of its central tendency (the mean, with its standard error and 90%, 95% and 99% confidence intervals, three robust estimators of the mean, the geometric mean, and the median, with its 95% confidence interval) and dispersion (quantiles, standard deviation, variance, mean deviation from the mean, and median absolute deviation from the median), and it performs the Grubbs test for outliers. The shape of the frequency distribution is appraised in terms of symmetry or skewness (Bowley's quartiles-based skewness coefficient, Randles-Fligner-Policello-Wolfe test, Wilcoxon signed-rank test of symmetry around the sample median) and peakedness or flatness (Moors octiles-based kurtosis coefficient, Kolmogorov-Smirnov test for an even distribution). The shape of the frequency distribution is pictured in box-and-whisker diagrams, for both raw and log-transformed data. Two tests for normality (Lilliefors and D'Agostini-Pearson tests) are applied to the raw and log-transformed data. The median or mean can be compared with a hypothetical value (using a t -test and Wilcoxon's signed-ranks test), and the Poisson dispersion test for heterogeneity is done (appropriate only if the values that were entered are counts). If a sequence of numbers is entered, it is tested for randomness (two runs tests, an up-and-down-runs test, and the mean square successive difference test), trend (Mann-Kendall and Cox-Stuart tests – including a test controlling for seasonal variation), a change-point, and centrifugality. The module provides Sen's estimator of slope, parametric and nonparametric linear regression analyses, and Spearman, Kendall's, and Pearson's correlation coefficients, and it smooths the curve, using procedures based on running medians and on Fourier transforms. Regression lines, smoothed curves, and the change-point are shown in a graph. Operating the Programs There is no special installation procedure; the programs need only be put in a folder of the user's choice. The appropriate program and module must first be selected. As an aid, a Pepi Finder (a Windows help file, FINDER.HLP) is provided; it is called up by clicking on its icon, and can be printed for easy reference. The Pepi Finder is an alphabetical index that shows which programs and modules deal with a specified procedure, measure, or kind of study. As seen in the excerpt shown in Figure 1 , the four WINPEPI programs are colour-coded. The Finder may point to more than one module; the entry for "Case-control study, unmatched", for example, is "COMPARE2 C,G". When COMPARE2 is opened (Figure 2 ) it is clear that its module G is designed for a case-control study with more than two exposure categories. The index also includes procedures provided by PEPI DOS programs (shown in italics) but not by WINPEPI programs. Figure 1 PEPI FINDER: Excerpt. Figure 2 COMPARE2: Opening screen. Each program has an opening screen (Figure 2 ) that displays a main menu and a top menu. Except in WHATIS, data entry is possible only after a selection has been made; a data-entry screen then appears. As an example, if option F of COMPARE2 is selected, i.e. "Categorical data (2 × k table)" (see Figure 2 ), the opening screen is replaced by the data-entry screen shown in Figure 3 . Figure 3 COMPARE2: Data-entry screen (for 2 × k table). The programs do not read data files, but require the entry of data that have already been counted or summarized, either manually or by using statistical software that processes primary data. The data can be entered at the keyboard, or (in multiple-entry boxes for the entry of tables) can be "pasted" from a file in which they are available. Once entered, tabular data can be pasted to a text file for future re-use by pasting. Alternative forms of data are often accepted, e.g. numerators instead of rates or proportions, and either individual or grouped observations. Warning messages are shown if obvious errors are made when entering data or if essential items are omitted. Simple on-screen instructions are provided, using simple language. For example, dichotomous variables are referred to as "yes-no" variables, and metric-scale observations, continuous or discrete, as "numerical". The term "rate" is used both for rates that have person-time denominators (e.g. incidence density) and for measures whose denominators are numbers of individuals (e.g. prevalence and risk); when the distinction is important, this is indicated. The instructions make use of terms well-known to epidemiologists, such as "case-control study", "exposed" and "not exposed", and "risk factor". (If the programs are used outside an epidemiological context, allowance must be made for their epidemiological labels.) To simplify operation, the program generally performs and reports all the prescribed procedures that the data will permit, without requiring choices by the user. But some options may be offered. In Figure 2 , for example, three options are shown: the categories may be nominal or ordinal, the scores allotted to the categories can be changed, and there is an option for performing a very specific kind of follow-up study. If "nominal" is checked instead of "ordinal", the instructions change, and the only option is for the partitioning of chi-square. Clicking on an option may modify the procedures a module performs, the manner in which the computation is done (e.g. depending on whether number-of-individuals or person-time denominators are entered, or whether a normal distribution can be assumed), and the data requirement (e.g. monthly or weekly or daily data for the appraisal of seasonal variation). Choice of an option may also modify the output. For example, the module that does a meta-analysis of studies of screening or diagnostic tests and produces forest plots for sensitivity, etc., permits optional display or suppression of the detailed numerical results for all studies. Pop-up hints and help screens are provided. Results are shown in an output screen (Figure 4 ), from which it is easy to return to the main menu or the previous screen. Results automatically go to the Windows clipboard, from which they can be pasted to other files. Clicking on "View" in the top menu displays all results obtained in the current session. "Print" options are offered. By clicking on "Note" in the top menu, it is possible to add comments to the results, for pasting, printing, or saving. A "Repeat" button is provided, permitting repeated analyses of the same data with changed options. Figure 4 COMPARE2: Results screen (for 2 × k table). All results are saved in a disk file, unless the user changes this default. The WINPEPI package contains a utility program (JOINTEXT) that can merge result files. DESCRIBE (but no other WINPEPI program) displays graphs – box-and-whisker plots, survival curves, seasonal peaks, regression lines, smoothed curves, forest plots, scattergrams, summary ROC curves, and graphs showing required sample sizes under different conditions. In most of the graphs, numerical values can be read by mouse-clicking at any location, optionally after magnifying a segment (zooming). Specimen graphs are shown in Figures 5 to 8 Figure 5 shows the number of clusters required for a cluster-based prevalence study (with stipulated requirements) for a true prevalence ranging from 5 to 20 per 100; the number can be read by clicking on the graph. Figure 6 shows a series of numerical observations, with regression lines, smoothed curves, and the change-point. Figure 7 shows post-test probabilities and net gain for a diagnostic test with a given likelihood ratio, for a range of pretest probabilities. Figure 8 shows a comparison of ROC curves, for use in appraising the effect of a covariate on the accuracy of a diagnostic test. Figure 5 Number of clusters required for a cluster-based prevalence study. Figure 6 A series of numerical observations. The straight lines are simple linear and nonparametric regression lines; the curved lines represent smoothing by two different methods; the red triangle marks the first point at which there is a significant change. Figure 7 Post-test probabilities and net gain for a diagnostic test. Positive likelihood ratio = 10. The net gain is the absolute difference between pretest and post-test probabilities. Figure 8 Comparison of ROC curves. Documentation Comprehensive manuals are provided. These furnish full information about each module, including explanations of the uses, limitations, and applicability of specific procedures, and formulae or references. The Pepi Finder serves as an index to the manuals. Discussion Criteria for the appraisal of statistical software for epidemiology [ 7 ] include not only its capabilities, but also "smoothness of the installation, simplicity of the interface, ease of use, completeness and statistical quality of the documentation, completeness and appearance of statistical graphics, accuracy of statistical computations". The WINPEPI programs are easy to install and easy to use (with the reservations discussed below). Their documentation is very detailed and (at the price of repetitiveness) includes a separate self-contained description of each module. A regrettable shortcoming of WINPEPI is that only one of the programs, DESCRIBE, presents graphic results. This is because DESCRIBE is the only 32-bit program, and the graph unit used by WINPEPI [ 8 ] is appropriate only for 32-bit programs. As for accuracy, the programs have been tested extensively, and all errors found have been promptly corrected; but (to cite the PEPI manual), it unfortunately remains a truism that no computer software can be entirely problem-free. But the WINPEPI programs do not provide data management facilities, and some other software package must be used if the data require processing. An epidemiologist or student whose data have been stored and maybe processed in another package, and who is well versed in the use of that package, may therefore have no need for the WINPEPI programs, despite their ease of operation, except when these do analyses not done by the other package. The WINPEPI programs aim "to complement – not replace – other statistics packages" [ 5 ]. Also (unlike the DOS-based PEPI programs for multiple logistic and Poisson regression analyses), the WINPEPI programs do not read data files. Data must be entered each time a program is used. This drawback is partly overcome by the possibility of pasting tabular data into data-entry boxes. But data entry can be tiresome, and users accustomed to programs that use data files may find it particularly vexatious. On the other hand, for some purposes keyboard entry may be seen as a boon: "Although conventional statistical software packages are adequate when you have a data set to work with, they are not always helpful when you need to do keyboard entry of data and rapidly perform simple analyses. For instance, you may want to replicate some analyses from a journal article and compute a Mantel-Haenszel odds ratio, or you may want to compute the sample size for your study while writing a grant proposal. Maybe you want to demonstrate to your students the impact of increasing sample size on the confidence intervals of a proportion. Perhaps you are a student and would like to do your epidemiology or biostatistics homework with some easy-to-use analytical routines... It is in this niche area that PEPI scores!" [ 6 ]. A criticism of version 3 of PEPI as being insufficiently user-friendly [ 9 ] led to a major revision in version 4. In the WINPEPI programs, user-friendliness is maximized by the provision of the Pepi Finder, simple on-screen instructions, pop-up hints and help screens, and warning messages, by streamlined data-entry procedures, which accept alternative forms of data, by the automatic saving of results, by the ease with which results can be recalled, annotated, printed, and pasted, and sometimes by the provision (in the output screens) of comments on the applicability of specific results. Unfortunately the wide variety of statistical procedures that is offered makes the WINPEPI programs less convenient to use; versatility carries a price. Even the provision made for the entry of alternative forms of data, meant as a convenience, necessitates a decision and may hence be an inconvenience – for example, a simple comparison of two proportions (using module A of COMPARE2) requires a choice between entry of four frequencies, of numerators and denominators, or of proportions and denominators. The DOS-based PEPI package elicited the comments "there are so many modules that sometimes it is difficult to remember which one to use" [ 10 ] and, with less restraint, "it is comprised of a large number of separate modules, which can make it a pain to use" [ 11 ]. The Pepi Finder was introduced (in version 3 of the package) to mitigate this problem. The advent of the WINPEPI programs, with their added statistical procedures, increased the potential for confusion and hence the value of the Finder, both for finding what program and module to use, and as an index to the detailed descriptions supplied in the manuals. The possibility of confusion is of course much reduced by the fact that related modules – for example, those concerning comparisons of two independent samples – are concentrated in the same WINPEPI program. Having opened the appropriate program, the user need only click on the kind of analysis that is required. But even that may tax some users. In COMPARE2, for example, a choice between modules B and D (see Figure 2 ) requires an awareness of whether the denominators are number-of-individuals or person-time ones. A further penalty for WINPEPI's versatility is that users may be confused by the large number of results in the output, some of them of little or no obvious relevance. As described above, module A of COMPARE2 (for a 2 × 2 table), for example, provides numerous "exact" and chi-square tests, and three measures of association, with confidence limits computed by different methods, as well as other results, including some that are valid only if inverse sampling was used. Similarly, module D1 of PAIRSetc (for paired numerical observations) provides three tests, six intraclass coefficients and a number of other measures of agreement, appropriate for different purposes. For this reason, every WINPEPI manual carries the admonition: "This program offers more options than most users will ever need, and will usually display more results than are needed. Ignore the options and results you don't require". (This of course assumes that the user knows what he or she wants.) But while all the results cannot be of interest to an ordinary user, each of them may be of interest to some users. As pointed out in a review of epidemiological software [ 11 ], "what one person might call 'statistical clutter' might be desirable to other people or even to that person if the person learned about that statistic". A review of PEPI says "Will you need all the programs in PEPI? Probably not. We have, for example, never used the Jonckheere-Tepstra test for trend or the Kullback-Leibler distances. However, more is good..." [ 6 ]. If a user wishes only to compute kappa , it can do no harm if the output provides extra results that draw attention to the fact that kappa has a ceiling value, or that its value can be adjusted to avoid paradoxical results. The user may be stimulated to use some of the additional procedures, after (if necessary) learning more about them. The manuals carry the warning: "It is unwise to use a statistical procedure whose use one does not understand. This manual cannot supply this knowledge, and it is certainly no substitute for the basic understanding of statistics and epidemiological thinking that is essential for the wise choice of methods and the correct interpretation of their results". The provision of alternative tests, and estimators based on alternative methods, may of course be confusing, whatever explanatory comments may be offered in the output or the manuals. But it may permit a knowledgeable user to select the method most appropriate in a particular situation, and it serves as a reminder to the less knowledgeable user that different methods exist, based on different assumptions and using different models, most of them yielding approximations, and none of them having absolute validity for all purposes, and as a warning that caution is indicated if different methods lead to very different conclusions. "Exact" results computed in different ways differ, and "exact" probabilities and confidence intervals are not always preferable to probabilities and confidence intervals computed in other ways. The length of the list in the Pepi Finder testifies to the wide variety of statistical routines offered. "The programs cover an amazing array of applications", says one review [ 6 ]. PEPI has repeatedly been called a "Swiss army knife" of utilities for epidemiologists and biomedical researchers [ 6 , 12 , 13 ]. One reviewer added, "one will find here more analytic options for a simple 2 × 2 or 2 × K table than will probably be needed during an entire epidemiology career" [ 13 ]. Another compared several packages when estimating sample size for a matched case-control study, and "found that PEPI provides an output richer than others do. This feature is common to other programs in PEPI" [ 14 ]. PEPI is of course very far from being a complete compendium of statistical routines for epidemiologists. It does not, for example, provide Cox regression, log-linear analysis, multiple regression analysis, procedures for the study of disease clustering, and many other procedures of interest to epidemiologists [ 7 , 11 ], which must be sought elsewhere. But it is a handy resource for many routines that are not very commonly used or very easily found, such as those concerned with misclassification, meta-analysis, reliability studies, the appraisal of screening and diagnostic tests, the equivalence of two proportions or means, cluster samples, inverse sampling, capture-recapture studies, serial correlation of residuals, skewed funnel plots, direct standardization using age intervals as weights, smoothing of curves, generalized odds ratios for ordinal data, quantitative measures of heterogeneity, harmonic analysis in the study of seasonality, and bias-adjusted and prevalence-adjusted bias-adjusted estimates of kappa (a feature picked out as "unique" in one comparison of PEPI with other epidemiological software [ 11 ]). From the viewpoint of veterinary epidemiologists, a shortcoming of WINPEPI is its use of "person-time" and not "animal-time". But they are doubtless used to this. WINPEPI's potential as a learning and teaching aid is worth stressing. Students welcome the facts that the package is free and requires no special installation procedure, and that (unlike major general-purpose statistical packages) it uses epidemiological language and provides results that are meaningful to epidemiologists. They rapidly learn to use the Pepi Finder and the programs themselves. They find the programs easy to use, although they may at first be confused by the multiplicity of modules and results; but they rapidly learn to focus on the specific modules and results that interest them, and to disregard others. At the same time, the rich output may serve to acquaint the student with other measures and tests, and excite interest in them. The weight the programs give to measures of association and their confidence intervals may help to counteract the belief that significance testing is the be-all and end-all of an analysis. "PEPI facilitates a ready understanding of important epidemiologic concepts, unfettered by the complexities of statistical programming", says a reviewer [ 6 ]. With appropriate data, for example, the Mantel-Haenszel results provided by COMPARE2's module B can serve as an object lesson on the assessment of confounding and effect modification, the control of confounding, and appraisal of the defensibility of a summary odds or risk ratio. The student can concentrate on analysis and interpretation, with no need to get involved in data management, sorting and tabulation. A useful feature is that, by clicking on the "Repeat" button and making changes to the data or options, students can easily do "what if?" exercises [ 15 ]. For example, they can easily learn, by manipulating data, how differences in prevalence or the number of controls per case can alter the required sample size, or how consideration of cost can alter sample size decisions in stratified sampling, or how the sensitivity or specificity of measures can alter a prevalence estimate or an odds or risk ratio. The sensitivity analysis provided by a module in COMPARE2 can demonstrate how markedly a single aberrant result can affect the results of a meta-analysis. Using the "misclassification" modules, it may be a salutary experience for students – and possibly also for some more experienced epidemiologists – to learn that an observed prevalence of 120 per 1000, using a measure whose sensitivity and specificity are 90%, points to a true prevalence of only 25 per 1000, or to find how inaccurate their guesses about the effect of misclassification on an odds or risk ratio can be. A recent epidemiology textbook makes frequent use of PEPI in its exercises "to relieve students from some of the tedium and anxiety of hand calculation, while opening up possibilities of using advanced techniques that might not otherwise be available. It is time to familiarize even introductory students to these essential tools of the trade" [ 16 ]. Conclusions WINPEPI complements other statistics packages. It is versatile, providing a wide variety of statistical routines commonly used by epidemiologists, but is far from being a complete compendium of such routines. It is a handy source of many procedures that are not very commonly or easily found. The programs are in general user-friendly, although some users may be confused by the large numbers of options and results provided. The main limitation is the inability to read data files, but tabular data can be entered by pasting, and for some purposes keyboard entry of data is an advantage. Only one of the programs presents graphic results. WINPEPI has a considerable potential as a learning and teaching aid. Availability and requirements The current version (at the time of this writing) of the software is available for free download as an additional file (WINPEPI.ZIP) attached to this article. It includes the programs, their manuals, and the Pepi Finder. Subsequent versions will be available at http://www.brixtonhealth.com for free download. Information about the latest WINPEPI version can be found at http://www.sagebrushpress.com/pepibook.html, where the DOS-based programs are available for free download. The programs and manuals are copyrighted, but may be freely copied and distributed for personal use; they may not be exploited commercially without permission. COMPARE2, PAIRSetc, and WHATIS are 16-bit programs (written in Delphi version 1) that can be run in any version of Windows. DESCRIBE is a 32-bit program (written in Delphi version 5), and can be run in any version of Windows except Windows 3. The manuals for DESCRIBE, COMPARE2, and PAIRSetc are in PDF format, and can be read or printed with Adobe Acrobat. WHATIS is documented in the version 4 manual [ 5 ]. Competing interests The author wrote the WINPEPI programs and manuals and is co-author of the DOS-based programs and manual, and hence may be biased in their favour. Supplementary Material Additional File 1 WINPEPI package. WINPEPI programs, with manuals and Pepi Finder. Click here for file

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555571

Influence of antioxidant (L- ascorbic acid) on tolbutamide induced hypoglycaemia/antihyperglycaemia in normal and diabetic rats

Background Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. Increased oxidative stress and decreased antioxidant levels are the leading cause of diabetes and diabetic complications. So it is felt that supplementation of antioxidants may be useful in controlling the glucose levels and to postpone the occurrence of diabetic complications. The objective of our study is to find the influence of antioxidant supplementation (L-ascorbic acid) on tolbutamide activity in normal and diabetic rats. Methods L- ascorbic acid/tolbutamide/L-ascorbic acid + tolbutamide were administered orally to 3 different groups of albino rats of either sex in normal and diabetic condition. Blood samples were collected from retro-orbital puncture at different time intervals and were analyzed for blood glucose by GOD-POD method. Diabetes was induced by alloxan 100 mg/kg body weight administered by I.P route. Results L-ascorbic acid/ tolbutamide produced hypoglycaemic activity in a dose dependant manner in normal and diabetic condition. In the presence of L-ascorbic acid, tolbuatmide produced early onset of action and maintained for longer period compared to tolbutamide matching control. Conclusion Supplementation of antioxidants like L-ascorbic acid was found to improve tolbutamide response in normal and diabetic rats.

Background Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. It requires life long treatment with drugs coupled with diet control and exercise. It may be due to decrease in the synthesis of insulin (Type-I diabetes) or due to decrease in the secretion of insulin from β-cells of islets of Langerhans of pancreas (Type-II diabetes). Insulin is the drug of choice in type – I diabetes and sulfonylureas are the drugs of choice in type II. Among sulfonylureas, tolbutamide is the drug of choice for geriatrics because of its short duration of action and lower incidence of hypoglycaemia in early hours of night. Diabetes is one of the stress related disorder. Diabetic subjects are shown to have increased oxidative stress and decreased antioxidant levels [ 1 - 3 ]. It was also shown that tight control of blood glucose is possible with decrease in oxidative stress [ 4 ]. Antioxidants are claimed to work as antistress agents by decreasing oxidative stress. L-ascorbic acid used in therapy for disorders like scurvy produces antioxidant activity. Earlier reports show that the relationship between scurvy and diabetes mellitus indicates the low levels of plasma ascorbic acid in diabetic rats compared to control rats [ 5 , 6 ]. Hence the present study was conducted to find the influence of L-ascorbic acid, a water soluble antioxidant and a free radical scavenger on the hypoglycaemic and antihyperglycaemic activities of tolbutamide in normal and diabetic rats. Methods All animal experiments were performed in accordance with our institutional animal ethics committee. Albino rats of either sex (Mahaveer Enterprises, Hyderabad) weighing between 125 – 175 g were used in the study. They were housed five per cage at temperature 22 ± 2°C and 12/12 h light/dark under controlled environment. Rats were fed with standard pellet diet. (Mahaveer Enterprises, Hyderabad) and water ad libitum . They were divided into 3 groups of five each. They were fasted for 18 h prior to the experiment allowing access to water only, and the water was withdrawn during the experiment. Blood samples were collected from the retro-orbital plexus of each rat at 0, 0.5, 1, 1.5, 2, 4, 6 hr after drug administration. Blood glucose levels were determined by using GOD – POD method [ 7 ]. Group I received L-ascorbic acid 60 mg / kg body weight, Group II received tolbutamide 20 mg/kg body weight and Group III was given L-ascorbic acid (60 mg / kg body weight) prior to the administration of tolbutamide 20 mg/kg body weight in normal rats. In clinical practice tolbutamide and vitamin C are administered orally hence in our study also this was administered orally. Induction of diabetes Albino rats of either sex weighing between 125 to 175 g were fasted overnight before injection with alloxan. Alloxan monohydrate was dissolved in saline solution and was administered at a dose of 100 mg/kg body weight intraperitonially. Animals were treated with 10% dextrose orally to combat the early phase of hypoglycaemia. Rats showing fasting blood glucose levels above 150 mg/dl were selected for the study. They were divided into 3 groups of five each. Group I received L-ascorbic acid 40 mg/kg body weight and Group II received tolbutamide 20 mg/kg body weight while Group III was given L-ascorbic acid 40 mg/kg prior to tolbutamide administration (20 mg/kg). L-ascorbic acid dose was fixed based on its response, which produced above 40%. Statistical analysis The significance of blood glucose reduction produced by L-ascorbic acid with tolbutamide compared to tolbutamide control was determined by applying students unpaired t-test and the significance is indicated by * mark. Results The presence of L-ascorbic acid upto 20 μg did not interfere with the blood glucose estimation when tested with different quantities in in vitro studies. In normal rats L-ascorbic acid at the dose of 60 mg/kg body weight administered orally produced 50.91% blood glucose reduction at 0.5 h and 20 mg/kg body weight of tolbutamide produced 33% at 4 h as peak effects. In the presence of L-ascorbic acid (60 mg/kg), the action of tolbutamide was early in onset and maintained for 6 h. In diabetic rats, oral administration of L-ascorbic acid alone at the dose of 40 mg/kg body weight produced 42.53% blood glucose reduction at 1.5 h and tolbutamide 20 mg/kg body weight produced 45.09 at 4 h. Administration of L-ascorbic acid 40 mg/kg body weight prior to tolbutamide produced antidiabetic activity at 0.5 h and was maintained for 6 h. The percent blood glucose reduction with L-ascorbic acid / tolbutamide/ L-ascorbic acid + tolbutamide in normal rats and diabetic rats were given in table 1 & 2 . Table 1 Percent blood glucose reduction (Mean ± SEM) with L-ascorbic acid/ tolbutamide / L-ascorbic acid + tolbutamide in normal rats (n= 5) Time in hours L-ascorbic acid 60 mg/kg bd.wt. Tolbutamide 20 mg/kg bd.wt. L-ascorbic acid 60 mg/kg bd.wt. + Tolbutamide 20 mg/kg bd.wt. 0 - - - 0.5 50.91 ± 0.49 10.08 ± 1.08 53.57 ± 2.01*** 1 20.26 ± 3.14 15.56 ± 1.48 28.70 ± 1.75*** 1.5 6.06 ± 1.3 18.75 ± 2.1 23.25 ± 1.72 2 1.65 ± 0.93 22.22 ± 2.13 29.78 ± 2.57 4 -2.83 ± 0.4 33.0 ± 0.69 45.21 ± 2.79** 6 - 10.28 ± 1.02 12.54 ± 2.5 Significance *** P < 0.001, **P < 0.01 Table 2 Percent blood glucose reduction (Mean ± SEM) with L-ascorbic acid/ tolbutamide / L-ascorbic acid + tolbutamide in diabetic rats (n = 5) Time in hours L-ascorbic acid 40 mg/kg bd.wt. Tolbutamide 20 mg/kg bd.wt. L-ascorbic acid 40 mg/kg bd.wt. + Tolbutamide 20 mg/kg bd.wt. 0 - - - 0.5 18.23 ± 1.88 3.03 ± 0.8 23.55 ± 2.37*** 1 35.45 ± 3.26 7.83 ± 1.84 35.59 ± 4.48*** 1.5 42.53 ± 1.78 18.58 ± 2.49 57.49 ± 1.63** 2 34.04 ± 2.22 37.97 ± 6.40 59.74 ± 1.22* 4 20.02 ± 3.32 45.09 ± 4.95 62.55 ± 0.64** 6 - 21.6 ± 1.94 39.43 ± 2.15*** Significance *** P < 0.001, **P < 0.01, *P < 0.05 Discussion The drug interaction studies are usually conducted in animal models to find out the mechanism before they are conducted in humans. We have selected rat as animal model since it is one of the animal, which synthesize ascorbic acid and can be maintained easily in the laboratory conditions. Reactive oxygen species (ROS) are thought to be implicated in the pathogenesis of diabetes as well as other diseases[ 8 ]. Reactive oxygen species usually comprise radicals that have the ability to oxidize and damage DNA, proteins and carbohydrates. Hyperglycaemia appears to induce oxidative stress on cells and this can cause an increase in the production of free radicals[ 9 ]. Human antioxidant enzymes are mobilized during hyperglycaemia, but they cannot meet the continued demand due to increased oxidative stress[ 10 ]. This problem is either due to decreased intake of needed precursors or an inability to synthesise the antioxidant enzymes[ 11 ]. Antioxidant supplementation may provide the only means to reverse this process[ 12 ]. Use of typical antioxidants alone or in combination may retard or even prevent the normal progression of diabetic complications. It was reported that L-ascorbic acid levels were decreased in diabetic patients and rats[ 13 ]. So it is felt that L-ascorbic acid supplementation may help in the treatment of diabetes mellitus. In the present study L-ascorbic acid and tolbutamide reduced blood glucose levels in normal & diabetic rats in a dose dependent manner. L-ascorbic acid when administered alone produced an early onset of action 0.5 & 1.5 h in normal and diabetic rats respectively. This early onset may be due to increase in the insulin secretion which support earlier reports that L-ascorbic acid supplementation increase the plasma insulin concentration[ 14 ]. Tolbutamide when administered in therapeutic dose produced the maximum effect at 4 h and was maintained up to 6 h in both normal and diabetic rats. In the presence of L-ascorbic acid the onset of action of tolbutamide was early and maintained for longer duration compared to tolbutamide control. Tolbutamide acts by stimulating insulin secretion (pancreatic)[ 15 ] and also by increasing tissue uptake of glucose (extra pancreatic)[ 16 ]. The early onset of action was noticed to be due to L-ascorbic acid, which was maintained later due to tolbutamide activity since both are reported to have influence on insulin secretion [ 14 , 15 ]. Conclusion The study indicates that additive action of L-ascorbic acid on pharmacodynamic response of tolbutamide may be useful to improve the tolbutamide activity in insulin resistant cases and to postpone the occurrence of diabetic complications. However further work on human patients is required to confirm the observation in diabetic condition and usefulness of L-ascorbic acid as supplemental agent for improved control of blood glucose levels when administered along with sulfonylureas. Competing interests The author(s) declare that they have no competing interests. Authors' contributions SS Conceived of the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. EK Participated in the design and coordination and in the standardization of methods. RJ Carried out the study in normal rats. VA Carried out the study in diabetic rats. All authors read and approved the final manuscript. Figure 1 Percent Blood Glucose Reduction with L-ascorbic acid / tolbutamide / L-ascorbic acid + tolbutamide in normal rats (n = 5) Figure 2 Percent Blood Glucose Reduction with L-ascorbic acid / tolbutamide / L-ascorbic acid + tolbutamide in diabetic rats (n = 5) Pre-publication history The pre-publication history for this paper can be accessed here:

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544865

Psychological health of family caregivers of children admitted at birth to a NICU and healthy children: a population-based cross-sectional survey

Background There is little information in the research literature on how parents of children who spend time in a neonatal intensive care unit (NICU) adapt psychologically to the demands of caregiving beyond the initial hospitalization period. Our aim was to compare parents of NICU children with parents of healthy full-term children, looking specifically at the relationship between parental psychosocial health and child characteristics, as well as the relationship between important predictor variables and psychosocial health. Methods A cross-sectional survey was sent to parents as their child turned 3 1/2 years of age. The setting was the province of British Columbia, Canada. The sample included all babies admitted to tertiary level neonatal intensive care units (NICU) at birth over a 16-month period, and a consecutive sample of healthy babies. The main outcome was the SF-36 mental component summary (MCS) score. Predictor variables included caregiver gender; caregiver age; marital status; parental education; annual household income; child health status; child behavior; birth-related risk factors; caregiver strain; and family function. Results Psychosocial health of NICU parents did not differ from parents of healthy children. Child health status and behavior for NICU and healthy children were strongly related to MCS score in bivariate analysis. In the pooled multivariate model, parental age, low family function, high caregiver strain, and child's internalizing and externalizing behavioral symptoms were independently associated with lower psychosocial health. In addition, female gender was associated with lower psychosocial health in the NICU group, whereas lower education and child's problem with quality of life indicated lower psychosocial health in the healthy baby group. Conclusions Overall, parental gender, family functioning and caregiver strain played influential roles in parental psychosocial health.

Background Neonatal intensive care is associated with a range of long-term health problems such as cerebral palsy, mental retardation, deafness, blindness and milder but more common problems such as learning disabilities and behavioral problems [ 1 - 13 ]. Although these problems create challenges for the parent responsible for the day-to-day provision of care to their child at home, the impact of caregiving on the health of parents of children discharged from neonatal intensive care units (NICUs) remains an under-explored research topic. There is a literature that focuses on the early hospitalization period. These studies show that mothers of preterm infants experience more severe levels of psychological distress in the neonatal period than do mothers of healthy full-term infants [ 14 - 17 ]. In the few studies that compare the impact of caregiving on parents of children discharged from NICUs with parents of healthy full-term children, the addition of a preterm infant into the family has been shown to have negative repercussions for the family in some studies [ 18 - 21 ], but not in others [ 22 - 24 ]. In one of the few NICU studies where parental mental health was the primary outcome measure, mothers of high and low-risk very low birth weight infants were compared with parents of healthy full-term infants [ 17 ]. The authors report that early differences between the groups at one month and two years were no longer apparent by the age of three, although parenting stress remained high throughout. In the present study, we sent a questionnaire booklet to mothers of all children admitted to a level III neonatal intensive care unit in the province of British Columbia (Canada) over a 16 month period to collect data on a range of factors in order to examine both neonatal and caregiver outcomes. Our study differs from other NICU follow-up studies in that it is population-based, focuses on preschool aged children and examines the full spectrum of NICU graduates. The aims of this paper are two-fold: (1) to compare psychosocial health of parents of NICU children with parents of healthy full-term children, looking specifically at the relationship between parental psychosocial health and child characteristics (i.e., health status, behavior problems, and birth-related risk factors); and (2) to identify predictors of parental psychosocial health (i.e., socioeconomic and demographic variables, child characteristics, caregiver strain, and family function). Methods Sample Ethical approval was gained from the University of British Columbia and participating hospitals. Our NICU sample included 2221 surviving babies admitted for more than 24 hours to one of three level III NICUs in British Columbia (BC), Canada over a 16 month period (March 1996 to June 1997). These 3 hospitals (Children's and Women's Health Centre of BC, Royal Columbian Hospital, Victoria General Hospital) provided 100% of the tertiary care NICU beds in the province. The birth mothers' name and contact details were obtained from the health records department at two hospitals and manually extracted from ledgers of the third hospital. Our list of babies was matched with provincial mortality records to exclude any babies that had died after discharge from the NICU and thereby prevent questionnaires being sent to bereaved parents. A comparison group of 718 healthy singleton full-term babies was recruited from the two hospitals with a hospital-based primary care unit (i.e., Children's and Women's Health Centre of BC and the Royal Columbian Hospital). This sample included all babies delivered over an 11 month period (March 1996 and January 1997) by primary care physicians at these two clinics. Babies with a sibling in the NICU sample and babies subsequently admitted to an NICU for more than 24 hours were excluded. Contact details for the mother were obtained from the health records department at one hospital, and directly from the primary care unit at the other. We excluded from the sample 150 babies (123 NICU; 27 healthy children) who did not meet our inclusion criteria for the following reasons: parent did not speak English (n = 95); baby died (n = 34); mother died (n = 6); and not applicable (n = 1). In addition, we excluded cases where the questionnaire was completed on the wrong child (n = 7) and where a comparison baby was subsequently admitted to a NICU (n = 7). The overall response rate (after exclusions), was 55% (54.3% NICU, 56.9% healthy baby group). The response rate for located families (82.8% of the sample was located) was 67.4% (n = 1140) for the NICU group, and 66.4% (n = 393) for the comparison group. Five NICU respondents returned a signed consent form without a completed questionnaire and were dropped from the analysis. Seventy-five percent of parents provided permission for data linkage between the questionnaire data and CNN database. The NICU sample included 181 children that were part of a multiple birth group: 171 twins; and 10 triplets. Table 1 contains sample characteristics. Most questionnaires (98%) were completed by a biological parent, most often the mother (96%). The NICU sample was composed of 1.8% fewer biological parents; 2.6% more male respondents, and 11.9% more families who earned less than $50,000 per year. Table 1 Characteristics of study sample Group; no. (%) of subjects NICU N = 1135 Comparison N = 393 Biological parent 1 1091 (97.7) 389 (99.5) Female 1 1070 (95.4) 383 (98.0) Married/common-law 962 (85.9) 344 (87.8) Age of parent, years 19–29 195 (17.8) 61 (15.7) 30–39 704 (64.1) 265 (68.3) ≥ 40 199 (18.1) 62 (16.0) Education level University 373 (33.4) 146 (37.4) Trade/technical school or community college 494 (44.3) 176 (45.1) High school graduation 185 (16.6) 50 (12.8) No high school diploma 64 (5.7) 18 (4.6) Household income, $ 2 511 (48.1) 136 (3) <30,000 247 (23.3) 58 (15.5) 30 – 49,999 264 (24.9) 78 (20.8) 50 – 79,999 333 (31.4) 145 (38.7) 80 > 218 (20.5) 94 (25.1) Male children in the sample 633 (55.8) 198 (50.6) Age of child, years 3 years 784 (69.3) 253 (64.4) 4 years 328 (29.0) 134 (34.1) 5 years 19 (1.7) 6 (1.5) 1 p < .05 (chi-square, Fischer's exact test); 2 p = .0018 (chi-square) Materials Our main measure of outcome was the SF-36 mental component summary (MCS) score [ 25 , 26 ]. The SF-36 is a well validated generic measure of adult physical and psychosocial health related quality of life (HRQL), which is composed of 36 items that measure 8 health domains. The MCS is computed from the following four domains: mental health (5 items); vitality (4 items); social functioning (2 items); and role limitations due to emotional problems (3 items). It has a mean of 50 and standard deviation of 10 and represents the mean and standard deviation of the general population (USA). Child health status was measured using the Health Status Classification Preschool Version (HSCS-PS) [ 27 ]. This measure asks about twelve health status (HS) problems that we have grouped into the following 4 categories: neurosensory (i.e., seeing and hearing); motor development (i.e., getting around, using hands and fingers, taking care of self); learning (i.e., speaking, learning/remembering and thinking/solving problems); and quality of life (i.e., pain/discomfort, feelings, behavior and general health). Each attribute has 3 to 5 levels of severity ranging from normal function to severe functional limitations. For each category of health problems, we recoded the data into the following: no problem; a mild problem; or a moderate or severe problem. Child behavior was measured with the Child Behavior Checklist 1.5–5 (CBCL/1.5–5) [ 28 ]. This questionnaire measures internalizing, externalizing and total problems, and scales can be scored categorically to indicate normal, borderline or clinical range scores. Data for birth-related risk data were obtained from the Canadian Neonatal Network Study [ 29 ] for the NICU children whose parents provided written consent for data linkage. The following variables were examined: birthweight; gestational age; small for gestational age; multiple birth, apgar score less than 7 at 5 minutes; congenital anomalies; the presence of a major morbidity (i.e., a composite score for the presence of at least one of the following: chronic lung disease (at 36 weeks); severe intraventricular hemorrhage (≥ grade 3); nosocomial infection; necrotizing enterocolitis; retinopathy of prematurity (≥stage 3)); and neonatal illness severity score [ 30 ]. Caregiver strain was measured using the Parental Impact-Time (PTT) scale from the Infant Toddler Quality of Life Questionnaire [ 31 ]. This 7-item scale asks parents to indicate limitations in the amount of time in the past 4 weeks they had for their own personal needs due to problems with their child's health (e.g., physical, emotional, cognitive, behavior, temperament). Scores on these scales can range from 0 to 100, with lower scores indicating greater caregiver strain. Family function was measured using the Family Assessment Device (FAD) [ 32 ]. Scores for this 12-item questionnaire can range from 0 to 36, with higher scores indicative of greater family dysfunction. Procedure A questionnaire booklet, which included the questionnaires described above, was sent to the address of the birth mother as her child turned 3 1/2 years of age. A consent letter was included to obtain permission to link the questionnaire data with hospital birth records. The primary caregiver in our study was defined as the person who, to that point in the child's life, had spent the most amount of time with the child. This could include the mother or father or another parent (e.g., grandparent, foster parent, guardian). We asked the primary caregiver (referred to in this paper as parent) to complete the questionnaire booklet and consent form. Non-respondents were sent a reminder letter, additional copies of the questionnaire booklet and a phone call as necessary. If the telephone number was not in service or reassigned, or a questionnaire booklet was returned to us from the post office, we implemented a comprehensive search strategy that involved searching the Internet and contacting the mothers' primary care physician. Data analysis To address the first objective, we compared the psychosocial summary score for the SF-36 questionnaire for parents of NICU children and parents of healthy children using student's T -test. T -test, ANOVA and the equivalent nonparametric tests, and Spearman correlation were used to explore relationships between MCS score and various child characteristics, including health status, behavior and birth-related risk factors. For health status and child behavior, we computed an effect size (mean difference divided by standard deviation of the group with no problems (health status) or with scores in the normal range (behavior)), to look at the magnitude of the difference in MCS score between subgroups for the NICU and healthy baby samples, and used the Cohen's guidelines for interpretation (0.2 is small, 0.5 is medium, 0.8 is large) [ 33 ]. To address the second objective, multiple regression analysis was used to examine the independent effects of, and proportion of variance in MCS scores explained by our predictor variables. For the analysis we examined a pooled model and a model where we stratified by group membership (i.e., NICU vs. healthy baby sample) to separately examine the contribution of each predictor variable for the two samples. Variables with significant (p < .05) or borderline p-values in bivariate analysis were included in the model. Certain birth-related risk factors (i.e., birthweight, congenital anomalies, illness severity score, and gestational age) were entered into the model on the basis of clinical rather than statistical importance, however, no effects were found. Potential predictor variables include the following: caregiver's gender; caregiver's age (continuous); marital status (married or common-law versus other), caregiver's education (less than high school graduation vs. other); annual household income (< or > $30,000); child health status (i.e., neurosensory; motor development; learning; and quality of life problems); child behavior; caregiver strain (continuous); and family function (continuous). For child health status and behavior variables, no problem (health status) and scores in the normal range (behavior) were the reference categories, with mild and moderate/severe (health status) or borderline and clinical range scores (behavior) entered separately, or combined and entered as dichotomous variables. We computed effect sizes to interpret the significance of beta coefficients. Results Psychosocial health comparing NICU and healthy children The unadjusted mean MCS score for parents of NICU children did not differ from parents of healthy children (48.2 versus 48.8; p = .305). We also compared MCS scores after adjusting for the three sample characteristics that differed between the two groups (i.e., proportion of biological parents; gender of subject; and those with lower household income), and no differences were found in the outcome variable. Psychosocial health by child health status problem On the HSCS-PS, 55.2% of healthy children had no health problems in any area, compared with 39.8% of NICU children (p < .001 on Chi-square). Table 2 shows the joint distribution of health status problems across the four categories for the NICU and healthy sample. These results show that the NICU sample had a higher proportion of children with more health status problems, as well as a higher proportion with moderate/severe versus mild problems. Table 2 Distribution of children with health status problems across the 4 health status categories for NICU and healthy children HSCS problems by domains NICU (N = 1104) Comparison (N = 386) no problem N 438 215 % 39.67 55.7 1 mild problem N 309 111 % 27.99 28.76 2+ mild problems N 183 37 % 16.58 9.59 1 moderate/severe problem only N 40 7 % 3.62 1.81 1 moderate/severe problem + any mild N 69 15 % 6.25 3.89 2–3 moderate/severe problems N 60 0 % 5.43 0 4 moderate/severe problems in all domains N 5 1 % 0.45 0.26 p < 0.0001, chi-square For parents of NICU children, for all 4 health status categories, parental MCS scores decreased as severity of the child health problem increased (see Table 3 ). Effect sizes comparing parents of children with no health status problems with parents of children with a moderate or severe health status problem were all moderate to large indicating important differences in parental mental health according to Cohen's benchmarks. The results for parents of healthy children show similar trends, with mainly moderate to large effect sizes. Table 3 Parental mental health summary score, 95% confidence intervals, number of subjects, p-value and effect size for child health status category Sample Type of HS problem None Mild Moderate or Severe p-value* Effect size NICU children Neurosensory 48.4 (47.7, 49.0) n = 975 46.3 (41.7, 51.0) n = 33 41.9 (36.0, 47.8) n = 17 .023 .040 .63 Motor development 49.1 (48.5, 49.8) n = 789 45.8 (44.0, 47.6) n = 174 42.0 (38.8, 45.2) n = 63 <.001 <.001 .74 Learning/remembering 49.3 (48.6, 50.0) n = 623 47.5 (46.3, 48.8) n = 298 43.4 (40.9, 45.9) n = 109 <.001 <.001 .63 Quality of life 49.8 (49.0, 50.5) n = 659 46.3 (45.1, 47.4) n = 313 39.4 (35.7, 43.2) n = 59 <.001 <.001 1.11 Healthy children Neurosensory 48.9 (47.9, 49.8) n = 361 57.0 n = 1 31.1 n = 1 .120 .154 1.89 Motor development 49.2 (48.2, 50.3) n = 333 45.6 (42.6, 48.7) n = 31 37.9 (22.6, 53.2) n = 3 .018 .002 1.18 Learning/remembering 49.6 (48.5, 50.7) n = 266 46.9 (44.7, 49.1) n = 89 45.8 (39.1, 52.5) n = 14 .037 .025 .41 Quality of life 50.1 (49.0, 51.2) n = 271 45.6 (43.5, 47.8) n = 86 36.2 (23.8, 48.6) n = 8 <.001 <.001 1.58 * first based on Anova, second based on Kruskal-Wallis non-parametric test (in italics) Psychosocial health by child behavior problem Child behavior was strongly related to parental psychosocial health in both groups of parents (see Table 4 ). Parents whose child scored in the clinical range for internalizing and externalizing symptoms and the total problem score on the CBCL/1.5–5 had the lowest mean (i.e., poorest) MCS scores. The differences between this group and the group with children scoring in the normal range resulted in large effect sizes, indicative of clinically important differences in parental psychosocial health. Table 4 Mean score, p-value and effect size for SF-36 psychosocial summary score comparing CBCL/1.5–5 normal with borderline and clinical groups CBCL scale Normal Borderline Clinical p-value Effect size NICU sample Internalizing 49.5 (48.9,50.2) n = 841 42.5 (39.6,45.5) n = 67 40.5 (37.6,43.4) n = 78 <.001 .95 Externalizing 48.9 (48.3,49.6) n = 925 41.6 (38.0,45.2) n = 45 35.0 (29.6,40.2) n = 32 <.001 1.43 Total 49.3 (48.6,49.9) n = 831 43.3 (39.7,47.0) n = 34 35.3 (31.0, 39.5) n = 39 <.001 1.45 Healthy children Internalizing 49.6 (48.6,50.6) n = 324 40.7 (36.8,44.6) n = 20 40.1 (34.0,46.2) n = 16 <.001 1.03 Externalizing 49.3 (48.3,50.3) n = 342 43.4 (37.0,49.8) n = 14 34.1 (22.5,45.8) n = 8 <.001 1.67 Total 49.4 (48.4,50.4) n = 330 41.5 (34.0,49.0) n = 12 36 (27.0, 44.0) n = 11 <.001 1.46 p-value based on Anova, (non-parametric tests: all p-values < .001) Parental psychosocial health by birth-related risk factors Within the NICU sample, MCS score did not vary by any birth-related risk factor (i.e., gestational age; small for gestational age; apgar score; multiple birth; the presence of a major morbidity; and neonatal illness severity score), with the exception of the presence of a congenital anomaly. For this variable, MCS scores were significantly lower in parents of children with versus without a congenital anomaly (mean difference = -3.8; p = .017; effect size = -.37). Children with a congenital anomaly (n = 87) had proportionally more mild and moderate/severe health status problems in all 4 categories (see Table 5 ). Table 5 Number (%) of NICU children with and without a congenital anomaly to report a problem for each health status category and p-value for Chi-square test of significance Type of HS problem Congenital anomaly None Mild Moderate or Severe p-value Neurosensory No 715 (95.6) 25 (3.3) 8 (1.1) <.001 Yes 70 (83.3) 8 (9.5) 6 (7.1) Motor development No 584 (78.3) 126 (16.9) 36 (4.8) <.001 Yes 48 (57.1) 17 (20.2) 19 (22.6) Learning/remembering No 457 (60.9) 222 (29.6) 71 (9.5) <.001 Yes 36 (42.9) 30 (35.7) 18 (21.4) Quality of life No 481 (64.2) 233 (31.1) 35 (4.7) <.001 Yes 38 (44.2) 34 (39.5) 14 (16.3) Correlates of psychosocial health in general In general, variables significantly associated with the MSC score in bivariate analysis were as follows: any health status problems (mean difference = -3.8; p < .001); neurosensory problems (mean difference = -3.7; p = 0.04); motor development problems (mean difference = -4.4; p < .001); learning/remembering problems (mean difference = -2.9; p < .001); poorer quality of life (mean difference = -4.8; p < .001); more internalizing behaviour symptoms (mean difference = -8.3; p < .001); more externalizing behavior symptoms (mean difference = -9.9; p < .001); household income below $30,000 per year (mean difference = -2.6; p < .001); female gender (mean difference = -2.6; p < .001);not living as common-law or married (mean difference = -3; p = .03); more caregiver strain (r = .41; p < .001); and lower family function (r = -.44; p < .001). Borderline significance was also found for less than high school education (mean difference = -2; p = .08). We examined a pooled model (both groups together) for a direct comparison of the NICU and healthy groups after adjustment for other variables. Due to the low number of male respondents in the healthy group, we restricted the pooled multivariable analysis to only female respondents. Predictors significantly associated with the outcome were the following: parental age (Beta = 0.15; p = 0.001); internalizing behavior (Beta = -2.06; p = 0.017); externalizing behavior (Beta = -3.24; p = 0.004); parental strain (Beta = 0.15; p < 0.001); and family function (Beta = -0.53; p < 0.001). The pooled model also showed an interaction effect between NICU admission and education (less than high school) (Beta-education = -5.94 with p = 0.009; Beta-interaction = 7.28 with p = 0.005)(see Table 6 .) For the NICU group, education did not show any effect in terms of difference in outcome, but for the healthy group, lower education was associated with a significantly lower mean MCS score. More specifically, for respondents with less than high school education, the healthy group reported lower MCS scores than did the NICU group. The results were not affected by exclusion of multiple births and cases of congenital anomalies from the analysis. Table 6 Beta coefficients, 95% confidence intervals, standardized beta coefficients and p-values for predictor variables in the multiple regression models for pooled model Pooled model Variable Beta CI-low CI-high St. beta p-value Intercept 34.21 30.26 38.17 <.0001 NICU -0.38 -1.32 0.55 -0.02 0.500 Parental age 0.15 0.08 0.23 0.08 0.001 Education -5.94 -9.67 -2.21 -0.13 0.009 Internalizing behavior -2.06 -3.47 -0.65 -0.07 0.017 Externalizing behavior -3.24 -5.08 -1.40 -0.08 0.004 Caregiver strain 0.15 0.13 0.18 0.26 <.0001 Family function -0.53 -0.60 -0.46 -0.32 <.0001 NICU-education interaction 7.28 3.01 11.56 0.14 0.005 Although other interaction terms with NICU status did not add any more significant results in the pooled model (non-significant partial F-test), we examined separate models for the NICU and the healthy baby group to further explore the association between gender and MCS score, and to evaluate the potential influence of congenital anomalies in NICU group. Correlates of psychosocial health for NICU sample Variables that were significantly associated with lower MCS scores at the bivariate level include the following: female caregivers (mean difference = -3.2; p = .037); household income below $30,000 per year (mean difference = -3.3 and p < .001); not living as common-law or married (mean difference = -5.1; p < .001); neurosensory problems (mean difference = -6.44; p = .011); motor development problems (mean difference = -7.1; p < .001); learning/remembering problems (mean difference = -5.9; p < .001); poorer quality of life (mean difference = -10.4; p < .001); more internalizing behaviour symptoms (mean difference = -9.03; p < .001); more externalizing behavior symptoms (mean difference = -13.9; p < .001); the presence of a congenital anomaly (mean difference = -3.8; p = .017); more caregiver strain (r = .411; p < .001); and lower family function (r = -.441; p < .001). Predictors that were significant in the final regression model appear in Table 7 . Female gender was an independent risk factor for lower MCS score: females scored on average 5.3 points (CI interval 2.5 to 8.0) lower, which represents a moderate effect size of 0.51 (when overall NICU parents group standard deviation (SD) 10.4 for MCS was used as the denominator). Scoring outside the normal range for internalizing and externalizing child behavior symptoms independently contributed to lower MCS scores (-1.9 and -2.8, both with wide confidence intervals), with the change representing small effect sizes of 0.18 and 0.27. More caregiver strain (i.e., lower PTT) was related with poorer MCS scores. A one point change in PTT corresponded to a 0.15 (CI: 0.11–0.19) change in MCS score. In NICU parents, the mean PTT was 86.9 and SD was 18.5. Therefore, 2 SD on the PTT would represent 5.5 points on the MCS, or an effect size of 0.53. The mean score for family function (FAD) was 8.1 and the SD was 6.4. A one point change in FAD corresponded to a 0.5 (CI: 0.62; 0.42) change in MCS. Therefore a 2 SD increase in family function score (i.e., poorer family functioning) would result in a 6.4 decrease (worsening) in MCS, representing a moderate effect size of .62. Overall, the adjusted R2 was .2884 (F = 73.96; df = 5; p = < .0001), with 5 out of 15 predictors included in the full model. Table 7 Beta coefficients, 95% confidence intervals, standardized beta coefficients and p-values for predictor variables in the multiple regression models for both samples NICU sample Healthy baby sample Variable Beta CI-low CI-high St. beta p-value Beta CI-low CI-high St. beta p-value Intercept 44.9 40.3 49.5 <.001 35.2 26.3 44.1 <.001 Parental age 0.3 0.1 0.5 0.1 .005 Female gender -5.3 -2.6 -8.0 -0.11 <.001 Education -5.00 -0.8 -9.1 -0.1 .019 Internalizing behavior -1.9 -0.1 -3.8 -0.06 .043 -4.0 -0.8 -7.2 -0.1 .014 Externalizing behavior -2.8 -0.4 -5.3 -0.07 .025 Caregiver strain 0.2 0.1 0.2 0.26 <.001 0.1 0.04 0.2 0.2 .003 family function -0.5 -0.4 -0.6 -0.32 <.001 -0.6 -0.4 -0.8 -0.4 <.001 Quality of life -6.9 -0.4 -13.4 -0.1 .039 Correlates of psychosocial health for healthy baby sample Variables that were significantly associated with poorer SF-36 MCS scores at the bivariate level include the following: younger parental age (r = .19; p < .001); household income below $30,000 per year (mean difference = -4.6; p = .005); less than high school education (mean difference = -6.22; p = 0.065); not living as common-law or married (mean difference = -6.1; p = .005); motor development problems (mean difference = -11.3; p = .043); learning/remembering problems (mean difference for any problems versus none = -2.68, p = 0.021); poorer quality of life (mean difference = -13.9; p < .032); more internalizing behavior symptoms (mean difference = -9.5; p < .001); more externalizing behavior symptoms (mean difference = -15.2; p < .018); more caregiver strain (r = .385; p < .001); and lower family function (r = -.438; p < .001). Predictors that were significant in the final regression model appear in Table 7 . The model for parents of healthy children did not include female gender (because of low numbers) and externalizing behavior symptoms, and included several variables not predictive in the NICU model (i.e., parental age; education; quality of life). Both models included internalizing child behaviors, caregiver strain and family function. In the healthy baby sample, younger parental age was related to poorer MCS score, with a one year change in age resulting in a 0.26 (CI: 0.08; 0.45) change in MCS. A ten year difference in age would correspond to a 2.6 difference in MCS, which would represent a small effect size of 0.27 (when the overall healthy baby parent group SD for MCS (9.6) was used as a denominator). Education was also associated with MCS. Compared with high school graduates, the MCS score for parents with less than a high school education were on average 5.0 lower (CI: 0.84; 9.1), which represents a moderate effect size of 0.52, although the effect could range from minimal to large due to lower precision of the beta estimate. Child internalizing symptoms, family function and caregiver strain were associated with parental MCS in a similar way as for NICU parents. However, due to lower numbers and resulting low precision in beta estimates, the effects ranged from minimal to large. Lower parent-reported child quality of life was also associated with a lower parental MCS. Parents who reported a problem with their child's quality of life had MCS scores that were 6.9 (CI: 0.37; 13.4) lower than parents who reported at least one quality of life problem compared with those who reported at least one problem. Again, due to the small numbers, the effect could range from minimal to large. In the final regression model, the adjusted R2 was .3046 (F = 25.97; df = 6; p < .0001), with 6 out of 16 predictors included in the full model. Discussion There is little information in the research literature on how parents of NICU children adapt psychologically to the demands of caregiving beyond the initial hospitalization period. We compared the psychosocial health of parents of NICU children with parents of a group of healthy full-term children using the SF-36, a popular generic measure of psychosocial HRQL. Although children admitted to a NICU at birth are at increased risk of a variety of long-term health problems, we did not find any difference in parental psychosocial health when the two groups were compared. This finding is in agreement with one of the few studies that measured mental health in parents of NICU children at preschool age. Singer et al. [ 17 ] reported that after the neonatal period, the mental health of mothers of low-risk infants did not differ from mothers of term infants, and by 3 years, they had lower levels of distress, which they suggest may be due to maternal relief after an initial period of fear and anxiety. Mothers of high-risk infants, in contrast, had more symptoms of distress at 2 years, more negative family impact at 2 and 3 years and more parental strains and illness stressors at 3 years. But by 3 years, their reported psychological distress did not differ from that of term mothers. The authors suggest that by 2 years, infant developmental scores are predictive of later outcomes, and many mothers of high-risk infants must relinquish their hopes for their children to "catch up" to healthy born children and that some psychological adaptation has taken place despite parental acknowledgment of greater family and parenting stressors. With our cross-sectional design, we are not able to confirm the trend noted by Singer, but given the lack of relationship between most birth-related risk factors and parental mental health, it is possible that the parents of high- and low-risk infants in our sample have adjusted over time. Current health status, in bivariate analysis, was strongly related with parental psychosocial health. In both groups of parents, those whose child had a neurosensory, motor development, learning/remembering or quality of life problem had poorer psychosocial health than those with children with no problems in these areas. Child behavior was also strongly related to parental psychosocial health. More specifically, parents of children who scored in the borderline or clinical range for internalizing, externalizing and total behavior problems on the CBCL/1.5–5 reported poorer psychosocial health than parents of children who scored in the normal range. These findings were consistent across both samples of parents. The only birth-related risk factor associated with parental psychosocial health was the presence of a congenital anomaly. Here the effect size was small, but points to the possibility that a congenital anomaly may affect parents mental health adversely. Researchers have reached a consensus that a minimally important difference in HRQL is close to one half of a standard deviation [ 34 ]. The differences that we found for health status and behavior were substantially larger and therefore represent clinically important differences in parental psychosocial health. However, not all of these variables showed a significant effect in the multivariate analysis, and it is possible that these variables influence other, more proximal, variables that showed stronger effects on parental psychosocial health. The factors associated with poorer psychosocial health in the multivariate models provide important information about correlates of adjustment for NICU and healthy baby families. In a more general pooled model, parental age, higher caregiver strain, lower family function, and child's internalizing and externalizing behavior were independently associated with poorer caregiver's mental health score. The effect of lower parental education was modified by NICU status of the child. In the healthy baby group, less than high school education indicated lower MCS score. Child externalizing behavior symptoms and female gender (parental) were associated with lower MCS scores in the NICU group, whereas lower parental age, less education and poorer child quality of life were associated with lower MCS in the healthy baby group. For both samples, as it is also seen in a pooled model results, low family function, high caregiver strain, and child's internalizing behavioral symptoms were independently associated with lower parental psychosocial health. For family function and caregiver strain, only a substantial departure from mean values (at least 2 SDs) would result in a clinically important moderate effect size for the NICU group. Our interpretation for the healthy baby sample is hampered by wide confidence intervals around the beta estimates, resulting in effect sizes that ranged from minimal to large. Internalizing behavior symptoms were associated with only a small effect on caregiver's MCS score, again with wide confidence intervals around the beta coefficients for both samples. A recent publication outlines the integration of a number of theoretical models into one multidimensional model that can be used to describe the caregiving process [ 35 ]. This model includes the following constructs: background and context; child characteristics; caregiver strain; intrapsychic factors; coping/supportive factors; and health outcomes. Fitting our findings within this framework, we found that poorer psychosocial health in parents was associated with background/context variables (i.e., female gender, younger age, less education); child characteristics (i.e., poorer quality of life, more child behavior problems); caregiver strain; and coping/supportive factors (i.e., family function). We suggest that future research with NICU parents be conceptually based and measure constructs found in other research to be important to caregiver health. Our study has several limitations. Because it is not possible to verify cause-effect using a cross-sectional design, we were only able to estimate the direct effect of a limited number of predictor variables on parental psychosocial health. While our study has helped to identify some possibly important caregiving variables, there are other variables important to caregiver health that we did not measure. For example, while it is possible that some parents of children with severe health problems may have received specialized or targeted services (health and/or social services) to help them cope with their child's health problems, we did not include measures to determine this. Another limitation concerns our response rate. Although it is within the range often obtained in a postal survey [ 36 ], non-response can introduce bias. Some non-respondents indicated (verbally or in writing) they were "too busy" to participate. It is also likely that some questionnaires returned to us blank were from non-English speakers. Where we had data and were able to explore response bias (NICU sample only), only a few differences in birth-related sample characteristics and outcome were found that suggests respondents had sicker babies [ 37 ]. However, our study findings about health outcomes of NICU graduates are in agreement with the larger NICU literature, so it is unlikely that the differences we found are entirely due to response bias. Conclusion Our findings would suggest that overall, parental gender, family functioning and caregiver strain played influential roles in parental psychosocial health. For child characteristics, current behavior was more influential than initial birth-related risk factors. List of abbreviations MCS – Mental Component Score NICU – Neonatal intensive care unit HS – Health status FAD – Family Assessment Device PTT – Parental Impact Time Competing interests The author(s) declare that they have no competing interests. Contributions of each author Anne Klassen contributed substantially to the study's conception and design, acquisition of data, analysis and interpretation of data; and she drafted and revised and gave final approval of the version to be published. Shoo Lee contributed substantially to the study's conception and design, acquisition of data, analysis and interpretation of data; and revised the article critically for important intellectual content and gave final approval of the version to be published. Parminder Raina contributed substantially to the analysis and interpretation of data; and revised the article critically for important intellectual content and gave final approval of the version to be published. Sarka Lisonkova contributed substantially to the analysis and interpretation of data; and revised the article critically for important intellectual content and gave final approval of the version to be published. Pre-publication history The pre-publication history for this paper can be accessed here:

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Recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis – a multicenter trial

The development of antifilarial antibody responses is a characteristic feature of infection with filarial parasites. It should be possible to exploit this fact to develop tools to monitor the progress of the global program to eliminate lymphatic filariasis (LF); however, assays based on parasite extracts suffer from a number of limitations, including the paucity of parasite material, the difficulty of assay standardization and problems with assay specificity. In principle, assays based on recombinant filarial antigens should address these limitations and provide useful tools for diagnosis and surveillance of LF. The present multicenter study was designed to compare the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. Coded serum specimens were distributed to five participating laboratories where assays for each antigen were conducted in parallel. Assays based on Bm14, WbSXP, or BmR1 demonstrated good sensitivity (>90%) for field use and none of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections. Limitations of the assays are discussed. Well-designed field studies are now needed to assess sampling methodology and the application of antibody testing to the monitoring and surveillance of LF elimination programs.

Background The exponential growth of the lymphatic filariasis elimination program has highlighted the need for tools that can be used to monitor progress toward programmatic endpoints (e.g. when to stop mass treatment) as well as to conduct surveillance to detect any potential resumption of transmission. Measurement of microfilaremia, the recognized gold standard for demonstrating the impact of community-wide interventions, is not an optimal monitoring or surveillance tool because of the requirement for nocturnal blood collection in much of the world and because it is a relatively insensitive test for infection [ 1 ]. For Wuchereria bancrofti , assessment of antigenemia offers the convenience of daytime testing and greater sensitivity than testing for microfilaremia; however, both microfilaremia and antigenemia develop from months to years after exposure, reducing their utility for detection of low levels of infection or recrudescence of transmission [ 2 - 6 ]. Entomologic methods can be used to monitor filarial infection in mosquitoes and to provide point estimates of transmission intensity; however, as infection levels decline, it may become more difficult to collect sufficient numbers of mosquitoes to demonstrate with confidence that infection is absent [ 7 ]. By providing a cumulative measure of exposure to filarial infection, antibody assays may circumvent many of the limitations of methods based on direct detection of the parasite, its antigens or its DNA. Antibody detection has served as the basis for diagnostic assays for filariasis for many decades. The best of these assays are sensitive for infection but are not specific, both because they cannot distinguish current infection from past infection or exposure to the parasite and because there is some degree of cross-reactivity with other helminth infections. On the positive side, prior studies have shown that virtually all residents of filariasis-endemic areas mount antifilarial antibody responses within the first few years of life. Thus, prevalence rates of antifilarial antibodies in children may be a useful index for assessing changes in transmission of the infection [ 8 , 9 ]. Antibody assays based on crude filarial extracts are limited by cross-reactions with other nematode antigens [ 10 ]. Recombinant filarial antigens should, in principle, be more useful as the basis of diagnostic or exposure assays because of their greater specificity. As a first step in the development and validation of such assays, we conducted a multicenter evaluation of antibody-based diagnostic assays using 3 recombinant antigens, Bm14, WbSXP and BmR1. Bm14 and WbSXP belong to a family of related genes that encode proteins that are strong immunogens in many parasitic nematode infections [ 11 ]. SXP and Bm14 were originally isolated from cDNA libraries based on their strong recognition by antibodies from microfilaremic persons, and both have been developed as candidates for diagnostic assays [ 12 , 13 ]. Assays based on detection of IgG4 antibodies to Bm14 have sensitivities of 85–90% when serum specimens from microfilaremic persons are tested [ 14 , 15 ]. This antigen is reported to be equally sensitive for sera from patients infected with Brugia malayi or Wuchereria bancrofti . Comparable results have been reported with assays for SXP [ 11 , 16 , 17 ]. BmR1 encodes a secreted antigen selected from a B. malayi cDNA library by antibody screening [ 18 , 19 ]. ELISA and dipstick formats of BmR1 assays have been reported to have a sensitivity of >90% when serum specimens from persons with B. malayi or B. timori microfilaremia were tested [ 18 - 22 ]. The present study was designed to compare objectively the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. We report here the results of this multicenter evaluation. Materials and Methods Serum Specimens Serum specimens from patients of known infection status (see Table 1 ) were sent to the U.S. Centers for Disease Control and Prevention (CDC). For persons with filariasis, infection was diagnosed by detection of microfilariae. Each specimen was assigned a code number and aliquotted into 5 tubes (100 – 200 μl per tube). A panel of coded serum samples was sent to each of the five participating laboratories along with antigens and assay kits for testing. Results were sent back to CDC for data analysis. Table 1 Sensitivity and Specificity of Antibody Assays 1 Infection Bm14 ELISA Positive/Tested (%) SXP Cassette Positive/Tested (%) BmR1 ELISA Positive/Tested (%) BmR1 Dipstick Positive/Tested (%) W. bancrofti (n = 35) 2 32/35 (91%) 30/33 (2 NC) (91%) 14/31 (4 NC) (45%) 17/30 (5NC) (56.7%) B. malayi (n = 28) 3 27/28 (96%) 7/18 (10 NC) (39%) 28/28 (100%) 27/27 (1NC) (100%) O. volvulus (n = 20) 4 11/16 (4 NC) (69%) 9/15 (5 NC) (60%) 0/20 (0%) 1/20 (5%) Loa (n = 10) 5 7/9 (1 NC) (78%) 3/7 (3 NC) (43%) 0/10 (0%) 0/9 (1 NC) (0%) Other (incl. S trongyloides, Echinococcus ; n = 20) 6 0/19 (1 NC) (0%) 0/19 (1 NC) (0%) 0/20 (0%) 0/20 (0%) 1 Specimens were collected from persons with documented infections with the listed parasites; for patients with filarial infections, microfilariae were detected. Abbreviations: NC, no consensus; specimens with a no consensus result were not included in the denominators for calculations. 2 Geographic source of specimens provided in Table 2. 3 Geographic source of specimens provided in Table 2. 4 Ten specimens were from Guatemala and 10 were from Ecuador. 5 Specimens were collected from patients in Benin. 6 Echinococcus specimens were collected in Kenya; Strongyloides specimens were from several settings where lymphatic filariasis was not endemic. Assay formats The Bm14 and BmR1 tests included in the evaluation are based on the detection of antifilarial IgG4 antibodies. The Bm14 assays were performed according to procedures described by Weil et al. [ 15 ]. Three different assay formats were used to test for BmR1: an ELISA [ 18 ], a dipstick [ 20 ] and cassette. All were produced by Malaysian Bio-Diagnostics, Research, Sdn, Bhd. A rapid cassette test for IgG antibody to WbSXP was produced by Span Diagnostics, Ltd (Sachin, India). All participating labs followed assay instructions provided by the assay or kit supplier. Analysis of Results Results of ELISA assays were determined using cut offs defined by the assay developer. For qualitative tests, each laboratory determined whether the appropriate band or spot was visible. To collate results for a given assay with a specific serum sample, a consensus result (either positive or negative) was defined on the basis of agreement among at least 4 of 5 labs. If only 3 of 5 labs obtained the same result or if 3 did and one of the two remaining laboratories did not obtain an interpretable result, this was considered to represent a lack of consensus (recorded as 'NC' or no consensus in Tables 1 and 2 ). Only two labs used the BmR1 cassette to test the specimens. Achieving consensus required two identical results for this test. Inter-laboratory agreement was assessed with Kappa coefficients, a measure of pair-wise agreement among observers making categorical judgments. For Bm14 and BmR1 ELISA, categorical assignments of positive or negative results were based on criteria established by the test developers. Table 2 Regional Differences in Antigen Recognition Infection Location (serum source) Bm14 ELISA SXP Cassette BmR1 ELISA W. bancrofti Cook Is. 10/10 9/9 (1 NC) 4/8 (2 NC) PNG 9/10 9/9 (1 NC) 6/10 India 9/10 9/10 4/8 (2 NC) Kenya 2/3 1/3 0/3 Haiti 2/2 2/2 0/2 B. malayi Indonesia 10/10 0/5 (5 NC) 10/10 India 7/7 4/6 (1NC) 7/7 Malaysia 10/11 3/7 (4 NC) 11/11 Results The Bm14 ELISA displayed comparable sensitivity for both W. bancrofti (91%) and Brugia (96%) infections, while the other two tests performed better with specimens from the homologous infections (Table 1 ). For example, the BmR1 ELISA was positive for 100% of the samples from Brugia patients, but displayed only modest sensitivity (45%) in terms of its performance with W. bancrofti samples. Results were comparable for both the BmR1 dipstick and cassette (data not shown). Similarly, the WbSXP assay was positive for 30 of 33 (91%) serum specimens from W. bancrofti patients, but only 39% of Brugia cases. BmR1 assays were remarkably specific for Wuchereria and Brugia infections, and there was little reactivity with specimens from persons with O. volvulus , Loa loa or other helminths (e.g. Strongyloides ) . The Bm14 assay, and to a lesser extent, the WbSXP assay, appeared to function as a 'pan-filaria' assay, showing reactivity with the specimens from persons with W. bancrofti , B. malayi , L. loa and O. volvulus . None of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections (Table 1 ) or with hyper-IgE syndrome (data not shown). When the geographic source of the serum specimens was considered, additional heterogeneity in responsiveness was noted. For example, although only a limited number of specimens were available for testing, 4 of 6 serum specimens from persons from India infected with B. malayi were positive using the WbSXP cassette; however, none of those from Indonesia were positive with this assay (Table 2 ). Inter-laboratory categorical agreement for the ELISA assays was quite good (Table 3 ). Rapid format tests, though convenient, often presented problems of interpretation, independent of the test. Some labs reported the presence of weak bands or dots with control sera. This resulted in a significant number of 'no consensus' results (Table 1 ) as well as the lower kappa scores associated with the rapid tests (Table 3 ). Table 3 Inter-lab Agreement for the Different Diagnostic Tests Assay Range of Kappa statistics Mean Bm14 ELISA 0.690 – 1.00 0.88 SXP Cassette 0.612 – 0.912 0.73 BmR1 ELISA 0.878 – 0.982 0.93 BmR1 Dipstick 0.817 – 0.965 0.87 BmR1 Cassette 0.546 – 1.00 0.80 Kappa statistics were derived from 10 pair-wise inter-lab comparisons. Discussion Assays based on Bm14, WbSXP, or BmR1 demonstrate adequate sensitivity for field use. The Bm14 assay appeared to function as a 'pan-filaria' assay, demonstrating antibody reactivity in the sera from patients with W. bancrofti , B. malayi , L. loa and O. volvulus . Although this cross reactivity makes the Bm14 assays useful for monitoring either bancroftian or brugian filariasis, cross reactivity with O. volvulus and L. loa may limit its utility in some areas of sub-Saharan Africa. The BmR1 assays were sensitive for B. malayi infection but relatively insensitive for W. bancrofti infection. They showed excellent specificity for Brugia and Wuchereria , with little reactivity with sera from persons infected with other parasites, including L. loa and O. volvulus . These results suggest that it may be useful to study the W. bancrofti homologue of BmR1 to determine if it is as specific for W. bancrofti as BmR1 is for Brugia . Unfortunately, recent work suggests that this is not the case [ 23 ]. For mapping the distribution of lymphatic filariasis, rapid antibody tests may provide acceptable sensitivity, depending on the geographic area where the mapping is to be done, but the potential for problems with specificity (both in distinguishing past exposure from present infection as well as differentiating filarial from non-filarial infection) still remains. For mapping W. bancrofti , there is minimal value in using antibody tests instead of the antigen tests that are currently used, but because there is no antigen test for Brugia , antibody tests might be an alternative for mapping the distribution of these infections. In Brugia -endemic areas, it will be important to demonstrate, however, the relationship between prevalence rates for microfilaremia and antibodies to validate the assay as a useful tool for programmatic decisions. At this point, it is not clear what antibody prevalence should be considered an indication to initiate mass treatment; no attempt was made in this study to distinguish between antibody responses associated with active infection and those triggered by exposure [ 24 , 25 ]. Antibody assays almost certainly will find other uses in the context of lymphatic filariasis (LF) elimination programs. For example, antifilarial antibody tests may be sensitive markers of transmission intensity or provide evidence of ongoing exposure to filarial infection long before the development of antigenemia or microfilaremia. Primates develop antibody responses to Bm14 within 4–8 weeks following B. malayi infection [ 26 ]. In a longitudinal study in Egypt, microfilaria-negative persons who were positive for Bm14 antibody at baseline were more likely to be microfilaria-positive after one year than were Bm14-negative persons [ 15 ]. Less is known about the kinetics of antibody responses to BmR1. Since antibody responses provide an early indicator of infection, assays for antifilarial antibodies should be useful for surveillance following initiation of LF elimination programs. As LF programs reach their planned end point (5 or more years of > 80% drug coverage in targeted populations), it will be necessary to determine whether or not transmission has been interrupted and whether mass drug administration can be stopped. Parasitologic testing, whether for microfilaremia or antigenemia, will require testing of thousands of persons to demonstrate that infection levels are below 0.1%, the level established by the Global Program as the end point for defining the elimination of LF. Since antibody responses develop in the absence of demonstrable infection, detecting incident antibody responses should provide a more sensitive measure of transmission than microfilaria or antigen detection. Children born following the cessation of transmission should be antibody-negative, while older children and adults may have evidence of residual antibody reactivity [ 8 , 9 , 27 ]. A testing strategy based on screening of children could be exploited for ongoing surveillance in the aftermath of LF programs and may not require screening of as many children as called for by current testing guidelines. The absence of antibody in appropriately chosen populations would strongly suggest that transmission has been interrupted. Additional studies are needed to test the value of antibody testing as a tool for certifying the elimination of filariasis transmission. Operational use of antibody assays requires far more practical experience with the assays than we now have. Of greatest concern is the specificity of the tests employed. For example, ELISA tests often use a statistical approach to establish cutoff values for positive results. A test that is 99% specific will predictably have some false positive results if large numbers of samples are tested. Further work will be needed to establish rates of antibody positivity that exceed the number that are likely to occur by chance. In addition, it will be necessary to develop and validate algorithms for confirming the presence of infection or ongoing transmission in situations where low antibody prevalence rates are detected. Despite these caveats, we believe that antibody tests based on antigens like Bm14, BmR1, and Wb-SXP will prove to be useful tools that can be used to facilitate decision making by program managers in the context of filariasis elimination programs. Competing Interests GW, RN, and PK have relationships with companies interested in developing commercial applications of the Bm14, BmR1 and WbSXP assays, respectively. Authors' Contributions GW, RN, PK, and VBL were responsible for the initial development of the assays. All of the authors participated in the planning of this multicenter study. DG was responsible for coordinating specimen shipment and database management. Participating labs included PL and DG from CDC, RN from the Universiti Sains Malaysia, PK and VBL from Anna Center for Biotechnology, CS from NIH and GW from Washington University School of Medicine. PL and EO were responsible for coordinating the study. PL wrote the first draft of the manuscript, but all of the authors participated in the editing of subsequent versions.

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555940

Heterologous expression of the filarial nematode alt gene products reveals their potential to inhibit immune function

Background Parasites exploit sophisticated strategies to evade host immunity that require both adaptation of existing genes and evolution of new gene families. We have addressed this question by testing the immunological function of novel genes from helminth parasites, in which conventional transgenesis is not yet possible. We investigated two such novel genes from Brugia malayi termed abundant larval transcript (alt) , expression of which reaches ~5% of total transcript at the time parasites enter the human host. Results To test the hypothesis that ALT proteins modulate host immunity, we adopted an alternative transfection strategy to express these products in the protozoan parasite Leishmania mexicana . We then followed the course of infection in vitro in macrophages and in vivo in mice. Expression of ALT proteins, but not a truncated mutant, conferred greater infectivity of macrophages in vitro , reaching 3-fold higher parasite densities. alt-transfected parasites also caused accelerated disease in vivo , and fewer mice were able to clear infection of organisms expressing ALT. alt -transfected parasites were more resistant to IFN-γ-induced killing by macrophages. Expression profiling of macrophages infected with transgenic L. mexicana revealed consistently higher levels of GATA-3 and SOCS-1 transcripts, both associated with the Th2-type response observed in in vivo filarial infection. Conclusion Leishmania transfection is a tractable and informative approach to determining immunological functions of single genes from heterologous organisms. In the case of the filarial ALT proteins, our data suggest that they may participate in the Th2 bias observed in the response to parasite infection by modulating cytokine-induced signalling within immune system cells.

Background Pathogens have evolved many ingenious mechanisms to manipulate innate and adaptive host immune responses [ 1 - 6 ]. The nematode parasite Brugia malayi is a causative agent of the disease lymphatic filariasis, which afflicts over 100 million people in tropical countries. Mosquito-borne infective stage larvae gain entry to the human body during a blood meal, and establish long-lived infections characterised by down-regulation of host T cell and macrophage reactivity [ 7 , 8 ]. We have studied the profile of genes expressed in infective larvae and reported that ~5% of the mRNA transcripts from this stage correspond to two closely related genes, which we have named abundant larval transcript (alt) -1 and -2 [ 9 , 10 ]. The two genes encode proteins with 79% amino acid identity, but no similarity to any gene of known function. alt -like genes are present in other filarial nematode species [ 11 , 12 ] and are characterised by a signal peptide, a variable acidic domain, and a conserved, cysteine-rich domain. A distantly-related gene is also present in the genomes of the free-living nematodes Caenorhabditis elegans and C. briggsae but in both cases the acidic domain is absent (Gregory, Maizels and Blaxter, unpublished observation). ALT proteins are stockpiled in the oesophageal glands of infective larvae [ 12 ] and are secreted by the parasites when they encounter mammalian culture conditions. Thus, their function may be to promote parasite survival within the host physiological or immunological environment. For example, ALTs may interfere with the critical first interaction between the innate immune system and the nematode invaders. It has been established that larval stages rapidly elicit a strong Th2 response in mice [ 13 ] and induce host macrophages to adopt a counter-inflammatory phenotype [ 14 ]. Although ALT antigens are not expressed on the parasite surface, they can induce protective immunity in animal models [ 9 , 15 , 16 ], indicating that neutralization of ALT function may be sufficient to protect the host from infection. Transgenesis and targeted gene deletion have yet to be established for parasitic helminths, so it is not possible to investigate the biological role of ALT proteins by conventional reverse genetics. We reasoned, however, that if ALT function is fulfilled within the host rather than within the parasite, we can validly study these proteins by transgenic expression in a more tractable carrier species. We chose to test this approach with the protozoal parasite Leishmania , several species of which are infective to laboratory mice. Leishmania can readily be modified genetically [ 17 - 20 ] to yield lines expressing high levels of exogenous transgenes. We selected L. mexicana as it establishes infections in murine macrophages in vitro , providing experimental access to a key cell type known to be modified in filarial infection [ 14 , 21 - 23 ]. L. mexicana will also infect mice, and although the immunological factors determining resistance and susceptibility are not as well-defined as in L. major [ 24 - 26 ], it offers the advantage of slower in vivo kinetics and consequently is anticipated to be more sensitive to altering factors. Using this model, we show here that transgenic L. mexicana expressing the ALT proteins are more virulent in macrophages in vitro , and that this property is abolished by deletion of the filarial-specific acidic domain. We also show that mice infected with alt -transgenic L. mexicana harbour higher parasite burdens than controls. By studying the responses of macrophages infected with transgenic parasites, we suggest that the ALT products modulate cytokine-induced signalling and render parasites more resistant to IFN-γ-induced killing. Results Expression of B. malayi ALTs in L. mexicana Bm-alt-1 and Bm - alt-2 genes were subcloned in their entirety, including endogenous signal peptide sequences, into the recombination vector pSSU, which contains flanking sequences homologous to the 18S small subunit (SSU) rRNA locus [ 19 , 27 ] (Figure 1 ). Electroporation of L. mexicana was undertaken, permitting homologous recombination of the alt-1 and -2 sequences downstream of the strong polymerase I promoter into the sequence for the small sub-unit rRNA, which is known to be transcribed in both stages of the life cycle of the parasite. Following puromycin selection, multiple transgenic lines were isolated for each alt gene, from which representative clones were selected containing the correct insert at an appropriate integration site. Transgenic ALT expression in the free-living culture promastigote stage was confirmed in both lines by Western blot (data not shown) and immunofluorescent staining (Figure 2A–D ) of permeabilized parasites with murine anti-ALT antibodies. Not only were ALT proteins found widely distributed in the transgenic protozoa, but staining of the membrane-rich flagella indicated surface expression in the promastigote stage. This was confirmed by flow cytometric analysis of anti-ALT-stained non-permeabilized transgenic promastigotes (Figure 2E, F ; negative controls panels G, H). In vitro infection of macrophages with transfected amastigotes Bone-marrow-derived macrophages were infected with axenic transformed amastigotes of each line. Both wild-type (Figure 2I ) and alt -transfected parasites (Figure 2J ) were fully infective to macrophages, and continued to express transgene-encoded protein reactive by immunofluorescence (data not shown). Within 24 hours, ALT-1 and ALT-2 expressing lines were able to infect significantly more host cells (Figure 3A , p < 0.001), a contrast that was sharply accentuated by day 7 of culture (Figure 3B ). Moreover, >90% of macrophages infected with alt -transgenic L. mexicana harboured at least 3 parasites, compared to <50% of cells infected with the wild-type, and overall there was a 3-fold difference in mean parasite load per macrophage. This enhancement was manifest in both alt-1 and alt-2 transgenic lines and did not affect the total number of macrophages surviving through the culture period. In contrast, transfectants expressing an unrelated filarial gene, the cystatin Bm -CPI-2 [ 28 ], have no effect on survival of L. mexicana in murine macrophages (Figure 3C, D ). Thus, the observed effect is gene-dependent and is not an attribute of the transfection system. Enhanced virulence of ALT-expressing L. mexicana promastigotes Analysis of transgene function in the more complex setting of in vivo infection was also performed, in order to test more stringently whether ALT products interfere with immunity. In two experiments, the alt -transfectants elicited larger lesions more quickly than either the parental wild-type strain of L. mexicana (Figure 3E ), or a transfectant encoding GFP (not shown). In both experiments, the effect of alt transfection was to accelerate lesion development to a plateau after 8–10 weeks, while control parasites reached similar levels only after 12–15 weeks. Recovery of parasites from the footpads of infected mice also showed marked exacerbation: parasites were detected in our assay in 94% (15/16) of animals given transfected L. mexicana compared to 50% (4/8) of those receiving wild-type parasites (p = 0.03, χ 2 test) (Figure 3F ). Nitric oxide production and susceptibility to NO-mediated killing Because nitric oxide generation is known to be a primary factor in the control of parasite survival in macrophages [ 29 , 30 ], we compared levels of the NO-metabolite nitrite from J774 macrophages infected with the different parasite lines. NO production was assayed 24- and 48-hours post-infection, as the differences in parasite numbers are apparent from an early stage. Irrespective of the transgenic status of the parasite, macrophages produced near-identical amounts of NO in culture medium, measured by nitrite, at both time points (Figure 4A ); thus the ALT products do not abolish the reactive nitrogen burst. To test whether there was a quantitative reduction in either NO responses or parasite sensitivity to destruction, we infected cells that had been previously stimulated with LPS and a range of doses of IFN-γ. Survival was measured by enumeration of infected macrophages 3 days later. Figure 4B shows that alt- transfected parasites not only achieve higher infection levels in unstimulated macrophages (no IFN-γ), but survive better in cells stimulated with intermediate IFN-γ doses (0.01–0.1 U/ml) than do wild-type organisms. Interestingly, the production of nitrite was comparable at each IFN-γ dose in all parasite types (Figure 4C ), indicating that the ALT products are likely to be interfering in an NO-independent pathway of immunity. Gene expression in infected macrophages To gain insight into what other mechanisms may be at play, we tested mRNA from macrophages infected with wild-type or transfected L. mexicana against an array of 514 genes associated with immune responsiveness formatted on the Mouse Cytokine Expression Array 1.0 (R & D Systems Inc.). Two independent experiments were performed, and hybridization of [ 33 P]-labelled cDNA was measured by phosphorimaging of spots of interest. Little difference was seen in hybridization for iNOS, IL-10, IL-12 (Table 1 ) or many other known players in the pro- and counter-inflammatory cascades. The constancy of iNOS expression following transfection is consistent with the measurements of nitric oxide production discussed above. However, other pro-inflammatory players (TLR6, LPS-BP, LTBP3 and TNFSF11 and 12) were down-regulated relative to wild type in both alt -1 and alt -2 transfection, in replicate experiments. Moreover, substantial shifts in mRNA expression were consistently observed with certain products known to modulate the type-1/type-2 balance. Bone-marrow-derived macrophages, infected 7 days earlier with wild-type L. mexicana infection, showed significant ablation of GATA-3 and SOCS-1, but expression was maintained or enhanced in cells infected with alt -transgenic parasites (Figure 5A and 5B ). These effects were reproduced with both alt genes in both experiments (Table 1 ). Because array hybridization may under certain conditions be non-linear with respect to mRNA concentrations, we performed quantitative RT-PCR with GATA-3- and SOCS-1-specific primers on replicate samples of infected macrophages taken at 24 h (Figure 5C, D ) and 7 days (E, F). For GATA-3, these data reinforce the conclusion that while wild-type infection results in a 2–3-fold loss of GATA-3 expression, alt -transgenic infected macrophages maintain the level of this transcription factor throughout the course of in vitro infection (Figure 5C and 5E ). Interestingly, SOCS-1 is sharply upregulated by 24 h in all infected macrophages, but elevated expression remains evident only where alt transgenic parasites are present (Figure 5D and 5F ). Although it is possible that larger parasite numbers in the alt- transfection system may in themselves alter macrophage gene expression, parasite densities were only slightly shifted at 24 h, when GATA-3 expression was markedly different in wildtype and transfected cell infections. Up-regulation of GATA-3 and SOCS-1 in filarial infection We then tested whether GATA-3 and SOCS-1 are up-regulated by live infection with B. malayi larvae in vivo . Mice were injected i.p with 200 larvae, and 7 days later peritoneal cell populations were recovered by lavage with medium. We then performed real time PCR on an adherent macrophage enriched cell population and a non-adherent cell population, predominantly lymphocytes. As shown in Figure 6A and 6C , the macrophage-rich adherent population showed a modest increase in both GATA-3 and SOCS-1 expression, whereas a very substantial rise in both was seen in the non-adherent cells (Figure 6B and 6D ). It seems that B. malayi infection results in up-regulation of the same genes as observed with ALT in macrophages, in both macrophage and non-macrophage subsets. The acidic domain of ALT is essential for biological function The enhanced virulence phenotype conferred by alt transfection, measured by in vitro infectivity to macrophages, provides a ready and tractable system for submolecular analysis of functional domains of the ALT protein. We investigated whether the N -terminal acidic domain, which is unique to the filarial genes, was important for the immunological activity of the protein. We constructed a truncated mutant of ALT-2, named acidic domain deleted (add) , which was cloned into the pSSU vector and electroporated into L. mexicana . These transfectants expressed immuno-reactive protein (Figure 7A–C ) and were able to infect bone marrow-derived macrophages. However, add -transfected parasites did not display the greater infectivity seen with alt -transfectants, but showed a phenotype indistinguishable from wild-type parasites (Figure 7D ). Thus, the acidic domain is required for functional expression of the immunological effects of the ALT proteins. Similar analyses will permit the future definition of the critical residues required for immunomodulatory activity. Discussion Long-lived helminth parasites have evolved highly effective strategies to evade host immunity, requiring both adaptation of existing genes and evolution of new gene families [ 6 ]. With genomes that encode many thousands of proteins, these parasites are likely to be repositories of numerous novel 'immune evasion genes' with no or only weak sequence similarity to known products [ 31 ]. The imminent completion of genome sequence information for major helminth parasites [ 32 - 34 ] accentuates the problem of how to identify functional immune modulators among numerous novel gene sequences. We hypothesized that transcripts for secreted immunomodulatory proteins would be among the most abundant mRNAs at key points in the parasite life cycle. Two such genes are those that encode the abundant larval transcripts (ALT) proteins, which are released by larval parasites ready to infect the mammalian host and represent 5% of the total mRNA at this stage [ 9 ]. To test whether the ALT proteins are functional immune evasion products, we transfected each alt gene into L. mexicana and showed that infection of macrophages in vitro is exacerbated by expression of either ALT protein. Moreover, mice infected with alt -transgenic parasites display more rapid lesion development and higher parasite burdens than controls. Our results also demonstrate that alt -transfected parasites are more resistant to INF-γ-induced killing by macrophages, supporting our hypothesis that ALT proteins act to modify host immune responses in filarial infection. These data validate the transfection strategy in general and, in highlighting changes in key intracellular factors resulting from ALT expression, justify the selection of a protozoal carrier to test the function of a helminth gene. A further advantage of the system we describe here is the facility with which selected mutants can be analysed with an in vitro read-out of infectivity to macrophages. We constructed a deletion mutant lacking the N-terminal acidic domain, which shows most variation between filarial species and is not present in distantly related genes from free-living nematode organisms. This deletion showed a clear-cut abolition of the alt phenotype, indicating the essential functional nature of this sequence and paving the way for a finer analysis of structure-function relationships in a tractable experimental system. We have also extended the use of this transfection system to the analysis of other parasite genes that are hypothesised to be immunomodulatory. For example, filarial parasites produce homologues of mammalian macrophage migration inhibitory factor, MIF [ 39 , 40 ], a cytokine generally considered to be an acute pro-inflammatory agent [ 41 , 42 ]. It is, however, paradoxical that long-lived tissue pathogens produce a potentially inflammatory mediator, and we therefore used the Leishmania transfection system to test the hypothesis that long-term MIF production may promote parasite survival. L. mexicana organisms transfected with B. malayi MIF homologues were tested first in vitro , in which setting they were less infective to macrophages, rarely exceeding one parasite per host cell. This result was consistent with an immediate pro-inflammatory action of MIF on macrophages. However, when tested in vivo over a 4- or 8-week-period, MIF-transfected parasites were able to survive better than wild-type, indicating that over the longer term, filarial MIF homologues are able to exert a down-modulatory effect on host immunity (Prieto-Lafuente, manuscript in preparation). We are now using this system to analyse the gene expression profiles of macrophages infected with MIF-transfected parasites in both in vitro and in vivo settings. These data illustrate the facility with which the Leishmania transfection system can be used in parallel for in vitro and in vivo experiments, and the importance of an in vivo read-out to assay gene effects within the immune system as a whole. In addition, our work has demonstrated that Leishmania transfection for helminth gene analysis is equally applicable for two completely unrelated, but immunologically important, gene families. The application of this new strategy to transfection of L. mexicana is ideally suited to the study of macrophage modulation by genes predicted to function in this environment. Parallel investigations can also be envisaged by transfection of L. major , which will permit a more thorough analysis of T cell modulation to be undertaken. For example, resistance to L. major is clearly associated with development of a parasite-specific Th1 response, and it is possible that parasite genes that inhibit Th1 differentiation will alter the course of L. major infection in vivo . These studies are now under way. Macrophages are known to be a critical cell population in the immune response to filarial parasites at successive points in time. First, they participate in innate defences against invading larvae [ 35 , 36 ]; second, if infection becomes established, they evolve an IL-4-dependent "alternatively activated" phenotype, which is broadly immunosuppressive [ 8 , 37 ]; and third, in late stage infection, they clear the bloodstream of microfilarial forms through a nitric oxide-dependent pathway [ 21 , 38 ]. Thus, the reduction in IFN-γ-responsiveness in macrophages harbouring alt -transfected parasites has resonance for initial host susceptibility, and longer-term propensity for chronic infection, as well as the ability to eliminate the blood-borne Mf stage. A key outcome of the present study is that ALT expression is associated with up-regulation of GATA-3 and SOCS-1. GATA-3 is a pivotal transcription factor for the development and function of the Th2 pathway [ 43 , 44 ] and has not previously been reported in mouse macrophages; hence, at the present time, downstream genes activated by GATA-3 in macrophages have not been defined. Significantly, GATA-3 is required for embryonic development and has recently been shown to be essential for the differentiation [ 45 ] and effector function [ 46 ] of murine eosinophils. Thus, GATA-3 expression in macrophages may fulfil an important role, possibly in conjunction with the altered phenotype of these cells in chronic parasite infections [ 8 , 47 ]. SOCS-1 is a member of the Suppressor Of Cytokine Signalling family of proteins, which regulate signal transduction by IFN-γ and structurally related cytokines [ 48 , 49 ]. SOCS-1 is particularly important in macrophage responsiveness to inflammatory stimuli [ 50 ], countermanding IFN-γ by directly inhibiting the JAK kinase associated with the IFN-γ receptor [ 51 ]. SOCS-1 is known to inhibit the IFN-γ-dependent killing of Leishmania parasites, because macrophages from SOCS-1 null mice require 100-fold less IFN-γ to clear infection [ 52 ]. Indeed, in the absence of SOCS-1, there is generalised activation of the immune system, causing autoimmune pathology [ 53 , 54 ]. Thus, by up-regulating SOCS-1, cells expressing ALT may have down-shifted responsiveness to inflammatory cytokines, requiring exogenous stimulation to induce Leishmania killing (Figure 5B ). In the context of Brugia infection, SOCS-1 induction by ALT proteins could explain why macrophages fail to develop the IFN-γ-activated phenotype, and instead express a counter-inflammatory profile [ 8 ]. Overall, these data suggest that the ALT proteins play a role in the evasion strategy of the filarial nematode, by directly amplifying Th2 responses and/or interfering with signals necessary for the development of pro-inflammatory Th1 populations. It is interesting to note that in vivo exposure to live infective larvae of B. malayi induces a prompt Th2 response measurable by 24 h and increasing to 10 days post-infection [ 13 ]. We have repeated these experiments by intraperitoneal inoculation of live L3 and find up-regulation of GATA-3 and SOCS-1 in both adherent and non-adherent peritoneal cells (Figure 6 ). Conclusion The novel approach we have described permits, for the first time, the elucidation of gene function for a major group of biologically and medically important parasites, which in the example presented here provides non-intuitive results linking parasite secretions to host cell signalling. The transfection strategy will also accommodate a mutagenesis analysis of structure-function relationships in unique gene families. In conclusion, our system provides a solution to one of the major obstacles facing helminth parasite immunology in the post-genomic era and offers a fascinating insight into the molecular and cellular intricacies of pathogen manipulation of host immune responsiveness. Methods Mice and parasites Six- to eight-week old female C57BL/6 and CBA mice were used. L. mexicana (strain MNYC/BZ/62/M379) promastigotes were cultured in vitro in semi-defined medium/10% heat inactivated foetal calf serum (hiFCS)/1% penicillin-streptomycin (complete SDM) at 26°C. Amastigotes were cultured axenically at 34°C in Schneider's Drosophila medium (Gibco BLR) supplemented with 20% hiFCS and 3.9 g/l 2-(N-morpholino)ethanesulfonic acid (Sigma, U.K.). Transgenic parasites were cultured under the same conditions with the addition of 20 μg/ml puromycin (Sigma, U.K). Cell culture The murine macrophage cell line J774 was passaged in DMEM containing 10% hiFCS/1% penicillin-streptomycin/1% L-glutamine and cultured at 37°C in 5% CO 2 . Leishmania expression construct for B. malayi ALTs Primers were designed to amplify the entire coding region of alt-1 and alt-2 from a B. malayi L3 cDNA library. The oligonucleotides used for alt-1 were 5'-CCGCTCGAG ATG AACAAATTGCTAATAGCA-3' (sense, initiating codon in bold) and 5'-TGCTCTAGA TTA CGAGCATTGCCAACTTTC-3' (antisense, terminating codon in bold); and for alt-2 , 5'-CCGCTCGAG ATG AATAAACTTTTAATAGCA-3' (sense) and 5'-TGCTCTAGAC TAT GCGCATT GCCAACCTGC-3' (antisense). After an initial denaturation step at 95°C for 5 min the PCR was cycled between 94, 55 and 72°C (1 min each) for 35 rounds, followed by 1 round at 72°C for 10 min. The fragments were digested with XhoI and XbaI and cloned into the pSSU vector (13), yielding pSSU- alt-1 and pSSU- alt-2 . Clones were fully sequenced on both strands. DNA was extracted from pSSU- alt-1 and pSSU- alt-2 using the Qiagen Miniprep kit following the manufacturer's instructions Site directed mutagenesis An ALT- 2 mutant, in which the acidic domain of the protein (amino acids 24–49) were deleted, was generated using the Exite PCR-based site-directed mutagenesis kit (Stratagene, USA) following the manufacturer's instructions. The pSSU- alt-2 construct (see above) was used as the template in a PCR reaction containing two oligonucleotides: 5'-TGATTCTGATACACACGGGAGTGT-3' (antisense, primer phosphorylated) and 5'-TATGTAACCAAAGGGAATTTGTT-3' (sense). The cycling parameters were as follows: 1 cycle of 1 min at 94°C, 4 min at 53°C and 2 min at 72°C; followed by 10 cycles of 1 min at 94°C, 2 min at 55°C (adding 10 s after each cycle) and 1 min at 72°C; and a final cycle of 5 min at 72°C. After the PCR the nonmutated parental plasmids were digested with Dpn restriction enzyme. The undigested linear DNA was then polished with Pfu DNA polymerase and ligated at 37°C for 1 h with T4 DNA ligase. The ligated DNA was then transformed into E. coli , yielding a pSSU-ADD (acidic domain deleted) construct. The insert was sequenced to verify that the intended mutation was correctly constructed. Transfection of Leishmania Logarithmic phase promastigotes (4 × 10 7 ) were electroporated with 10 μg of PmeI linearized fragments of either pSSU- alt-1 , pSSU- alt-2 or pSSU- add . Clones were selected on 24-well plates in complete SDM supplemented with 20 μg/ml of puromycin and further propagated in a culture volume of 10 ml. Immunofluorescence Promastigotes were washed in PBS, fixed in 2% paraformaldehyde for 30 min at room temperature, and quenched in NH 4 Cl for 10 min. Wild-type parasites were incubated with dilution buffer (0.1% saponin, 2% goat serum) in the presence of anti- L . mexicana rabbit serum or anti-ALT mouse serum (from C57BL/6 mice) for 45 min. Following 3 washes with wash buffer (0.1% saponin, PBS) the parasites were incubated with either an anti-rabbit-FITC or an anti-mouse-FITC secondary antibody (DAKO, Denmark) for 45 min. They were then washed 3 times before mounting with Cityfluor (Cityfluor Ltd, UK) for microscopy. Flow cytometry Promastigotes were washed in PBS, resuspended at 1 × 10 6 /ml and fixed without permeabilization in 2% paraformaldehyde for 15 min at room temperature. After two washes in PBS, 10% FCS (FACS wash), the parasites were stained with anti-ALT-1 antibody or normal mouse serum diluted in FACS wash. This was followed by incubation with a FITC anti-mouse secondary antibody (DAKO, Denmark). Flow cytometric analysis was performed using a FACScan (Becton Dickinson) and analysed using CellQuest 3.1 software. Preparation of bone marrow-derived macrophages (BMM) BMM were obtained from femurs and tibias of 6- to 8-week old CBA mice by flushing the bones with DMEM (Gibco, BLR) containing 10% hiFCS/1% penicillin-streptomycin/1% L-glutamine. Cells were centrifuged and plated out in non-tissue culture Petri dishes at a density of 5 × 10 5 /ml in complete DMEM, supplemented with 20% (v/v) L929 cell-conditioned medium as a source of M-CSF and 20% hiFCS. After 6 days at 37°C, the cells were detached by incubation in PBS containing 3 mM EDTA and 10 mM glucose, plated and cultured in small non-tissue culture Petri dishes at 2.5 × 10 5 /ml in complete DMEM for 24 h at 37°C. Infection of BMM with L. mexicana Day 7 BMM were infected for 24 h or 7 days at 34°C with wild-type or alt -transfected amastigotes at a ratio of 10 parasites per macrophage. After the period of infection macrophages were harvested as described above, washed with PBS and fixed and permeabilized using a "Fix & Perm kit" (Pharmigen); 1 × 10 5 cells were centrifuged on to glass slides with a Cytospin. Intracellular Parasites were detected and counted by immunofluorescence as described above. Between 100 and 200 macrophages were counted for each time-point. Infections in vivo For L. mexicana in vivo infections, groups of 8 female C57BL/6 mice were injected subcutaneously in the footpad with 3 × 10 6 stationary-phase wild-type L. mexicana , alt-1 transfectants and alt-2 transfectants. Lesion size was measured weekly during the course of infection with a dial micrometer and expressed as the difference in size between the infected footpad and the contralateral uninfected footpad. For B. malayi in vivo infections, groups of 5 female C57BL/6 mice were injected intraperitoneally with 200 infective larvae recovered from crushed Aedes aegypti mosquitoes. After the experimental period the mice were euthanized by terminal anaesthetic, and peritoneal cells (PEC) were harvested by thorough washing of the peritoneal cavity with 15 ml of ice-cold RPMI supplemented with 10% FCS. The harvested PEC were plated in 24-well culture plates at 2 × 10 6 cells/well. Following 3 h at 37°C to allow cells to adhere, both the non-adherent and the adherent macrophage-enriched cell populations were harvested. Parasite quantification The number of parasites in the footpad was estimated by limiting dilution assay. Infected footpads were harvested in cold PBS after removal of the skin. Footpad tissue was dispersed through a cell strainer and resuspended in PBS-1% penicillin/streptomycin. After centrifugation the pellet was resuspended in complete SDM. The cell suspension was then serially diluted in 10-fold steps, in quadruplicate, in 96-well plates. The plates were incubated for 4 days at 27°C; the wells were then observed for parasite growth. Measurement of nitrite production J774 macrophages were plated out on 96-well plates (1 × 10 5 /well) and incubated at 37°C for 24 h. The medium was then removed, the cells were washed twice, and L. mexicana promastigotes were added for 4 h at a ratio of 10 parasites per well. IFN-γ (40 U/ml) and LPS (10 ng/ml) were then added, with or without the arginine analogue N -monomethyl-D-arginine (D-NMMA), 1 mM. Nitrite accumulation in medium over the subsequent 24–48 h was used as an indicator of NO production and was assayed by the Griess reaction in which 100 μl of Griess reagent [ 55 ] was added to 100 μl of each supernatant in triplicate wells in a 96-well plate. Plates were read at 490 nm against reference wavelength 620 nm using an ELISA plate reader. NaNO 2 was used to make a standard curve for each plate reading. Leishmanicidal assay BMM (5 × 10 4 ) were plated out on glass coverslips in 24-well plates, allowed to adhere for 24 h, and stimulated with 100 ng/ml LPS and the indicated concentrations of IFN-γ. Cells were incubated for 6 h at 37°C before adding stationary phase promastigotes of wild-type L. mexicana or transfected alt-1 and alt-2 parasites at a ratio of 10 parasites per cell. After 72 h at 37°C the cells were stained with Giemsa. The percentage of macrophages infected with parasites was determined by counting 4 samples of 100 cells. RNA isolation Total RNA was isolated from non-infected and infected BMM as well as adherent and non-adherent cell populations using TRIzol Reagent (Invitrogen, Life Technologies) according to the manufacturer's instructions. The RNA was subjected to DNase treatment (Ambion, INC) to eliminate genomic contamination, according to the manufacturer's instructions Gene array analysis Gene expression was analysed using the Mouse Cytokine Expression Array (R&D systems). The mouse cytokine-specific primers were first annealed to the total RNA, which was then reverse transcribed in the presence of SuperScript II (Invitrogen, Life Technologies) and [α- 33 P]dCTP. The radiolabelled cDNA probes generated from non-infected and infected cells were hybridized to identical membranes containing the mouse cDNA arrays. Following hybridization, high stringency washes were performed and the membranes were subjected to autoradiography. Quantification was carried out using a PhosphorImager and data analyzed with ImageQuant v1.2. Real Time RT-PCR analysis Total RNA was extracted in Trizol, as described above, and single-stranded cDNA was synthesized using MMLV reverse transcriptase (Stratagene). Relative quantification of the expression of the genes of interest was measured by real-time PCR using the LightCycler (Roche Molecular Biochemicals). PCR amplifications were performed in 10 μl volumes containing 1 μl cDNA, 2.5 mM MgCl2, 3 μmM primers and the LightCycler-DNA SYBR Green I mix (Qiagen). The reaction was performed in the following conditions: 15 min activation step at 95°C for one cycle, 15 s denaturation at 95°C, 20 s annealing of primers at 50°C and 15 s elongation at 72°C, for 50 cycles. The fluorescent DNA binding dye SYBR Green was monitored after each cycle at 80°C. Five serial 1:2 dilutions of alt-2 infected macrophages cDNA were used to produce a standard curve in each reaction. The abundances of GATA-3 and SOCS-1 were expressed as ratios of amplified product to the control, mouse S29 ribosomal protein. Primers for RT-PCR analysis were as follows: GATA-3: 5'-CTA CGG TGC AGA GGT ATC C-3' and 5'-GAT GGA CGT CTT GGA GAA GG-3'; SOCS-1: 5'-ACC TTC TTG GTG CGC GAC AGT CGC CAA-3' and 5'-GGA ACT CAG GTA GTC ACG GAG TAC-3'; and S29 ribosomal protein: 5'-ATG GGT CAC CAG CAG CTC TAC-3' and 5'-GTC CAA CTT AAT GAA GCC TAT-3'. Abbreviations ALT, abundant larval transcript protein; alt , abundant larval transcript gene or mRNA; BMM, bone marrow-derived macrophages; FACS, fluorescence-activated cell sorter; L3, third-stage infective larva; NO, nitric oxide. Authors' contributions NG-E designed and led the experimental work, including molecular constructs, cell transfection, immunological assays, array and RT-PCR analyses. She also drafted the manuscript. CB and LP-L made constructs in pSSU, maintained Leishmania cultures, participated in electroporation procedures, macrophage infection, in vivo infection and dilution assays. TA designed the transfection system and devised the Leishmania transfection methodology. TA, CCB and RMM jointly conceived the transfection strategy for Brugia genes, critically assessed progress, and made revisions to the draft manuscript. The overall study was co-ordinated by RMM who also completed the draft of the manuscript. All authors read and approved the final manuscript.

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543449

Coccidioides posadasii infection alters the expression of pulmonary surfactant proteins (SP)-A and SP-D

Background Coccidioidomycosis or Valley Fever is caused by Coccidioides in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in Coccidioides infection are not known. Methods In this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of Coccidioides posadasii infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of C. posadasii (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays. Results We found that in lavage fluid samples of C. posadasii infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % (SEM 3.5, p < 0.001), 38 % (SEM 5.8, p < 0.001) and 4 % (SEM 1.3, p < 0.001) of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of C. posadasii protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens. Conclusion Our results suggest that the C. posadasii infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.

Background Coccidioidomycosis or Valley Fever is a fungal disease caused by the biphasic, highly virulent, soil-fungus Coccidioides immitis or posadasii [ 1 ]. It is endemic in the southwest regions of US, Northern Mexico and parts of Central America [ 2 ]. C. posadasii or C. immitis , are the most virulent fungal pathogens enlisted in Select Agent list and pose a risk for bioterrorism [ 3 ]. The primary infection is acquired by inhalation of air-borne, mycelial phase arthroconidium that converts into endosporulating spherule in the lung. Clinical manifestations of the disease range from pulmonary infection to a more severe fatal mycosis involving extra-pulmonary tissues in 1–10% of the infected people [ 1 - 4 ]. Previous studies suggest that Th1 cell mediated immunity protects individuals against Coccidioides [ 5 , 6 ]. However, information is lacking regarding the pulmonary innate immune components that may play a critical role in regulation of immune responses against Coccidioides . At alveolar level in the lung, the innate immune system is composed of many cell types and chemical mediators, including surfactant. The pulmonary surfactant is a complex mixture of lipids (88–90%) and proteins (10–12%), synthesized by type II epithelial cells and Clara cells. It lines the alveoli, and helps in maintaining normal lung function [ 7 ]. Among four different surfactant proteins, surfactant proteins-A (SP-A) and D (SP-D) are members of the "Collectin" family [ 8 ]. In the past, several studies have suggested that both SP-A and SP-D play an important role in innate host defense against various viral, fungal and bacterial pathogens [ 9 , 10 ]. More evidence for the pulmonary collectins' role in host defense comes from studies on SP-A- deficient mice that are susceptible to intra-tracheal Group B Streptococci [ 11 ], Pseudomonas aeruginosa [ 12 ], and Respiratory Syncytial Virus [ 13 ]. Also, intranasally administered SP-D has been found to reduce replication of Respiratory Syncytial Virus in the lungs of infected mice [ 14 ]. Both SP-A and SP-D, have been classified as secretory pattern-recognition receptors that can bind to a variety of pathogens and help in clearance [ 9 , 15 ]. Recent evidences indicate that in addition to their pathogen recognition property, SP-A and SP-D also play an important role in stimulating immuno-regulatory pathways [ 15 ]. However, the collectins' role in coccidioidomycosis is not known. This study focuses on analyzing the changes in amounts of the SP-A and SP-D in the bronchoalveolar lavage fluid (BALF) samples from mice infected with lethal dose of C. posadasii and C. posadasii protected mice after immunization with protective formalin killed spherule (FKS) vaccine, and binding of pulmonary collectins to Coccidioidal antigens. Methods Mice BALB/c and C57BL6 mice (6 weeks old female) from Jackson Laboratory (Bar Harbour, ME) were used in this study. Both mouse strains are susceptible to C. posadasii infection. The BALB/c mice were used to study the changes in pulmonary surfactant after intranasal challenge with C. posadasii . And, the C57BL6 mice were used to study the changes in pulmonary surfactant after vaccination with protective FKS vaccine. Mice were housed in Biosafety Level-3 animal facility at UTHSCSA and provided with food and water ad libitum. All experimental animal care and treatment protocols were reviewed and approved by Institutional Animal Care and Use Committee. Coccidioides posadasii C. immitis (now posadasii ) Silveira strain, cultured on 1 % glucose-0.5 % yeast extract agar (GYE), was used for infecting the mice [ 1 ]. The arthroconidia were harvested in endotoxin-free 0.15 M saline (Baxter Health Care Products, Deerfield, IL) from 6–8 weeks old mycelial phase cultures grown on GYE plates. The arthroconidia suspension was passed over a sterile cotton column to remove hyphal elements and arthroconidia were enumerated by hemacytometer counts. The viable cfu counts were confirmed pre- and post-infection by plate cultures on GYE agar. All the experiments with C. posadasii were carried out in Biosafety Level-3 facility at UTHSCSA. Intranasal Challenge with C. posadasii Arthroconidia Mice were anaesthetized after intramuscular injection of ketamine- xylazine (75 μg/g body weight ketamine and 10 μg/g body weight xylazine) and were then challenged intranasally with a lethal dose of arthroconidia (n = 30, fresh harvest of C. posadasii arthroconidia) suspended in endotoxin-free 0.15 M NaCl (Baxter Health Care Corp, Deerfield, IL) using sterile pyrogen-free microtip. Mice were held in an upright position for 1–2 min to resume normal breathing after injection. Control mice were challenged with equal volume of endotoxin-free 0.15 M NaCl. Preparation of Coccidioide -FKS vaccine and Immunization of Mice C. posadasii (strain Silveira )arthroconidia were used to prepare FKS as described earlier [ 16 ]. Briefly, arthroconidia were inoculated in modified Converse medium containing Tamol and cultured while shaking at 180 rpm at 40°C in 20 % CO 2 incubator. The spherules were collected from the harvested culture, washed in endotoxin-free water and killed with 1 % formalin. FKS preparation was checked for sterility and lyophilized. C57BL6 female mice (age 6 weeks old) were immunized intramuscularly twice and subcutaneously once at one week interval with FKS (0.7 mg/dose each time). The mice in FKS immunized, infected group were then challenged intranasally with 30 C. posadasii arthroconidia, 15 days after last immunization, as described above. Fungal Burden Assay Mice were anaesthetized as mentioned above, prior to sacrifice on day 10, post intranasal infection. This standard procedure was used for intranasal injection since it does not cause respiratory depression during anaesthesia. The lung and spleen tissues were collected in sterile 0.15 M NaCl for studying fungal load. The fungal burden was studied by plating ten fold dilutions of lung and spleen homogenates in 0.15 M saline on Mycosel agar plates (BD Biosciences, Franklin Lakes, NJ) and incubating for 72 h at 30°C. The cfu counts were recorded and normalized with organ weight. Collection and Processing of BALF At the time of necropsy, we collected BALF by injecting 1 ml endotoxin-free 0.15 M NaCl solution (Baxter Health Care Corp, Deerfield, IL) three times, via an angiocatheter (BD Biosciences, San Diego, CA) placed in the trachea. The volume of the input solution was kept constant (3 ml total) and approximately, 90–95 % of the solution was recovered consistently. The BALF was centrifuged at 500 rpm for 10 min at 4°C to remove cells. The cell free BALF supernatant was filtered through 0.2 μm syringe filters (Nalge Nunc International, Rochester, NY) and stored at -80°C for further analysis. Total Protein and Lipid Analysis The total protein concentration was measured in BALF specimens using micro bicinchonic acid protein assay kit (Pierce, Rockford, IL) against bovine serum albumin (BSA) standard protein. The total phospholipid content in lipid extracts of BALF specimens was determined using the method of Stewart, against Dipalmitoyl-phosphatidylcholine (DPPC, Avanti Polar Lipids, Alabaster, AL) standard solutions [ 17 , 18 ]. Briefly, the lipid extract of BALF specimens and DPPC standard solutions was completely dried under compressed nitrogen gas. The dried lipids were dissolved in chloroform and mixed with 1 ml of 2.7% ferric chloride and 3% ammonium thiocyanate in glass tubes. The mixture was vortex mixed for 1 min and centrifuged at 200 rpm for 5 min. The bottom red lower layer of phospholipids and ammonium ferro-thiocyanate complex was collected and absorbance was read at 488 nm. Western Blotting The BALF and lung tissue homogenate samples (total protein 10–50 μg) were run on 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) running gel and transferred on nitrocellulose membranes (Schleicher & Schuell, Keene, NH) overnight at 15 mA current. The nonspecific sites on the membrane with transferred proteins were blocked by 15% nonfat milk in Tris-buffered saline containing 0.05% tween 20 (TBST). The membrane was washed and incubated for 1 h with diluted (1:500) primary anti-human SP-A polyclonal antibody raised in rabbit (obtained from Dr. Richard J. King, UTHSCSA, San Antonio, TX) or anti-mouse SP-D antibody (kindly provided by Dr. Jo Rae Wright, Duke University Medical Center, Durham, NC). After washing the membrane with TBST, the membrane was incubated for 1 h with 1:10,000 diluted alkaline phosphatase conjugated anti-rabbit IgG raised in goat (Sigma Chemical Co, St. Louis, MO). The immunoreactive bands were observed by alkaline phosphatase conjugate system (Biorad, Hercules, CA). Purified human SP-A (kindly provided by Dr. Richard J. King, UTHSCSA, San Antonio, TX) and recombinant human SP-D (kindly provided by Dr. Erika C. Crouch, Washington University in St. Louis, St. Louis, MO) were run with the samples. The Coccidioidal antigens: lysates and filtrates of Coccidioidin (CDN), prepared as a toluene-induced lysate of young C. posadasii mycelia (obtained from Dr. Rebecca A. Cox, UTHSCSA, San Antonio, TX, [ 19 ]) were also run to check the cross-reactivities of anti-SP-A and SP-D antibodies to fungal antigens. Enzyme-Linked Immunosorbent Assay (ELISA) for SP-A and SP-D The concentrations of SP-A and SP-D were measured in BALF samples as described earlier [ 20 ]. The antibodies against SP-A and SP-D reacted with 34 kDa (SP-A) and 43 kDa (SP-D) immunroreactive bands in BALF and lung tissue homogenates (Fig 1 ). For measuring the lavage concentrations of SP-A and SP-D, the indirect ELISA procedure was used [ 20 ]. Briefly, the wells of Immulon 4 strips (Dynatech, Chantilly, VA) were coated overnight with purified human SP-A or recombinant human SP-D antigens (standards) and diluted BALF (three different dilutions) in 0.1 M NaHCO 3 , pH 9.6. The wells were washed three times with deionized water, and nonspecific sites were blocked with a buffer containing 0.25% BSA, 0.05% tween 20, 0.17 M boric acid and 0.12 M NaCl, pH 8.5. The wells were washed and incubated for 2 h with rabbit anti-human SP-A or rabbit anti-mouse SP-D antibody. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent (Sigma Chemical Co. St. Louis, MO). The reaction was stopped by adding 50 μl of 2 N H 2 SO 4 and read at 450 nm spectrophotometrically. The regression coefficient for a least-square linear fit to the standard curve of SP-A and SP-D was 0.99. The limits of detection for SP-A and SP-D were 2 ng/ml. Figure 1 Western blot for (A) SP-A and (B) SP-D proteins in mouse lung. Lanes (a, b): 2.5 μg total lavage fluid protein (c, d): 100 μg of total lung tissue homogenate protein from two healthy, non-infected BALB/c mice, and (e): 10 ng purified human SP-A or recombinant SP-D protein. Binding of SP-A and SP-D to Coccidioidal Antigens A microtiter well based method [ 21 ] was used to study the SP-A and SP-D interactions with Coccidioidal antigens (CDN-lysate and CDN-filtrate). Briefly, microtiter wells (Immulon 4; Dynatech, Chantilly, VA) were coated with 50 μl of CDN-lysate (10 μg/ml diluted in 0.1 M NaHCO 3 buffer, pH 9.6) or CDN-filtrate (10 μg/ml diluted in 0.1 M NaHCO 3 buffer, pH 9.6) or BSA (10 μg/ml diluted in 0.1 M NaHCO 3 buffer, pH 9.6) at room temperature. The nonspecific binding was blocked with phosphate buffered saline (pH 7.4) containing 0.1% triton-X 100 and 3% nonfat milk (buffer A). The purified human SP-A and recombinant human SP-D diluted in 20 mM Tris (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl 2 and 1 mg/ml BSA were then added to the wells and incubated for 3 h at 37°C. The wells were then washed with buffer A and incubated for 1 h at room temperature with diluted (1:1000 in buffer A) anti-SP-A and anti-SP-D antibodies. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent (Sigma Chemical Co. St. Louis, MO). The reaction was stopped by adding 2 N H 2 SO 4 and read at 405 nm spectrophotometrically. The coating of Coccidioidal antigens (CDN-lysate and CDN-filtrate) to the plates was confirmed using a positive control antibody that recognizes Coccidioidal antigens as described earlier [ 22 ]. The alkaline phosphatase-conjugated rat anti-mouse IgG antibody (Zymed, San Francisco, CA) served as secondary detection antibody. Statistics Statistical analyses of the data (t-test or ANOVA) were done using Prism Software (Graphpad Software, San Diego, CA). The p value <0.05 was considered significant. Results Pathological status All of the C. posadasii infected mice survived till the day of sacrifice (day 10 post infection). However, the mice were lethargic and lost body weight (Table 1 ). Abscess like lesions were quite evident on gross examination of the lung. The total wet lung weights were increased in C. posadasii infected mice. Table 1 Body weights (g) of C. posadasii infected and non-infected BALB/c mice (n = 10 of each type). Values are shown as Mean (SEM) of one representative experiment of two independent experiments. Days post challenge → Mice ↓ Day 0 Day 10 Non-infected 18.36 (0.29) 19.46 (0.23) ** C. posadasii infected 17.77 (0.39) 16.35 (0.53) *, # ** p < 0.01 as compared to non-infected control mice at day 0. *p < 0.05, # p < 0.0001 as compared to C. posadasii infected mice at day 0 and non-infected mice at day 10, respectively (t-test). The mean protein content of BALF samples from infected mice was 788 μg versus 326 μg protein in BALF samples from non-infected saline injected control mice, after 10 days of intranasal infection (p < 0.05, Table 2 ). In contrast, the phospholipid concentration was reduced in BALF samples from C. posadasii infected mice (58 μg) when compared to non-infected saline injected controls (165 μg, p < 0.05). Table 2 Total protein and phospholipid contents in BALF samples from non-infected and C. posadasii infected BALB/c mice (n = 5 of each type). Values are shown as Mean (SEM) from one represenative experiment of two independent experiments. Mice Total protein (μg) Total phospholipid (μg) Non-infected 326.0 (26.9) 165.5 (10.8) C. posadasii infected 788.7 (248.6)* 58.7 (15.7)* * p < 0.05 as compared to non-infected control mice (t-test). The amounts of SP-A, SP-D and phospholipid are reduced in BALF samples from C. posadasii infected mice The anti-human-SP-A and anti-mouse SP-D antibodies recognized 34 kDa and 43 kDa monomer bands of SP-A and SP-D in BALF and lung tissue samples (Fig 1 ). The upper bands of approximately 68 kDa and 85 kDa size (dimer of SP-A and SP-D protein) were also visible in lanes e of Figure 1A and 1B due to incomplete reduction, respectively. There were no differences in the detectable isoforms of SP-A or SP-D in the BALF samples from infected mice as compared to non-infected control mice (data not shown). The antibodies did not cross-react with Coccidioidal antigens in CDN lysate or CDN filtrate (Fig 2 ). The amounts of SP-A and SP-D were significantly reduced in BALF samples from C. posadasii infected BALB/c mice when compared to saline injected, non-infected control mice after 10 days of intranasal challenge (p < 0.001) (Fig 3A ). No significant changes were observed in the amounts of SP-A and SP-D in BALF samples collected from BALB/c mice, 5 days after intranasal challenge with C. posadasii (data not shown). Figure 2 Western blot of CDN-lysate and CDN-filtrate for crossreactivity with (A) anti-human SP-A and (b) anti-mouse SP-D antibodies. Lanes (a): 20 μg, (b): 10 μg and (c): 1 μg CDN-filtrate protein. Lanes (d): 20 μg, (e): 10 μg and (f): 1 μg CDN-lysate protein and last lane: 10 ng purified human SP-A protein or recombinant SP-D protein. Figure 3 SP-A and SP-D levels in BALF samples from (A) C. posadasii infected BALB/c mice (ng/μg protein, % of non-infected control mice, n = 5 of each type) (B) FKS immunized, C. posadasii infected C57BL6 mice (protected mice) (ng/μg protein, % of FKS immunized non-infected mice, n = 5 of each type). The data are shown from one representative experiment of two independent experiments. * p < 0.001 (ANOVA) The fungal colonies of C. posadasii were recovered from both lung and spleens of infected animals indicating the presence of active infection (Fig 4 ). Recovery of fungus in the spleen provides evidence of dissemination of C. posadasii to extra-pulmonary organs. Figure 4 Fungal load in the lung and spleen tissues of C. posadasii infected BALB/c mice (n = 5). The numbers of fungal colonies (CFU) were normalized with organ weight (g). Lavage SP-A and SP-D levels are unaltered in C. posadasii protected mice No significant changes were seen in SP-A or SP-D levels in BALF samples of protected (FKS immunized, C. posadasii arthroconidia infected) C57BL6 mice when compared to FKS immunized, non-infected mice (Fig 3B ). Also, there was no significant change in total protein content in BALF samples of FKS immunized, C. posadasii arthroconidia infected mice (890.9 μg) versus FKS immunized, non-infected mice (538.4 μg). SP-A and SP-D bind to Coccidioidal antigens We further examined the binding of SP-A and SP-D to Coccidioidal antigens (CDN-lysate and CDN-filtrate) coated onto microtiter wells (Fig 5 ). Both SP-A and SP-D bound to coccidioidal antigens, but not to BSA in a concentration-dependent manner (Fig 5 ). Binding of SP-A to CDN-lysate and CDN-filtrate antigens was saturable, and maximum SP-A binding was reached between 2.5–5 μg/ml and 5–10 μg/ml, respectively (Fig 5A ). Similarly binding of SP-D to CDN-lysate and CDN-filtrate antigens was also saturable, and maximum SP-D binding was reached between 5–10 μg/ml (Fig 5B ). Figure 5 Binding of (A) SP-A and (B) SP-D to Coccidioidal antigens (CDN-lysate and CDN-filtrate). The binding of 1–10 μg/ml purified human SP-A or recombinant SP-D proteins was detected in CDN-lysate or CDN-filtrate or BSA coated wells (0.5 μg/well). Results are from one representative experiment of two independent experiments performed in duplicate. Values are shown as mean+SEM. In some cases, the error bars are smaller than the symbols. Discussion In the present study we found that the levels of pulmonary surfactant collectins were altered in the lungs of C. posadasii infected mice, but were intact in lungs of C. posadasii protected mice after immunization with protective FKS vaccine. Furthermore, our results suggest that both SP-A and SP-D bind to Coccidioidal antigens. This is the first study where the amounts of SP-A and SP-D were measured in BALF samples from mice infected with lethal dose of C. posadasii and the binding of pulmonary collectins to Coccidioidal antigens was assessed. Since lung is the first organ of the body that comes into contact with air-borne C. posadasii arthroconidia, we hypothesized that the pulmonary surfactant may play an important role in regulating the immune response against C. posadasii . Among four surfactant proteins, SP-A and SP-D, interact with most of the clinically important fungal pathogens including Pneumocystis carinii [ 23 ] Cryptococcus neoformans [ 24 ], Aspergillus fumigatus [ 25 ] and Candida albicans [ 26 ]. SP-D has been shown to bind to C. albicans and directly inhibit growth by aggregation of the organism without involvement of macrophage dependent phagocytosis [ 27 ]. On the other hand, surfactant proteins also induce phagocytosis, activation and killing of A. fumigatus conidia and C. neoformans by alveolar macrophages and neutrophils [ 28 , 29 ]. In support of these findings, further evidence comes from a study by Madan et al., that suggests that the introduction of recombinant SP-D improves the lung function and increases the survival rate of mice infected with A. fumigatus [ 25 ]. The decrease in amounts of SP-A and SP-D during C. posadasii infection versus the unaltered amounts in the lungs of protected mice and binding of collectins to Coccidioidal antigens indicate that pulmonary collectins may be involved in uptake/phagocytosis of C. posadasii by antigen presenting cells and downstream immune regulation. Besides changes in amounts of SP-A and SP-D, a decrease in the amount of BALF phospholipids was also observed in C. posadasii infected mice (Table 2 ). Earlier, Sheehan et al., [ 29 ] and Hoffman et al., [ 30 ] have reported similar findings of reduced surfactant phospholipid level in BALF samples of rats and humans infected with Pneumocystis carinii [ 29 , 30 ]. To date, however, the information is lacking concerning how surfactant phospholipids may be involved in host defense [ 31 ]. Likewise, the mechanisms underlying the decrease of BALF surfactant in murine model of Coccidioidomycosis remain to be defined. We speculate that the reduction in the collectins and phospholipids could be either due to metabolic dysfunction of pulmonary type II epithelial cells during C. posadasii infection or due to their utilization in the binding and uptake of C. posadasii by local antigen presenting cells. The metabolic pathways of pulmonary type II epithelial cells may be affected by secondary inflammatory mediators, such as TNF-α or IL-1β, secreted by inflammatory cells during C. posadasii infection. A variety of host inflammatory mediators and substances such as, cytokines (TNF-α) and growth factors are released during infection and inflammation. These cytokines and growth factors affect the synthesis and secretion of pulmonary surfactant by pulmonary epithelial cells [ 32 , 33 ]. In the present study we found slightly increased levels of SP-D in C. posadasii protected mice, but not of SP-A (Fig 2 ). We speculate that the difference could be due to diverse mechanisms for regulation of SP-A and SP-D expression. Probably the cytokines and chemokines that are released as a result of FKS vaccination (protective immune response) increase the SP-D expression, but do not affect the SP-A expression. As reported earlier, the expression of SP-A and SP-D is differentially regulated during lung infection [ 34 ]. In future, more studies are warranted to understand the mechanism of the alterations in levels of surfactant phospholipids, SP-A and SP-D. At present, the treatment of C. posadasii infected patients with disseminated disease is not very effective [ 35 ]. Treatment with anti-fungal agents is most often related to relapse of the infection and side effects on the body. Earlier, the FKS based immunization has been found protective against Coccidioides infection in murine model of coccidioidomycosis, but failed in humans [ 35 ]. More pre-clinical studies on developing different vaccine strategies are underway. Since C. posadasii and C. immitis are highly virulent organisms, cause endemic infection, and pose a risk for bioterrorism, there is an urgent need for discovery of improved therapeutic drugs and regimens or preventive vaccines [ 3 , 35 ]. Conclusions In future, clearance experiments after in vivo administration of artificial or natural surfactant in C. posadasii infected mice may be useful in determining their therapeutic usefulness. We speculate that the findings from our present study would initiate similar studies to understand the role of pulmonary innate immune components in infectious diseases caused by C. posadasii or other virulent respiratory pathogens. Authors' Contributions SA designed and co-ordinated the study, performed assays and statistical analysis and drafted the manuscript. JJC assessed lung pathology. DMM prepared and immunized mice with FKS. All authors read and approved the final manuscript.

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554109

Rural Indian tribal communities: an emerging high-risk group for HIV/AIDS

Background Rural Indian tribes are anthropologically distinct with unique cultures, traditions and practices. Over the years, displacement and rapid acculturation of this population has led to dramatic changes in their socio-cultural and value systems. Due to a poor health infrastructure, high levels of poverty and ignorance, these communities are highly vulnerable to various health problems, especially, communicable diseases including HIV/AIDS. Our study sought to assess knowledge, attitudes and practices regarding sexuality, and the risk factors associated with the spread of HIV/AIDS and STDs among these communities. Methods A nested cross sectional study was undertaken as part of the on going Reproductive and Child Health Survey. A total of 5,690 participants age 18–44 were recruited for this study. Data were obtained through home interviews, and focused on socio-demographics, knowledge, attitudes and behaviors regarding sexuality, HIV/AIDS and other STDs. Results The study revealed that only 22% of adults had even heard of AIDS, and 18 % knew how it is transmitted. In addition, only 5% knew that STDs and AIDS were related to each other. AIDS awareness among women was lower compared to men (14% vs.30 %). Regarding sexual practices, 35% of the respondents reported having had extramarital sexual encounters, with more males than females reporting extramarital affairs. Conclusion Lack of awareness, permissiveness of tribal societies for premarital or extra-marital sexual relationships, and sexual mixing patterns predispose these communities to HIV/AIDS and STD infections. There is a dire need for targeted interventions in order to curtail the increasing threat of HIV and other STDs among these vulnerable populations.

Background India is the second most populous nation in the world and has changing sociopolitical and demographic characteristics as well as varied morbidity and mortality patterns [ 1 ]. These changes, in conjunction with the country's high population growth rate, have exacerbated the prevailing and emerging public health challenges the country is facing. Since 1986 when the first case of human immunodeficiency virus (HIV) was reported in India [ 2 ], it has become imperative to include acquired immunodeficiency syndrome (AIDS) on its long list of public health issues that need to be addressed. As a direct result of these challenges, India has begun to assess and monitor the impact of HIV/AIDS throughout the country's various states and regions with the assistance of several international health organizations. According to 2003 estimates from UNAIDS, approximately 5.1 million individuals in India are infected with the HIV virus [ 3 ]. Furthermore, recent studies indicate that transmission of HIV is no longer confined to high-risk urban populations, but is spreading across rural settings as well [ 4 ]. This trend is a cause for concern as AIDS is increasingly hampering social and economic development throughout the country. For effective control of the spread of HIV/AIDS, it is crucial to have data on knowledge, attitudes and behavioral practices for specific population as research has shown that socio-cultural influences, traditional lifestyles, societal norms, and traditions influence HIV/AIDS transmission rates [ 5 , 6 ]. Because India's HIV/AIDS transmission pattern is predominantly heterosexual (85% of all newly reported cases) [ 7 ], subcultures that have relaxed marital structures or are tolerant of high-risk sexual practices (e.g., sex with a commercial sex worker) are particularly vulnerable to the spread of HIV/AIDS and STDs within their communities [ 7 , 8 ]. With more Indian men reporting premarital and extramarital sexual activity, women who marry as teenagers are vulnerable to HIV/AIDS infection and STDs [ 9 , 10 ]. The rapid spread of HIV/AIDS in rural Indian communities has been attributed to the country's poor health infrastructure, poverty and lack of awareness [ 4 , 10 ]. Despite these indicators, little is known about the risk factors, transmission rates, or the impact AIDS will have in these areas in the future. Traditionally, there has been little research and only a paucity of health-related research conducted among this potentially high-risk vulnerable population. Throughout India, approximately 8% of the population lives within rural tribal communities, which are collectively referred to as 'Tribes'. These communities are geographically distinct; with each tribe having its own unique customs, traditions, beliefs and practices. Even within a particular tribal entity, differences in dialect, health practices, unique customs, values, and traditions are apparent. In rural Indian communities indices of reproductive health are typically very poor: maternal mortality rate is about 230 per 100,000 live births and 61.2% of the women suffer from at least one gynecologic pathology [ 11 ]. Because tribal groups have existed on the fringe of Indian society, they may still be unaware or indifferent to the potential health threats from HIV/AIDS. Ascertaining whether or not tribal communities are potentially a high risk group warranting intervention is a necessary step in India's war on AIDS. Accordingly, we undertook this study to explore the risks for this special group of people. Methods We conducted a cross-sectional study nested within an existing enumerative study referred to as the Reproductive and Child Health Survey (RCHS). The RCHS was initially designed to enumerate and ascertain basic demographic and health profiles for all tribal members. Data collection for this particular study was done in two phases. Phase one involved adding additional questions to the original RCHS study to assess risk factors (knowledge, attitudes and behavioral practices) associated with the transmission of HIV/AIDS and other communicable diseases. Study population The study population comprised of tribal communities living in the southern region of Karnataka (Figures 1 , 2 ). Members of these tribes have traditionally derived much of their livelihood from the country's vast reserve of natural forest resources. However over the years, these tribes have been forced to migrate from their ancestral land and are currently living within poor rural communities throughout the state. The initial displacement was as result of the submergence of their traditional homelands through the construction of the Kabini dam, and the second displacement was as a result of 'Project Tiger,' a wildlife conservation project that displaced them to their current location in the southern region of Karnataka, where this study was conducted. Figure 1 Map of India showing the region of Karnataka where the study was conducted. Used with permission from India-tourism.com Figure 2 The reproductive and child health program area in the southern part of Karnataka. Used with permission from the Reproductive Child Health Survey (RCHS) project Sampling Because of the enumerative nature of the RCHS, all persons within the age group 18–44 years who participated in the RCHS study were included for phase one of this study. This age group was selected as the focus of the initial study was on reproductive health issues. Survey instruments A semi-structured questionnaire with both open- and close-ended questions was developed to collect information on knowledge, attitudes and behaviors regarding HIV/AIDS, as well as other relevant demographic information not included in the RCHS. The instrument was developed in English, translated to the native language, and subsequently back translated to English for content and language verification. The survey instrument was field-tested for validity purposes and modified accordingly. Data collection A team of ten interviewers from the local tribal communities with a minimum of high school education were selected and trained for two weeks to ensure uniform and high-quality data collection. All adults were interviewed separately to ensure confidentiality. Each interviewer read out each of the questions and response choices (where appropriate) to the interviewees and recorded all answers directly on the questionnaire. Verbal informed consent was obtained from each respondent prior to starting the interview. Data analysis Data were entered into an electronic database using Sybase Central Software (Sybase, Inc. Dublin, CA). To ensure confidentiality, all respondent identifiers were expunged to create a secondary data set that was used for the final analysis. Frequency tables were generated for selected demographics and health related categorical variables. In addition, univariate analysis was performed on relevant continuous variables. The findings are presented below. Ethical consideration Ethical clearance was obtained from ethical boards within each of the tribal communities, and appropriate government agencies were informed about the study objectives. Permission was also obtained from the Institutional Review Board (IRB) at the University of South Florida in Tampa, Florida. Verbal informed consent was obtained from the local leaders, and the individual participants. Because of the high levels of illiteracy, it was not feasible for us to obtain written consent. Results Demographic profile A total of 11,379 individuals from the 5 tribal communities had been enumerated as part of the RCHS. Of these, 5,690 were within the study age range (18–44 years) and formed the basis for this analysis. Table 1 shows the demographic profile of the study participants. The mean age of the study group was 31 years. There were more males than females; 53% vs.47%. Eighty four percent were married (91% females & 78% males). The average age at marriage was 13 years for females, and 22 years for males. Only 28% (27% female, & 30% male) of the population was literate i.e. able to read and write in any of the Indian languages. The majority of respondents (67 %) reported living in tiled roof houses with mud flooring, while only 40 % indicated easy access to potable water. Agriculture was the major source of income in these communities. The reported average daily income ranged from US $1.50 to $2.00. Approximately 35% of the respondents migrated on average three to four months each year to nearby areas for work. Table 1 Demographic profile of the study participants Characteristics Number (%) (N = 5,690) Mean age 31 years Sex Men 3,016 (53) Women 2,674 (47) Currently living in tiled-roof housing 3,812 (67) Access to potable water 2,276 (40) Migrated to find work 1,992 (35) Currently married Men 2,353 (78)* Women 2,433 (91)** All 4,786 (84) Literacy*** Men 814 (27)* Women 802 (30)** All 1,616 (28) Mean age of first marriage Mean years (95% CI) Men 22.0 (19.5–24.5) Women 13.0 (11.2–14.7) All 15.0 (11.2–18.8) *Percentages are based on the total number of men in the study. **Percentages are based on the total number of women in the study. ***Literacy as defined by census operations of India is the ability to read and write in any of the Indian languages. CI = Confidence interval Unique sexual practices among tribal members The findings revealed that these tribal communities did not have a structured marital system; instead members practiced a form of serial monogamy in which they change partners and remarry every four to five years. Regarding sexual practices, 35% of the respondents reported either premarital affairs or extramarital affairs (Table 2 ). However such practices were more common in men compared to women. Furthermore, 20% of the male participants reported having had sex with a commercial sex worker (CSW) during the period the wife had had a child. Table 2 Self reported sexual practices of respondents Characteristics N = 5,690 Age at first sexual activity Mean years (95% CI) Men 17.0 (13.4–20.6) Women 13.0 (11.5–14.5) Premarital or extramarital sexual encounters Number (n) (%) Males 1,434 72.0 Females 558 28.0 Total 1,992 35* Sex with commercial sex worker within a year after spouse giving birth (men only n = 3,016) 470 20 * Percent based on the total sample of 5,690 respondents Knowledge and beliefs about HIV/AIDS and STDs Among these communities, there was a low level of knowledge on HIV/AIDS; only 22 % of all study participants (n = 1,252) had heard of AIDS (Table 3 ). Among those who have heard of AIDS, less than 20 % (n = 250) knew how HIV/AIDS was transmitted (16.8 % male vs. 8% females). About 98 % were not aware of the methods to prevent HIV/AIDS transmission. As many as 30 % (n = 376) of those who had heard of AIDS believed that "sinners" will get AIDS, while 10 % (n = 125) believed that AIDS and STDs could be prevented by the sterilization of women. Fifteen percent (n = 188) thought "AIDS is acquired by looking at a person who has AIDS," and 18 % (n = 225) believed that "AIDS is acquired by talking to a person who has AIDS." Only 5 % knew that a relationship exists between HIV/AIDS and STDs. Interestingly, 4 percent (n = 51) believed that there was a cure for AIDS. Most had not heard of STDs, and of those who had heard of them only 1 percent (n = 16) were aware of associated symptoms. Table 3 Knowledge and beliefs about HIV/AIDS and STDs Knowledge and beliefs about HIV/AIDS No. of study subjects N = 5,690 No. of study subjects who have ever heard of HIV/AIDS N = 1,252 N Percent of 5,690 Percent of 1,252 Have ever heard of HIV/AIDS 1,252 22.0 100.0 Know how HIV/AIDS is transmitted 250 4.4 20.0 Know methods to prevent the transmission of HIV/AIDS 114 2.0 9.1 Believe "sinners" will get AIDS 376 6.6 30.0 Believe AIDS and STDs can be prevented by sterilization of women 125 2.2 10.0 Believe AIDS is acquired by looking at an infected person with AIDS 188 3.3 15.0 Believe AIDS is acquired by talking to a person who has AIDS 225 4.0 18.0 Believe there is a cure for HIV/AIDS 51 0.9 4.1 Discussion In today's modern world, it is difficult to imagine societies that are still socially and culturally isolated from the rest of civilization; however, they do exist. The tribal societies throughout India have remained socially and culturally alienated from mainstream Indian society until developmental and conservation activities in tribal areas forced interactions between them. Displacement of the tribal people of southern Karnataka has led to a complex process of rapid acculturation and loss of cultural identity; as they struggle to maintain their traditional social structure, they must adopt new skills, beliefs, and practices necessary for success in their new environment. During this acculturation process, they have been faced with a myriad of public health challenges complicated by poverty, ignorance, and reluctance to abandon traditional beliefs and practices that would allow them to assimilate successfully. To date this is the first health related study among the displaced tribal communities of southern Karnataka that has attempted to assess risk threshold for the transmission and spread of HIV/AIDS and other STDs. It is not surprising that knowledge and awareness about HIV/AIDS and STDs was very low among tribal communities compared to the national figures given the degree of isolation, low literacy rates, and minimal access to information. The high level of poverty, inadequate health resources, ignorance and high-risk beliefs and practices among the tribal communities has contributed to the vulnerability of this population. As such it has created a highly susceptible population for the rapid spread of HIV/AIDS and other STDs' as well. Because tribal members are forced to migrate outside of their communities in search for work and increased wages, this may contribute to the spread of HIV/AIDS as many engage in extramarital affairs, seek commercial sex partners, or are under the threat of sexual harassment (females). While there has been limited scientific research exploring the cultural context of extramarital sexual behaviors, it is generally noted that in these communities, extramarital affairs are condoned and widely practiced especially during periods when women are pregnant or nursing or during period of travel for work [ 8 ]. This kind of behavior creates a fertile ground for HIV transmission and spread. Our data indicate that tribal women are particularly vulnerable for HIV/AIDS in this population since many of them commence sexual activity at an early age, and get married early as well. Also, they are in a culture that condones extramarital sex, and this exposes the women to a particularly precarious situation, increasing their risk for acquiring HIV. Conclusion It is evident from this study that the Indian tribal community is experiencing a latent phase that is potentially a precursor for an HIV/AIDS epidemic. There is a high prevalence of behavioral risk factors, coupled with ignorance, and inadequate health infrastructure thus creating a potential risk for rapid spread of HIV/AIDS, as well as other related diseases. In a country that is struggling to contain the spread of HIV, it is particularly important for concerned parties to pay attention to this population. Currently, virtually no resources are allocated toward the treatment of those infected with HIV/AIDS; the main stay of management is through education and preventive measures to control the spread of the scourge as they represent the most practical and cost effective strategies in this developing nation. It therefore becomes imperative and urgent to address the health concerns revealed in our study in order to formulate effective, culture-sensitive and appropriate intervention programs so that an imminent disaster (i.e. HIV/AIDS epidemic) in this remote and isolated communities can be averted. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All the authors were involved in the design of the study, analysis, interpretation and development of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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374243

Accelerated Evolution of the ASPM Gene Controlling Brain Size Begins Prior to Human Brain Expansion

Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume. The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates. Mutations in ASPM , which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH. Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast. We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change. The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years. Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM . Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals. We report the first direct application of TAR cloning technology to the study of human evolution. Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.

Introduction The human brain, particularly the cerebral cortex, has undergone a dramatic increase in its volume during the course of primate evolution, but the underlying molecular mechanisms that caused this expansion are not known. One approach shedding light on the molecular mechanisms of brain evolution is the analysis of the gene mutations that lead to defects in brain development. Among the best examples of such defects is the human primary microcephaly syndrome. Primary microcephaly (MCPH) is an autosomal recessive neurodevelopmental disorder in which the brain fails to achieve normal growth. The affected individuals have severe reduction in brain size; however, the gyral pattern is relatively well preserved, with no major abnormality in cortical architecture ( McCreary et al. 1996 ; Mochida and Walsh 2001 ). Moreover, there are no recognizable abnormalities in the organs other than the central nervous system. The most common cause of MCPH appears to be mutations in the ASPM gene ( Roberts et al. 2002 ). The ASPM gene encodes a 10,434-bp-long coding sequence (CDS) with 28 exons, and spans 65 kb of genomic DNA at 1q31. ASPM contains four distinguishable regions: a putative N-terminal microtubule-binding domain, a calponin-homology domain, an IQ repeat domain containing multiple IQ repeats (calmodulin-binding motifs), and a C-terminal region ( Bond et al. 2002 ). Though the exact function of the human ASPM in the brain needs to be clarified, the homologue in the fruit fly, Drosophila melanogaster, abnormal spindle (asp), is localized in the mitotic centrosome and is known to be essential for both the organization of the microtubules at the spindle poles and the formation of the central mitotic spindle during mitosis and meiosis. Mutations in asp cause dividing neuroblasts to arrest in metaphase, resulting in reduced central nervous system development ( Ripoll et al. 1985 ; do Carmo Avides et al. 2001 ; Riparbelli et al. 2001 ). In the mouse (Mus musculus) brain, the Aspm gene is expressed specifically in the sites of active neurogenesis. Expression in the embryonic brain was found to be greatest in the ventricular zone, which is the site of cerebral cortical neurogenesis ( Bond et al. 2002 ). This expression profile suggests a potential role for Aspm in regulating neurogenesis. Interspecies comparisons of ASPM orthologs have shown their overall conservation, but also a consistent correlation of greater protein size with larger brain size ( Bond et al. 2002 ). The increase in protein size across species is due mainly to the increased number of IQ repeats, suggesting that specific changes in ASPM may be critical for evolution of the central nervous system. In an attempt to reconstruct the evolutionary history of the ASPM gene, we isolated large genomic clones containing the entire ASPM gene in several nonhuman primate species. Sequence analysis of these clones revealed a high conservation in both coding and noncoding regions, and showed that evolution of the ASPM gene might have been under positive selection in hominoids. These clones could also provide important reagents for the future study of ASPM gene regulation in its native sequence context. Results Comparison of Genomic Organization of the ASPM Genes in Primates Homologues from chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) were isolated by transformation-associated recombination (TAR) cloning in yeast (Saccharomyces cerevisiae), the technique allowing direct isolation of a desirable chromosomal region or gene from a complex genome without constructing its genomic library ( Kouprina and Larionov 2003 ). The method exploits a high level of recombination between homologous DNA sequences during transformation in the yeast. Since up to 15% divergence in DNA sequences does not prevent selective gene isolation by in vivo recombination in yeast ( Noskov et al. 2003 ), for cloning purposes, a TAR vector was designed containing short human ASPM -gene-specific targeting hooks specific to the exon 1 and 3′ noncoding regions (see “Materials and Methods” ). The TAR cloning scheme for isolating the ASPM gene homologues from nonhuman primates is shown in Figure 1 . The yield of ASPM -positive clones from chimpanzee, gorilla, orangutan, and rhesus macaque was the same as that from the human DNA, suggesting that most homologous regions from nonhuman primates can be efficiently cloned by in vivo recombination in yeast using targeting hooks developed from human sequences. Figure 1 Isolation of the Syntenic Genomic Regions Containing the ASPM Gene from Human, Chimpanzee, Gorilla, Orangutan, and Rhesus Macaque by TAR Cloning The method exploits a high level of recombination between homologous DNA sequences during transformation in the yeast Saccharomyces cerevisiae . For isolation, genomic DNA is transformed into yeast spheroplasts along with a TAR vector that contains targeting hooks homologous to the genomic DNA sequence. CEN corresponds to the yeast Chromosome VI centromere; HIS3 is a yeast selectable marker. Recombination between the vector and the genomic DNA fragment results in cloning of the gene/region of interest as YAC. Chromosomal regions with sizes up to 250 kb can be isolated by TAR cloning. For cloning purposes, TAR vector was designed containing a 5′ hook specific to exon 1 and a 3′ hook specific to the 3′ end of the human ASPM . Transformation experiments were carried out with freshly prepared spheroplasts for each species. To identify ASPM -containing clones, the transformants were combined into pools and examined by PCR for the presence of the unique ASPM sequences not present in the vector. The yield of ASPM -positive clones from primate species was the same as that from the human DNA (3%). Because the TAR procedure produces multiple gene isolates, six independent TAR isolates for each species were checked. The detectable size of the cloned material corresponded to that predicted if the entire ASPM gene had been cloned, i.e., all gene-positive clones contained circular YACs with approximately 65-kb DNA inserts. Alu profiles for each species were determined and found to be identical for each species, suggesting that the isolated YACs contained nonrearranged genomic segments. Finally the YACs were retrofitted into BACs, and their restriction patterns were examined by three restriction endonuclease digestions. No differences between ASPM clones for each species were found. We have compared complete gene sequences from primate species with a 65-kb, full-size human ASPM gene. All the analyzed genes are organized into 28 exons encoding a 3,470–3,479-amino-acid-long protein. ASPM genes start with an approximately 800-bp-long CpG island, that harbors promoter sequences, 5′ untranslated regions, and the first exon ( Figure 2 ). ASPM sequences share a high degree of conservation ( Figure 2 H), and pairwise DNA identity ranges from 94.5% for macaque and gorilla to 99.3% for the human–chimpanzee comparison ( Table 1 ). Multiple alignment of the genes revealed a low proportion of indels. Only ten insertions/deletions equal to or longer than 50 bp have been found, all of them located within introns ( Figure 2 B). Seven detected insertions were mainly associated with repetitive DNA: two (AT) n microsatellite expansions, three Alu insertions, including retroposition of AluYi9 in the orangutan–gorilla–chimpanzee–human clade, and retroposition of a new macaque-specific AluY subfamily similar to human AluYd2 . Analysis of eight different macaque individuals showed that this particular insertion is polymorphic in the macaque population (data not shown), and thus the insertion appears to be very recent. One macaque-specific 245-bp-long insertion is linked to expansion of a 49-bp-long, minisatellitelike array. The remaining macaque-specific insertion (50 bp) is nonrepetitive. A closer analysis suggests that the insert is not a processed pseudogene of known genes (data not shown). Figure 2 Structure and Evolution of the ASPM Gene in Primates The scale of all plots corresponds to the consensus sequence obtained based on a multiple alignment of five ASPM genes. (A) Schematic representation of the alignment. Promoter regions, exons, and introns are marked in gray, red, and blue, respectively. White segments correspond to gaps. (B) Positions of long (50 bp or longer) insertions/deletions. “O” denotes orangutan, “M” macaque, “OGCH” the orangutan–gorilla–chimpanzee–human clade, and “GCH” the gorilla–chimpanzee–human clade. (C) Positions of polymorphic bases derived from the GenBank single nucleotide polymorphism (SNP) database. (D) Positions of the CpG island. The approximately 800-bp-long CpG island includes promoter, 5′ UTR, first exon, and a small portion of the first intron. (E) Location of an approximately 3-kb-long segmental duplication. (F) Positions of selected motifs associated with genomic rearrangements in the human sequence. Numbers in parentheses reflect number of allowed differences from the consensus motif (zero for short or two ambiguous motifs, two for longer sites). (G) Distribution of repetitive elements. The individual ASPM genes share the same repeats except of indels marked in (B). (H) DNA identity and GC content. Both plots were made using a 1-kb-long sliding window with 100-bp overlaps. The GC profile corresponds to the consensus sequence; the individual sequences have nearly identical profiles. Table 1 Pairwise Identity of Aligned Primate ASPM Genes The pairwise identities were calculated for five complete ASPM genes and therefore include all promoter regions, introns, and exons. The values above the diagonal show DNA identities (in percent) calculated after removing indels. Under the diagonal are values for comparisons with gaps Of the two detected deletions, the macaque-specific 72-bp-long deletion appears to be associated with nonrepetitve DNA. The second one, an 818-bp-long deletion in orangutan, was probably caused by homologous Alu–Alu recombination (see below and Figure S1 ). The remaining indels are related to expansion/contraction of a short minisatellite array. It was caused either by a 53-bp expansion in the gorilla–chimpanzee–human clade or by two independent deletions/contractions in the macaque and orangutan lineages. An approximately 3-kb-long intronic segment between exons 4 and 5 is present in several copies in the genome ( Figure 2 E; Figure S2 ). Closer analysis of the human genome confirmed that copies of this region are homologous to 24 segmental duplications located mainly in telomeric regions of Chromosomes 1–8, 10, 11, 16, 19, 20, and Y. Based on the sequence similarity and the presence of an L1P4 LINE insertion at the 5′ end, the most closely related are three duplications at 7q11–13. The most similar copy is located on Chromosome 7 and shares 93% identity with the ASPM intronic segment. Five duplications are located on Chromosome 1; the closest copy is found 27 Mb away from the ASPM gene. We looked for several common motifs associated with genomic breakpoints in cancers ( Abeysinghe et al. 2003 ). Figure 2 F shows the positions of such potentially unstable oligonucleotides. Interestingly, the orangutan-specific deletion ( Figure 2 B) has its 5′ breakpoint located just 1 bp upstream of a sequence 100% identical to the chi-like consensus motif GCWGGWGG (see Figure S1 ). The chi motif is recognized by the RecBCD-mediated recombination pathway in prokaryotes and seems to be associated with rearrangements in the human genome ( Dewyse and Bradley 1991 ; Chuzhanova et al. 2003 ). Both deletion breakpoints in the orangutan deletion are located within 5′ parts of two Alu sequences, suggesting that the deletion was created by homologous Alu–Alu recombination. Similar homologous recombinations with breakpoints located near chi-like motifs in 5′ regions of Alu sequences were described previously ( Chen et al. 1989 ; Rudiger et al. 1995 ). In summary, despite the presence of a few indels, coding and noncoding regions of ASPM homologues show a marked degree of conservation. Evolution of the ASPM Protein We have analyzed ASPM CDSs from six primate species: human, chimpanzee, gorilla, orangutan, rhesus macaque, and African green monkey (Cercopithecus aethiops) . Except for orangutan and rhesus macaque, two or more ASPM CDSs were used for analysis. ASPM proteins showed the same overall length and domain structure ( Figure 3 A). The IQ repeat domain contains the same number of repeats, suggesting that their expansion occurred in early primate evolution. The CDSs are, as expected, more conserved than the complete gene sequences with promoter and intronic regions ( Table 2 ; Table 3 ). Only six short indels were identified ( Figure 3 B). Figure 3 Structure of ASPM CDSs and Evolution in Primates The scale of all plots corresponds to the 3,480-amino-acid-long protein alignment; positions in the CDS were scaled accordingly. (A) Structure of the human ASPM CDS and protein. The first scheme shows positions of major domains in the ASPM protein ( Bond et al. 2002 ). The putative microtubule-binding domain is in gray, the calponin-homology domain in orange, IQ repeats in blue, and the terminal domain in black. Positions of exons in the CDS are drawn in the second block. To separate individual exons, odd numbered exons are colored in black and even numbered ones in white. (B) Positions of insertions/deletions in the protein sequences. Coordinates correspond to the human protein sequence. “O” denotes orangutan, “G” gorilla, “M” macaque, “Gm” African green monkey, and “OGCH” the orangutan–gorilla–chimpanzee–human clade. (C) Substitutions in hominoid CDSs relative to the common ancestor. The expected ancestor CDS was derived using ML codon reconstruction implemented in PAML. African green monkey and rhesus macaque were outgroups. Nonsynonymous/synonymous (ω = Ka/Ks) ratios were free to vary in all branches. Positions marked in green correspond to synonymous changes relative to the ancestral sequence; the red bars indicate nonsynonymous changes. (D) Synonymous (red) and nonsynonymous (green) changes in ancestral lineages leading to human. aOGCH–aGCH is the ancestral lineage from the orangutan divergence to the gorilla divergence; aGCH–aCH represents the lineage from the gorilla divergence to the chimpanzee common ancestor. aCH–human corresponds to the human lineage after the chimpanzee divergence. There are seven synonymous and 19 nonsynonymous human-specific substitutions. Methods and description are the same as in (C). (E) Positions of polymorphic bases for different CDSs of African green monkey, gorilla, chimpanzee, and human. Positions marked in green correspond to synonymous polymorphisms, and the red bars indicate nonsynonymous sites. Numbers of compared sequences are in parentheses; in the case of human we show nine polymorphic positions (four synonymous and five nonsynomous) from the GenBank SNP database. ASPM mutations detected in MCPH patients are shown separately in (F). (F) Positions of 19 mutations reported for MCPH patients ( Bond et al. 2002 ; Bond et al. 2003 ). All the reported mutations introduce premature stop codons. Mutation sites located within CpG dinucleotides are highlighted in red. (G) Positions of CpG dinucleotides in the human CDS. (H) Comparison of Ka and Ks rates with codon adaptation index (CAI). Ka and Ks values are for all branches (fixed ω ratio); CAI is an average for all five primates (note that CAI differences are very small between the five species). The window was set to 300 bp (100 amino acids) with a 30-bp (10-amino-acid) step. (I) Conservation at the nucleotide and protein level in primates. Y-axis corresponds to proportions of conserved (identical) positions in the CDS and the protein alignment. The plot was obtained using 100-amino-acid-long, overlapping windows, and the step was set to 10 amino acids. In the case of CDS conservation, the window was 300 bp and step 30 bp. Table 2 Pairwise Identity of ASPM CDSs The pairwise identities were calculated for six CDSs. The values above the diagonal show DNA identities (in percent) calculated after removing indels. Under the diagonal are values for comparisons with gaps Table 3 Pairwise Identity of ASPM Proteins The pairwise identities were calculated for six protein sequences. The values above the diagonal show DNA identities (in percent) calculated after removing indels. Under the diagonal are values for comparisons with gaps From the DNA and protein conservation profiles ( Figure 3 I), ASPM segments evolve differently along the length of the CDS. N- and C-terminal regions and the region corresponding to exons 5–15 are conserved. In contrast, exons 3 and 4 and the complete IQ repeat domain (positions 1,267–3,225) are more variable. Indeed, nonsynonymous substitutions in hominoid primates ( Figure 3 C) and in ancestral lineages ( Figure 3 D) and nonsynonymous polymorphism ( Figure 3 E) are nearly absent in the conserved central (exons 5–15) and C-terminal regions. This pattern indicates different rates of evolution along the ASPM protein, visualized by plots of synonymous Ks and nonsynonymous Ka rates ( Figure 3 H) and supported by phylogenetic analysis (see below and Figure 4 ). It is notable that the comparison of the primate and mouse proteins also revealed the same pattern of conservative and nonconservative regions along ASPM protein ( Figure S3 ). Figure 4 Phylogenetic Trees and ω ratio for Complete ASPM and Three Selected Segments Trees and ω (Ka/Ka) ratios were computed using the ML method for codons implemented in PAML. Branch lengths represent ML distances for codons, i.e., using both synonymous and nonsynonymous nucleotide sites, and in all branches the ω ratio was set free to vary. All trees are drawn to the same scale. Branch labels mark the ω ratios for corresponding branches. Values in square brackets show ω for additional cDNA sequences whenever available. Default values and branch lengths were calculated from genomic copies. Selected tested hypotheses are listed. ω H stands for the ω rate in the human lineage, ω C in the chimpanzee lineage, ω CH in the common human–chimpanzee ancestral lineage after the gorilla divergence, ω G in the gorilla lineage, and ω 0 in all other branches. Single asterisks indicate p < 0.05, χ 2 1 = 3.84; double asterisks indicate p < 0.01, χ 2 1 = 6.63. (A) Phylogeny for the complete ASPM CDS. In addition to testing different ω values in the human lineage, we also tested the hypothesis that the complete gorilla–chimpanzee–human clade evolved at a constant rate, different from the rest of the tree (compared to the one-ratio model, boxed). (B) The ASPM phylogeny derived from a conserved segment from exon 5 to the beginning of the IQ domain (amino acids 676–1,266). The branch connecting the human and chimpanzee common ancestor with the gorilla–chimpanzee–human common ancestor had no substitutions, therefore the ω ratio could not be calculated. (C) IQ domain (amino acids 1,267–3,225). We also tested the hypothesis that the gorilla and human lineages evolved at the same ω rate, different from the rest of the tree (compared to the one-ratio model, boxed). (D) Phylogeny of eight primate sequences from a 1,215-amino-acid-long segment of exon 18 (amino acids 1,640–2,855). We also tested the hypothesis that the gorilla and human lineages evolved at the same ω rate, different from the rest of the tree (compared to the one-ratio model, boxed). Analysis of the nonsynonymous/synonymous substitution ratio (ω = Ka/Ks) revealed an elevated value in the human branch ( Figure 4 A). According to the likelihood ratio test, the human ω rate is significantly different from the rate in the rest of the tree ( p < 0.05). Also the model that the complete gorilla–chimpanzee–human clade is evolving at one ω rate different from that in the rest of the tree is well supported ( p < 0.01). Because ASPM consists of regions with different degrees of sequence conservation (see Figure 3 ), we separately analyzed a conserved region (exons 5–15 plus a small part of exon 16) and a variable IQ repeat domain. As can be seen ( Figure 4 B) the conserved region has all branches shorter, indicating overall a slower rate of evolution. In the human lineage, the ω ratio equals zero; however, the test for whether the human branch has a different (lower) ω rate than the rest did not yield significant values. In contrast, the tree based on the variable IQ repeat domain exhibits ω values greater than one for the human and gorilla branches ( Figure 4 C). The likelihood ratio test supports the model in which human and gorilla lineages evolved under a significantly higher ω ratio than the rest of the tree. Similar results were obtained for exon 18 with additional sequences from two New World monkeys ( Figure 4 D). As seen from Figure 4 A– 4 D, different sequences from African green monkey, gorilla, and chimpanzee individuals result in different ω values for their corresponding terminal branches. One chimpanzee sequence also produced an ω ratio greater than one for exon 18 ( Figure 4 D). It is worth noting that neither codon bias nor selection on third codon positions seemed to influence the synonymous rate Ks strongly ( Table S1 ). Therefore, the high Ka/Ks ratios in human and gorilla are likely to be products of adaptive evolution. Sequencing of two CDSs in African green monkey, three in gorilla, and three in chimpanzee allowed us to look for ASPM polymorphism in those species (see Figure 3 E). Human polymorphism data from ASPM mutant haplotypes are not representative of wild-type variation so were not used in these comparisons. For African green monkey, five synonymous and five nonsynonymous changes were found between two sequences. The gorilla and chimpanzee CDSs in particular showed an apparently high degree of replacement polymorphism. Gorilla polymorphism included 35 point mutations (15 silent mutations and 21 replacements). Chimpanzee sequences differed in five synonymous and 11 nonsynonymous sites. In order to interpret this seemingly high level of observed polymorphism, intraspecific diversity was compared to interspecific diversity using the McDonald and Kreitman test ( McDonald and Kreitman 1991 ). In the case of chimpanzee polymorphism compared to divergence with human, we could not reject the null hypothesis that polymorphism and divergence between species were significantly different (William's adjusted G statistic = 0.083, chi-square with 1 d.f., not significant; values based on PAML-generated Ka and Ks values using the free ratio model). Gorilla polymorphism was compared to divergence between the gorilla common ancestor and the human–chimpanzee common ancestor. In this case we can reject the null hypothesis (William's adjusted G statistic = 122.45, chi-square with 1 d.f., p < 0.001) to conclude that the pattern of gorilla polymorphism is therefore different from the divergence pattern. Indeed gorilla polymorphism is less than variation resulting from divergence: within species, the ω ratio is 1.43 for gorillas compared to 2.2 for the divergence between the gorilla common ancestor and the human–chimpanzee common ancestor. Intraspecific variation, although seemingly unusual in showing so many replacement substitutions in both chimpanzee and gorilla, is less than or in line with what we have observed for ASPM divergence between species. Therefore, relaxation of selection cannot explain the high nonsynonymous/synonymous substitution ratios among African hominoids, further supporting the idea that adaptation has occurred in ASPM. Discussion In this study, we applied TAR cloning technology to investigate molecular evolution of the ASPM gene, which is involved in determining the size of the human brain and in which mutations lead to MCPH. The ASPM homologue in the fruit fly is essential for spindle function, suggesting a role for this gene in normal mitotic divisions of embryonic neuroblasts. Complete gene homologues from five primate species were isolated and sequenced. In agreement with the predicted critical role of ASPM in brain development, both coding and noncoding regions of ASPM homologues showed a marked degree of conservation in humans, other hominoids, and Old World monkeys. The differences found in noncoding regions were small insertions/deletions and lineage-specific insertions of evolutionarily young Alu elements into introns. Analysis of nonsynonymous/synonymous substitution ratios indicates different rates of evolution along the ASPM protein: part of ASPM evolved under positive selection while other parts were under negative (purifying) selection in human and African ape lineages. Such “mosaic” selection has been previously described for other proteins ( Endo et al. 1996 ; Crandall et al. 1999 ; Hughes 1999 ; Kreitman and Comeron 1999 ). When our work was completed, the paper by Zhang supporting accelerated evolution of the human ASPM was issued ( Zhang 2003 ). However, because the author did not analyze the gorilla gene homologue, he concluded that accelerated sequence evolution is specific to the hominid lineage. Our finding that selection on ASPM begins well before brain expansion suggests that the molecular evolution of ASPM in hominoids may indeed be an example of a molecular “exaptation” ( Gould and Vrba 1982 ), in that the originally selected function of ASPM was for something other than large brain size. In the case of ASPM, rapidly evolving residues are mainly concentrated in the IQ repeat domain containing multiple IQ motifs, which are calmodulin-binding consensus sequences. While there is no direct evidence yet, it is likely that the function of human ASPM is modulated through calmodulin or calmodulinlike protein(s). Previous interspecies comparisons of ASPM proteins have shown a consistent correlation of greater protein size with larger brain size mainly because of the number of IQ repeats ( Bond et al. 2002 ). For example, the asp homologue of the nematode Caenorhabditis elegans contains two IQ repeats, the fruit fly — 24 IQ repeats, and the mouse—61 IQ repeats, and there are 74 IQ repeats in humans ( Bond et al. 2002 ). ASPM homologues in the nonhuman primates examined here contain the same number of IQ repeats as human, supporting the idea that repeat expansion occurred prior to the anthropoid divergence (which gave rise to New World monkeys, Old World monkeys, and hominoids) and possibly even earlier in primate evolution. IQ motifs are seen in a wide variety of proteins, but the ASPM proteins in primates are unique, because they have the largest known number of IQ repeats. Given the proposed role of ASPM in regulating divisions of neuronal progenitors, both the number of repeats and the particular amino acid substitutions in the IQ repeats may be strongly related to brain evolution. Human ASPM gene mutations which lead to MCPH provide a direct link between genotype and phenotype. ASPM is yet another example on the growing list of positively selected genes that show both accelerated evolution along the human lineage and involvement in simple Mendelian disorders ( Clark et al 2003 ). However, ASPM is unique because its distinctive pattern of accelerated protein evolution begins several million years prior to brain expansion in the hominid lineage. Absolute brain size in orangutans (430 g in males; 370 g in females) is barely different from that in gorillas (530 g in males; 460 g in females) and common chimpanzees (400 g in males; 370 g in females) ( Tobias 1971 ), yet accelerated ASPM evolution began in the common ancestor of gorillas, chimpanzees, and humans, approximately 7–8 million years ago. Only much later did brain expansion begin in hominids, starting at 400–450 g roughly 2–2.5 million years ago and growing to its final current size of 1350–1450 g approximately 200,000–400,000 years ago ( Wood and Collard 1999 ). Therefore genotypic changes in ASPM preceded marked phenotypic changes in hominoid brain evolution, at least at the level at which they have currently been studied. The molecular changes in ASPM may predict the existence of differences in early neurogenesis between orangutans, on the one hand, and gorillas, chimpanzees, and humans, on the other, which may manifest as more subtle differences in brain anatomy than gross changes in brain volume. How might evolutionary changes in the ASPM protein affect cerebral cortical size? One potential mechanism might be that changes in ASPM induce changes in the orientation of the mitotic spindle of neuroblasts. Normally, neural precursor cells can have mitotic spindles oriented parallel to the ventricle or perpendicular to the ventricle. Mitoses in which daughter cells are oriented next to one another at the ventricular zone are typically “symmetric” in that a single progenitor cell generates two progenitor cells, causing exponential expansion of the progenitor pool. In contrast, mitoses that generate daughter cells that are vertically arranged are typically “asymmetric” so that one daughter cell becomes a postmitotic neuron, whereas the other daughter cell remains as a progenitor, causing only a linear increase in cell number. Control of this proliferative symmetry can cause dramatic alterations in cerebral cortical size ( Chenn and Walsh 2002 ), and so changes in ASPM could regulate cortical size by making subtle changes in spindle orientation. Alternatively, evolutionary changes in ASPM may not themselves have led to increase in the size of the brain, but instead perhaps ASPM might be essential to insure faithful DNA replication and proper chromosome segregation. In rodents, a surprising number of cerebral cortical neurons are aneuploid ( Rehen et al. 2001 ). Perhaps directed selection of specific domains of ASPM helps insure faithful chromosome segregation to allow a larger number of cerebral cortical neurons to be formed without an unduly high incidence of chromosome aneuploidy. Functional genomics studies are clearly needed to elucidate the exact nature of the molecular mechanisms affected by ASPM gene evolution in hominoids. Here, we have demonstrated the utility of TAR cloning for evolutionary sequence comparisons among humans and other primates. In addition, the ASPM TAR clones isolated in these studies could provide valuable reagents for studying ASPM gene regulation in its natural sequence context. Overall, we anticipate this technology will be extremely useful in studying the evolution of other genes that may be responsible for uniquely human traits. Note The related paper by Evans et al. (2004 ) was published in Human Molecular Genetics shortly after this paper was submitted. Materials and Methods TAR cloning of the ASPM gene homologues by in vivo recombination in yeast To isolate the full-size ASPM gene from the human (Homo sapiens), chimpanzee (Pan troglodyte s ), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) genomes, a TAR vector containing two unique hooks was constructed. Two targeting sequences were designed, 131 bp 5′ and 151 bp 3′, from the available human genomic sequence of ASPM (positions 155,758–155,888 and 92,922–93,071 in the BAC RP11–32D17 [GI:16972838]). The targeting sequences were PCR amplified from the genomic DNA using two specific primers ( Table S2 ). PCR products were cloned into a polylinker of the basic TAR vector pVC604 as Apa I– Sal I and Sal I– Xba I fragments. Before transformation experiments, the TAR cloning vector was linearized with Sal I to release targeting hooks. Genomic DNA samples were prepared from chimpanzee, gorilla, orangutan, and rhesus macaque fibroblast culture cell lines (Coriell Institute for Medical Research, Camden, New Jersey, United States) in agarose plugs. Spheroplast transformation experiments were carried out as previously described in Kouprina and Larionov (1999 ). To identify clones positive for ASPM , yeast transformants were examined by PCR using diagnostic primers specific for exon 2 and exon 27 of ASPM ( Table S2 ). Integrity of yeast artificial chromosomes (YACs) and the issue of their stability during propagation in yeast were examined. DNA was isolated from ten subclones carrying the ASPM YACs for each primate, and their size was analyzed by Not I digestion followed by CHEF. Each subclone carried a YAC of similar size, indicating that these clones were stable in yeast. Alu profiles of the clones were checked by Taq I digestion of 1 μg of total yeast DNA isolated from transformants. Samples were run by electrophoresis, transferred to a nylon membrane, and hybridized with an Alu probe. YACs were retrofitted into bacterial artificial chromosomes (BACs) by homologous recombination in yeast using a BAC/Neo R retrofitting vector, BRV1, and then transformed into a recA DH10B E. coli strain ( Kouprina and Larionov 1999 ). Before sequencing, the integrity of inserts in BACs was confirmed by Not I, Hin dIII, Eco RI, and Pst I digestions. The promoter regions of the chimpanzee, gorilla, orangutan, and rhesus macaque (approximately 3.2 kb) and exon 18 of the red-chested mustached tamarin (Saguinus labiatus) and black-handed spider monkey (Ateles geoffroyi) (approximately 4.7 kb) were PCR amplified using a pair of specific primers ( Table S2 ) from primate genomic DNAs (Coriell Institute for Medical Research) and then TA-subcloned for further sequencing. RT-PCR of ASPM coding regions RNAs were extracted from primate cell lines (African green monkey [Cercopithecus aethiops] kidney, COS-7 [American Type Culture Collection, Manassas, Virginia, United States], chimpanzee peripheral lymphoblast, EB176 [JC], and gorilla peripheral lymphoblast, EB [JC] [European Collection of Cell Cultures, Wiltshire, United Kingdom]) using TRIzol reagent (Invitrogen, Carlsbad, California, United States). Reverse transcription and 5′- and 3′-RACE reactions were performed using SMART RACE cDNA Amplification Kit (BD Biosciences, San Jose, California, United States). Sequencing Chimpanzee, gorilla, orangutan, and rhesus macaque TAR clones containing full-size ASPM genes were directly sequenced from BAC DNAs ( Polushin et al. 2001 ). Forward and reverse sequencing of the promoter and exon 18 as well as fragments of coding regions of the ASPM homologues were run on a PE-Applied Biosystem 3100 Automated Capillary DNA Sequencer (Applied Biosystems, Foster City, United States). Primer pairs for cDNA sequencing were designed based on the human ASPM mRNA sequence. Primer sequences are available upon request. All sequences were named and numbered according to the clone/accession identifier. Sequence analysis Genomic sequences were aligned using MAVID ( http://baboon.math.berkeley.edu/mavid/ ) ( Bray and Pachter 2004 ); proteins and protein-coding DNA sequences were aligned by DIALIGN2.1 ( http://bibiserv.techfak.uni-bielefeld.de/dialign/ ) ( Morgenstern 1999 ). Alignments were manually edited in the SEAVIEW editor ( http://pbil.univ-lyon1.fr/software/seaview.html ) ( Galtier et al. 1996 ). We have used a number of programs from the EMBOSS package ( http://www.hgmp.mrc.ac.uk/Software/EMBOSS/ ) for sequence analysis. Short nucleotide patterns associated with genome rearrangements were searched using FUZZNUC (EMBOSS). We searched for the following recombinogenic motifs: chi-like octamer (GCWGGWGG), immunoglobulin heptamer (GATAGTG), translin (ATGCAGN(0,4)GCCCWSW and GCNCWSCTN(0,2)GCCCWSSW), topoisomerase II (RNYNNCNNGYNGKNYNY), topoisomerase IId (GTNWAYATTNATNNR), topoisomerase IIi (YYCNTASYGGKYYTNNC), and V(D)J recombinase (CACAGTGN(12/23)ACAAAAACC). For short or highly ambiguous patterns (topo-isomerase II), no mismatches were allowed; for longer motifs (translin, V(D)J recombinase) up to two mismatches were permitted. Prediction of CpG islands was performed by CPGPLOT (EMBOSS) with default parameters (length ≥ 200; CpG/GpC ≥ 0.6; GC ≥ 0.5). CENSOR ( http://www.girinst.org/Censor_Server-Data_Entry_Forms.html ) ( Jurka et al. 1996 ) and REPEATMASKER ( http://repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker ; developed by A.F.A. Smit and P. Green) were used for identification of repetitive elements. Minisatellites were detected by TANDEM REPEAT FINDER ( Benson 1999 ). ASPM segmental duplications in the human genome were detected by local BLAT searches ( http://genome.ucsc.edu/cgi-bin/hgBlat ) ( Kent 2002 ). First, we used ASPM genomic sequences with all repeats masked to detect segmental duplications. Full-size duplications were then obtained by BLAT alignment with full (i.e., repeat-containing) ASPM sequence. Primate CDSs were deduced from the ASPM gene alignment with human sequences. Synonymous and nonsynonymous substitutions were detected by SNAP ( http://www.hiv.lanl.gov/content/hiv-db/SNAP/WEBSNAP/SNAP.html ). Codon maximum likelihood (ML) in CODEML in PAML v. 3.13 ( http://abacus.gene.ucl.ac.uk/software/paml.html ) ( Yang 1997 ) has been applied for reconstruction of phylogenetic trees, reconstruction of ancestral sequences, and detection of positive selection. Branch lengths and ancestral sequences were reconstructed using a free ω ratio for individual branches. The methodology of likelihood ratio tests is described elsewhere ( Yang 1998 ). For large alignments, the initial input trees for PAML were estimated by ML implemented in PHYLO_WIN ( http://pbil.univ-lyon1.fr/software/phylowin.html ) ( Galtier et al. 1996 ). Segmental duplications were clustered by a neighbor-joining method implemented in the same program. Distance measurements for examining intraspecific/interspecific diversity were calculated in PAUP (Swofford, D. L. 2003. PAUP v. 4.0b10; Sinauer Associates, Sunderland, Massachusetts, United States; http://paup.csit.fsu.edu/index.html ) and corrected for multiple substitutions using the Tamura-Nei algorithm. Supporting Information Commentary Selection operating on codon usage may increase the ω ratio by lowering the rate of synonymous substitutions ( Sharp and Li 1987 , 1989). Therefore, we tested the correlations between the CAI ( Sharp and Li 1987 ) and the rate of synonymous substitutions (Ks). We found no significant association between the tested variables. Moreover, interspecies comparisons disclosed that CAI is nearly identical for all compared species, and no CAI increase over other species was detected for human or gorilla (data not shown). On the other hand, there was a significant negative correlation between CAI and both protein and DNA identity. A partial correlation analysis revealed that the significant positive linear correlation between Ka and CAI was merely caused by the strong negative correlation of Ka with DNA and protein identity. When we controlled for identity, the correlation between Ka and CAI disappeared (data not shown). These results may indicate that at positively selected sites, protein changes are preferred over optimization of codon usage, and thus mutations causing amino acid replacements do not immediately produce optimal codons. It should be noted that selection on codon usage seems to be generally relaxed in mammals ( Duret and Mouchiroud 2000 ). Mammalian codon usage as well as the rate of nonsynonymous substitutions can be potentially biased by selection favoring a high GC content (or even saturation by G and C) at the third codon positions (GC3) ( Bernardi and Bernardi 1985 ; Aota and Ikemura 1986 ). However, ASPM is an AT-rich gene (GC content 36.4%–37%) and, as expected ( Bernardi and Bernardi 1985 ; Aota and Ikemura 1986 ), the third codon positions are also AT-rich (GC3 content, 30.6%–31.4%) and thus far from saturation. In summary, neither the codon bias nor selection on the third codon seems to strongly influence the synonymous rate Ks. Therefore the high Ka/Ks ratio in human and gorilla is likely to be the product of adaptive evolution. Figure S1 Recombination Breakpoints in the Orangutan-Specific 818-bp-Long Deletion Both orangutan breakpoints are located within 5′ portions of two Alu elements. The sequence conservation is marked by different shades of gray. Both Alu elements are compared to their corresponding AluSp and AluSz subfamily consensus sequences. Gorilla, chimpanzee, and human sequences located 1 bp downstream of the 5′ breakpoint share a perfect match with the chi-like octamer consensus sequence GCWGGWGG (first box, positions matching the chi consensus are shown in black). On the other hand, the 3′ breakpoint sequences are diverged from the chi consensus (second box). Both Alu elements in the alignment are shown from the first position and end at the same position, and thus positions in one element correspond to positions in the other Alu copy. As can be seen, the breakpoint position in the first AluSp repeat exactly corresponds to the breakpoint position within the second AluSz element, suggesting homologous recombination between the two repeats. (163 KB PDF). Click here for additional data file. Figure S2 Segmental Duplications of the Fourth Internal Intron From left to right: phylogeny, chromosomal position, band name, identity to ASPM segment (percent same), and a schematic alignment of segmental duplications. The ASPM segment (black) shares similarity with 24 segmental duplications that contain additional sequences and are present on several human chromosomes. The ASPM copy and three duplications on Chromosome 7 share the same L1P4 terminal insertion, which is absent from all other duplications. The tree on the left shows evolutionary relationships among the duplications estimated by the neighbor-joining method. (169 KB PDF). Click here for additional data file. Figure S3 Comparison of Mouse and Human ASPM Proteins The amino acid identity in the conserved regions is 85.44%, 49.39%, and 68.74% for exon 3, exon 4, and the IQ domain, respectively. In addition, while the alignment of conserved regions is completely gap-free, the variable domains exhibit several gaps including a large deletion in the mouse IQ domain (human positions 2655–2943). (97 KB PDF). Click here for additional data file. Table S1 Primers Used in This Work Upper case letters indicate sequences homologous to ASPM and lower case letters indicate cloning sites. (118 KB PDF). Click here for additional data file. Table S2 CDS and Protein Correlations All correlations were obtained for the same 100-amino-acid-/300-nucleotide-long, nonoverlapping windows. The first value shows the correlation coefficient; p -value is in parentheses. The section over the diagonal is calculated using the Pearson (linear) correlation coefficient; under the diagonal are correlations obtained using the Spearman's rank coefficient—nonparametric). Nontrivial or interesting significant correlations are shown in bold and italics. The CAI represents the mean for all species (the CAI values are nearly identical for individual species). The ω ratio, Ka, and Ks (rows/columns 2, 3, and 4) correspond to all branches of the phylogenetic tree. They were obtained using a ML model with one fixed ω ratio for all branches. Click here for additional data file. Accession Numbers The GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession number for the human ASPM mRNA sequence used in this study is NM_018136. The sequence data from chimpanzee, gorilla, orangutan, and rhesus macaque full-length ASPM have been submitted to GenBank under accession numbers AY497016, AY497015, AY497014, and AY497013. The sequence data from chimpanzee, gorilla, and African green monkey ASPM cDNA have been submitted to GenBank under accession numbers AY508452, AY508451, and AY486114. The sequence data from spider monkey and tamarin exon 18 have been submitted to GenBank under accession numbers AY497017 and AY497018.

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555559

Activation of p53, inhibition of telomerase activity and induction of estrogen receptor beta are associated with the anti-growth effects of combination of ovarian hormones and retinoids in immortalized human mammary epithelial cells

Background A full-term pregnancy has been associated with reduced risk for developing breast cancer. In rodent models, the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P). However, the effects of pregnancy levels of E/P in humans and their underlying mechanisms are not fully understood. In this report, we investigated the growth inhibitory effects of pregnancy levels of E/P and both natural and synthetic retinoids in an immortalized human mammary epithelial cell line, 76N TERT cell line. Results We observed that cell growth was modestly inhibited by E/P, 9-cis-retinoic acid (9-cis RA) or all-trans-retinoic acid (ATRA), and strongly inhibited by N-(4-hydroxyphenyl) retinamide (HPR). The growth inhibitory effects of retinoids were further increased in the presence of E/P, suggesting their effects are additive. In addition, our results showed that both E/P and retinoid treatments resulted in increased RARE and p53 gene activity. We further demonstrated that p53 and p21 protein expression were induced following the E/P and retinoid treatments. Furthermore, we demonstrated that while the telomerase activity was moderately inhibited by E/P, 9-cis RA and ATRA, it was almost completely abolished by HPR treatment. These inhibitions on telomerase activity by retinoids were potentiated by co-treatment with E/P, and correlated well with their observed growth inhibitory effects. Finally, this study provides the first evidence that estrogen receptor beta is up-regulated in response to E/P and retinoid treatments. Conclusion Taken together, our studies show that part of the anti-growth effects of E/P and retinoids is p53 dependent, and involve activation of p53 and subsequent induction of p21 expression. Inhibition of telomerase activity and up-regulation of estrogen receptor beta are also associated with the E/P- and retinoid-mediated growth inhibition. Our studies also demonstrate that the potency of retinoids on cell growth inhibition may be increased through combination of estrogen and progesterone treatment.

Background It is well documented that women who experience a full-term pregnancy early in their lives have a significantly reduced risk for developing breast cancer [ 1 , 2 ]. The mechanisms by which pregnancy affects maternal breast cancer incidences are not fully understood. Previous studies suggest that the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P) in rodent models [ 3 , 4 ]. However, the effects of pregnancy levels of E/P in human and their underlying mechanisms have not been investigated. Retinoids are a promising class of chemopreventive agents against breast cancer because of their antiproliferative and proapoptotic properties [ 5 , 6 ]. Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are nuclear transcription factors that modulate the biological effects of retinoids. Most retinoid forms, including 9-cis-retinoic acid (9-cis RA) and all-trans-retinoic acid (ATRA), activate RAR family members, whereas RXR family members are activated by 9-cis RA. N-(4-hydroxyphenyl) retinamide (HPR), a synthetic derivative of ATRA, may weakly interact with retinoid receptors [ 7 ]. The 76N TERT cells were derived from a reduction mammoplasty specimen [ 8 , 9 ]. They are normal human mammary epithelial cells immortalized by plasmids containing hTERT, the human catalytic subunit of the reverse transcriptase protein of telomerase [ 10 ]. hTERT-expressing normal cell clones have been shown to have an extended life span without any change in karyotype [ 11 ]. The 76N TERT cells in culture could continuously grow about 60 population doublings [ 8 ], and the level of p53 protein has been shown to remain consistent at early or late passages [ 9 ]. Unlike the tumor cell lines widely used in breast cancer researches such as MCF-7 and MDA-MD-231 cells, which have undergone several steps in tumorigenesis, the 76N TERT cell line represents a system that is immortal but does not yet have the capacity to form a tumor. Hence, it is potentially a better model to study the genetic changes, and to test the effects of carcinogenic or chemopreventive agents on the development of mammary tumors. In this study, we investigated whether E/P induce growth inhibition in 76N TERT cells; and the molecular mechanisms by which E/P inhibited 76N TERT cell growth. For comparison purpose, the anti-growth effect of both natural and synthetic retinoids was examined in parallel in this immortalized mammary epithelial cell line. We also investigated whether we could increase the responsiveness of retinoids by using retinoids in combination with E/P. Our studies demonstrate that 1) inhibition of cell growth by E/P and retinoids could be partially mediated through a p53-dependent mechanism; 2) induction of p21 expression, inhibition of telomerase activity, or up-regulation of estrogen receptor beta (ERβ) by E/P and retinoids may contribute to their anti-growth effects; 3) combination of retinoids with E/P lead to increased inhibitory effects on cell growth. Results Expression of RARs, RXRs, estrogen receptors and progesterone receptors in 76N TERT cells As the ability of estrogen, progesterone and retinoids to influence cell proliferation is mediated by their respective receptors in most cases, we first examined the expression of these receptors in 76N TERT cells. Using Western blot analysis, the proteins of RAR (RARα, RARβ and RARγ), RXR (RXRα, RXRβ and RXRγ), and ER (ERα and ERβ) were observed as suggested by the manufactures (Figure 1 ). The antibody for the progesterone receptor (PR) detected a protein between 85 and 125 KDa (Figure 1 ). The existences of RARβ and ERβ in 76N TERT cells were further confirmed by quantitative RT-PCR at the mRNA level (RARβ data not shown, ERβ data see Figure 6B ). Figure 1 Expression of RAR, RXR, ER and PR in 76N TERT cells. Whole cell lysates were analyzed by using anti-RAR, RXR, ER, and PR antibodies as described under "Materials and Methods". Blots shown are representative of 2–3 experiments with similar results. Figure 6 Induction of ERβ expression by E/P and retinoids in 76N TERT cells . Cells were treated with indicated retinoids with or without E/P for 72 hours. A. Western blot analysis of ERβ protein. Relative ERβ expression was normalized to actin protein and expressed as fold changes compared to vehicle treatment. Blot shown is representative of 3 experiments with similar results. B. Quantitative RT-PCR analysis of ERβ genes. Data are the means ± SE from 3 experiments. *, significant differences from their own controls. Inhibition of 76N TERT cell growth by E/P and retinoids We then tested the influence of E/P or retinoids on 76N TERT cell growth, using [ 3 H]thymidine incorporation assay. The concentrations of E/P and retinoids used in the experiments were chosen based on previous studies and are clinically or physiologically relevant [ 3 , 4 , 12 ]. Treatment of cells with E/P or all three retinoids resulted in decreases in cell proliferation. As shown in Figure 2 , treatment of 76N TERT cells with 1 μM 9-cis RA or 2 μM ATRA significantly decreased the cell growth by 28.8% and 24.5% respectively. In comparison, cells were more responsive to HPR. Treatment with 2 μM HPR exhibit a significant 71.4% decrease in cell growth relative to controls. Combination of 1 ng/ml of β-estradiol and 1 μg/ml of progesterone, a regime that mimics the protective effects of pregnancy, also resulted in a significant 32.2% decrease in cell proliferation. In the presence of E/P, the inhibitory effects on cell growth of 9-cis RA, ATRA and HPR were further increased to a respective 56.6%, 53.3% and 86.8%, indicating that the anti-proliferative effect may be additive between E/P and retinoids. Figure 2 Inhibition of 76N TERT cell growth by E/P and retinoids . Cells were treated with indicated retinoids with or without E/P for 24 hours and then labelled with [ 3 H]thymidine as described under "Materials and Methods". Data are the means ± SE from 3–4 experiments. *, significant differences from control. Activation of RARE and p53 gene by E/P and retinoids Many biological responses to retinoids are thought to be mediated through receptors by binding to retinoic acid response elements ( RARE s) and regulation of transcriptional activity [ 5 , 6 ]. In addition, cross-talk between ER and RAR pathways has been previously reported [ 13 , 14 ]. Given this, a comparison of RARE gene activation in response to E/P and retinoids was carried out using cells transfected with a RARE -luciferase reporter gene construct. As shown in Figure 3A , 76N TERT cells exhibited a respective 3.3-, 5.4-, and 2.5-fold activation of RARE gene in response to 9-cis RA, ATRA or E/P alone. In contrast, HPR caused no significant change in luciferase activity relative to the control. In the presence of E/P, the effects of 9-cis RA and ATRA on RARE gene activation were essentially the same as without E/P. Figure 3 Activation of RARE and p53 gene by E/P and retinoids in 76N TERT cells . Cells were transiently transfected with the RARE ( A ) or p53 ( B ) reporter plasmid, and then treated with retinoids for 24 hours with or without E/P. Luciferase activity was measured and normalized as described under "Materials and Methods". Results are the means ± SE from 3 experiments. *, significant differences from control. Functional p53 provides a protective effect against tumor growth [ 3 , 15 ]. We next examined whether E/P and retinoids could enhance the transcriptional activity of p53 using 76N TERT cells transfected with a p53 -responsive luciferase reporter gene construct. As shown in Figure 3B , a 2.3-, 1.8-, 1.8- and 2.2-fold induction of luciferase activity was observed by treatment of cells with 9-cis RA, ATRA, HPR and E/P respectively. Co-treatment with E/P and retinoids showed no additional activation of p53 gene as compared to their treatments alone. Induction of p53 and p21 protein expression by E/P and retinoids We then performed Western blot analysis to test whether increased p53 gene activity is paralleled by increased p53 protein expression. Treatment with 9-cis RA, ATRA, HPR or E/P alone slightly increased (about 1.5-fold) the expression of p53 protein. Consistent with the data on p53 gene activation, no additive effects between E/P and retinoids on induction of p53 protein were observed (Figure 4 ). Figure 4 Induction of p53 and p21 protein by E/P and retinoids in 76N TERT cells . Cells were treated with indicated retinoids with or without E/P for 72 hours. Whole cell lysates were subjected to Western blot analysis using anti-p53, p21 and Bax antibodies. Relative expression of each protein was determined using the same membrane, and normalized to actin protein. Data are fold changes compared to vehicle treatment. Blot shown is representative of 4 experiments with similar results. Activated p53 could induce the transcription of either p21 to cause growth arrest, or Bax to induce apoptosis [ 15 ]. We therefore investigated whether increased p53 protein expression can modulate the expression level of p21 or Bax proteins in 76N TERT cells. In Figure 4 , exposure to retinoids or E/P alone did moderately increase the p21 protein level, with a 1.7-, 2.7- and 2.1-fold increase in p21 expression for ATRA, HPR and E/P respectively. The increases of p21 protein expression with combined E/P and retinoids were similar to that observed when E/P and retinoids were used alone. In addition, no significant effects by E/P or retinoids on Bax protein expression were observed in 76N TERT cells. Inhibition of telomerase activity by E/P and retinoids Activation of telomerase is an early event in the development of breast cancers that may lead to cellular immortality, a critical and rate-limiting step in oncogenesis [ 16 , 17 ]. Activated p53 has been associated with regulation of the telomerase activity [ 18 - 20 ]. To evaluate the effects of E/P and retinoids on telomerase activity in 76N TERT cells, cells were treated for different time periods with E/P and retinoids, and the levels of telomerase activity were determined by a quantitative real-time PCR method. As shown in Figure 5 , treatment with E/P decreased telomerase activity in a time-dependent manner with a 63.3% inhibition at 72 hours, whereas vehicle treatment had no effect at any time during the experiment. The maximum inhibition on telomerase activity was observed at 24 hours for both 9-cis RA and ATRA treatments, with a respective of 68.9% and 69.4% decrease. In comparison, the effects of HPR occurred more rapidly, with a complete inhibition at 16 hours, and persisted throughout the treatment. The inhibitory effects of 9-cis RA, ATRA or HPR and E/P seemed to be additive, as in the presence of E/P, 9-cis RA, ATRA or HPR showed increased inhibitions at various time points. These effects correlate well with their observed growth inhibitory effects in 76N TERT cells, suggesting that inhibition of telomerase activity by E/P and retinoids may contribute to their additive effects on inhibition of cell growth. Figure 5 Inhibition of the telomerase activity by E/P and retinoids in 76N TERT cells . Cells were treated with indicated retinoids with or without E/P for the indicated time periods. Telomerase activity was determined as described under "Materials and Methods". Data are the means ± SE from 2–3 measurements. *, significant differences from their own controls. Induction of ERβ expression by E/P and retinoids There is growing evidence that ERβ could be an inhibitor of tumorigenesis of breast cancer [ 21 - 23 ]. We examined whether there were any changes in the expression of ERβ in response to E/P and retinoid treatment in 76N TERT cells. After treatment of cells with retinoids or E/P for 72 hours, the amount of ERβ protein was determined by Western blot analysis. As shown in Figure 6A , the normalized ERβ protein showed a respective 2.1-, 2.3-, and 1.5-fold increase in response to ATRA, HPR and E/P, as compared to the vehicle treatment. We also carried out a quantitative analysis of ERβ mRNA expression in response to E/P and retinoid treatment in 76N TERT cells using a real-time PCR assay. As shown in Figure 6B , there was a 2.0-, 2.3- or 2.8-fold induction of ERβ mRNA levels in HPR, E/P or combination of HPR and E/P treated cells, respectively. Discussion Similar to retinoids, a full-term pregnancy has been associated with beneficial effects on breast cancer risk over the long term [ 1 , 2 ]. The mechanisms by which pregnancy affects maternal breast cancer incidences are not fully understood. Studies have showed that higher concentrations of progesterone elicit a growth-inhibiting response from normal and cancerous breast cells [ 24 , 25 ], and are inversely related to breast cancer incidence [ 26 ]. In this study, we examined the ability of pregnancy levels of E/P and retinoids to affect the growth of the immortalized normal mammary epithelial cells. Our results demonstrated that three isoforms for RAR and RXR (α, β, γ), two isoforms for ER (α and β), and PR receptor proteins are expressed by 76N TERT cells. Treatment with 9-cis RA, ATRA, HPR or E/P inhibited 76N TERT cell proliferation and resulted in the activation of p53 gene, followed by increased expression of p53 protein and p21 protein, and inhibition of telomerase activity. Additionally, we first report here that the expression of ERβ is induced in response to E/P and retinoid treatment at both the transcriptional and translational levels. Importantly, we demonstrate that the inhibitory effects of retinoids on cell growth are more effective in the presence of E/P, and correlate well with their inhibitory effects on telomerase activity in 76N TERT cells. Our data suggest that the growth inhibitory effects of E/P and retinoids may involve the activation of p53 pathway in 76N TERT cells. First, our results showed that both E/P and retinoid treatments lead to the increased p53 gene activity. Secondly, we demonstrated that the protein expression of p53 and p21 were increased following the treatment. It has been shown that p21 can inhibit cyclin A/cdk2 kinase activity and subsequently result in cell cycle arrest [ 27 , 28 ]. Our data is in line with the previous findings that in normal mammary epithelial cells, retinoids induce cell cycle arrest which is associated with an increase in p21 expression [ 29 ]; and that in both rats and mice, p53 is activated in response to E/P and this activation is sustained to induce p21 [ 3 ]. Thirdly, our data showed that treatment with E/P or retinoids decreased the telomerase activity in 76N TERT cells. Although a few reports suggest that telomerase activity appears to be independent of p53 expression or mutation [ 30 , 31 ], the majority of the evidence to date support the involvement of p53 in regulation of telomerase activity in mammary epithelial cells and breast cancer [ 18 - 20 ]. The molecular mechanisms of regulation of telomerase activity by p53 may involve down-regulation of hTERT transcription or the interaction of p53 with other transcription factors [ 19 ]. However, our data also suggest the possibility that inhibition of cell growth by E/P and retinoids may be independent of p53 pathway in 76N TERT cells. Our data show that the enhancing effects of retinoids on p53 gene activation or on the p53 and p21 protein expression were not further augmented by the addition of E/P, unlike their inhibitory effects on cell growth, indicating that other mechanisms besides the p53 pathway are likely to be involved. A p53-independent cell cycle arrest by retinoids has been previously suggested in a number of breast carcinoma cells [ 27 , 32 ]. The p53-independent inhibitory effects of retinoids on cell growth can be exerted through various mechanisms including regulation of other genes that play critical roles in cell cycle progression such as c-myc [ 18 ], inhibition of activator protein mediated transcription [ 33 ], or induction of caspase-independent cell death via calcium and calpain [ 34 ]. However, the mechanisms of E/P-mediated p53-independent cell growth inhibition are still unknown, and are currently under investigation using cell lines with different functional p53 systems. Clearly, our data suggest that there are some overlaps between E/P- and retinoid-mediated growth inhibition in 76N TERT cells, considering that in response to E/P and retinoid treatments, same effectors such as RARE and p53 gene, p53 and p21 protein, and the telomerase activity were affected. Additionally, there also seems to be cross-talk between the E/P- and retinoid-mediated growth inhibition. Previous studies have suggested that there is cross-talk between ERβ and RAR pathways [ 13 , 14 , 35 ]. Here, we demonstrated that the RARE gene activity was increased in response to E/P treatment. Furthermore, for the first time, we showed that treatment of immortal cells with E/P or retinoids could induce the expression of ERβ, both at the mRNA and protein level. The expression of ERβ often is found to be decreased markedly in the early stages of mammary carcinogenesis [ 22 ]. Loss of ERβ expression has been suggested as one of the events leading to the development of breast cancer [ 36 ]. Our data may reveal another important mechanism by which E/P and retinoids produce their anticancer function, indicating ERβ may represent a possible therapeutic target in breast cancer prevention. More importantly, our data show that the growth-inhibitory effects of retinoids were potentiated by co-treatment with E/P in 76N TERT cells. These observations indicate that different mechanisms may be involved in E/P- and retinoid-mediated inhibition of cell growth. Our results of their differential inhibitory effects on telomerase activity may provide some explanation for this. Although E/P and all three retinoids inhibited telomerase activity, the time courses of their actions were different. While retinoids produced their maximal inhibitory effects around 16 to 24 hours after treatment, E/P required 72 hours to reach its maximal inhibition, suggesting different mediators may be utilized to decrease telomerase activity by E/P and retinoids. ATRA and 9-cis RA have been previously reported to inhibit cell growth and decrease telomerase activity through down-regulation of the expression of hTERT telomerase gene [ 37 ]. On the other hand, progesterone treatment has been shown to down-regulate telomerase activity by modulation of cell cycle phases [ 38 ]. Previous studies have also provided evidence that the function of p53 in suppression of telomerase activity is separable from its cell cycle checkpoint function [ 20 ]. Therefore, it is likely that even though E/P and retinoids treatments both activate p53 pathway, they may use different mechanisms to inhibit telomerase activity. The different mediators involved in the inhibitory effects of E/P and retinoids on telomerase activity may contribute to their additive effects on inhibition of 76N TERT cell growth. The detailed mediator mechanisms down-stream of p53 and up-stream of telomerase activity for both E/P and retinoid pathways remain to be defined. Several lines of evidence suggest that the mechanisms through which HPR regulates cell growth seem different than those by 9-cis RA and ATRA in 76N TERT cells. In the [ 3 H]thymidine incorporation experiments, our results showed that whereas only 25–30% inhibition was observed for 9-cis RA and ATRA, 70% inhibition was reached by HPR. In addition, in the RARE -luciferase activity assay, 9-cis RA and ATRA induce about 3- to 5-fold activation on RARE gene activity. In contrast, HPR treatment resulted in no significant change. Finally, our data showed that 9-cis RA or ATRA treatment caused a moderate inhibitory effect on telomerase activity. In comparison, the telomerase activity is almost completely abolished by HPR treatment. The time courses of their inhibition of telomerase activity were different as well. While 9-cis RA and ATRA maximally inhibited the telomerase activity around 24 hours after treatment, HPR produced its maximal effect at 16 hours post-treatment. An obvious explanation for these different responses observed between 9-cis RA, ATRA and HPR is that these retinoids most likely possess different mechanisms for their actions. As suggested by numerous investigators, 9-cis RA and ATRA may function through the classical retinoid pathways involving the RARs and RXRs. On the other hand, in addition to activation the retinoid receptors [7a], HPR may also function through alternative pathways such as down-regulation of the IGF system [ 39 ], activation of TGF-beta [ 40 ], induction of genes which have antiproliferative activity [ 41 ], inhibition of aromatase activity and expression [ 42 ], and involvement of cellular signals such as reactive oxygen species [ 43 ] and the sphingolipid ceramide [ 44 ]. Conclusion In summary, our data demonstrate that 76N TERT cells express RAR, RXR, ER and PR, and represent a potential useful model to investigate the genetic changes, and the carcinogenic or chemopreventive effects of new agents on the development of mammary tumors. In addition, our data clearly suggest that part of the anti-growth effects of E/P is mediated through a p53-dependent pathway, as well as the involvement of the inhibition of telomerase activity and induction of ERβ. Comparing the E/P- and retinoid-mediated inhibitory effects on cell growth, there are overlaps, cross-talks and distinct effectors between these pathways. Furthermore, our studies suggest that retinoids may be more effective when combined with E/P to prevent breast cancer development. This increased potency and sensitized response to retinoids with combined E/P treatment might have several important clinical implications for anti-cancer agents that mimic E/P effects. Firstly, it might allow the currently used RA regimens to show improved response in cancer prevention. Secondly, it may be sufficient to overcome some acquired or intrinsic RA resistance in cancer cells. Finally, it may lower the required does of either classes of anticancer agents used, leading to less side effects or toxicity. Overall, our studies better the understandings of the common and the unique mechanisms by which E/P and retinoids regulate cell growth, and may help us to design or to improve the clinical applications of anti-cancer agents. Methods Chemicals ATRA, 9-cis RA, HPR, β-estradiol and progesterone were all purchased from Sigma (St. Louis, MO, USA) and dissolved in ethanol. The final concentration was 2 μM for ATRA and HPR, 1 μM for 9-cis RA, 1 ng/ml for β-estradiol and 1 μg/ml for progesterone. These concentrations were chosen based on previous studies and are clinically or physiologically relevant [ 3 , 4 , 12 ]. Culture of 76N TERT cells Cell line was originally supplied and cultured as described by Band et al. [ 8 ]. The culture medium D-MEM/F-12, fetal bovine serum, penicillin, streptomycin, and gentamicin were from Gibco (Carlsbad, CA, USA). All the other cell culture reagents were from Sigma (St. Louis, MO, USA). Cells were grown in D-MEM/F-12 mixture (1:1, vol/vol) containing 15 mM HEPES buffer and 2.5 mM L-glutamine, supplemented with 1% fetal bovine serum, 12.5 ng/ml epidermal growth factor, 10 nM triiodothyronine, 50 μM freshly made ascorbic acid, 2 nM estradiol, 1 μg/ml insulin, 2.8 μM hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphorylethanolamine, 10 μg/ml transferrin, 15 nM selenite, 1 ng/ml cholera toxin, 35 μg/ml bovine pituitary extract, 100 units/ml penicillin, 100 mg/ml streptomycin, and 20 μg/ml gentamicin. Cells were maintained in 95% humidified air plus 5% CO 2 and sub-cultured weekly. All experiments were performed on cells with passage numbers from 6 to 15. [ 3 H]Thymidine Incorporation Assay Cells were seeded into 24-well plates at 5 × 10 4 cells per well and incubated at 37°C overnight. Cells were then treated in triplicates with indicated retinoids in the presence or absence of E/P for 24 hours. After labelling cells with 1 μCi/ml of [ 3 H]thymidine (Amersham, Arlington Heights, IL, USA) for 24 hours, cells were harvested by washing with PBS and 10% TCA, solubilizing with the mixture of 0.1% SDS and 0.1N NaOH. Aliquots were taken for the quantification of radioactivity by the Tri-Carb 2900 TR Liquid Scintillation Analyzer (Perkin Elmer, Wellesley, MA, USA). Incorporation of [ 3 H]thymidine was expressed as a fold change from vehicle control under the same conditions. Luciferase Reporter Assays Cells were seeded in 24-well plates at 5 × 10 4 cells per well. Cells were transiently transfected with 0.5 μg of either a RARE -luciferase or a p53 -luciferase plasmid along with 0.05 μg of pCMV-Renilla luciferase using the SuperFect transfection reagent (QIAGEN, Valencia, CA, USA), following the manufacturer's recommended procedure. Twenty-four hours after transfection, triplicate cultures were treated with retinoids for 24 h in the presence or absence of E/P. The cells were then washed and lysed. The luciferase activities were measured using the DUAL-luciferase Assay System (Promega, Madison, WI, USA), and normalized by pCMV-Renilla luciferase activity for each sample. Real-Time PCR Telomerase Activity Assay Cells were lysed in CHAPS lysis buffer (Chemicon International, Temecula, CA, USA) and incubated at 4°C for 30 min. The lysate was then centrifuged at 12000 × g for 20 min at 4°C, and the supernatant was collected. The protein concentration was measured in each extract using the BCA protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Telomerase activity was determined in duplicates by a quantitative real-time PCR telomerase detection Kit (Allied Biotech, Ijamsville, MD, USA) according to the manufacturer's protocol, using Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, USA). Quantitative RT-PCR for ERβ gene analysis Cells at subconfluence were treated with retinoids in the presence or absence of E/P for 72 hours. Total RNA was extracted with TRIZOL Reagent from Gibco (Carlsbad, CA, USA) according to the instructions of the manufacture. Single-stranded cDNA was made from 1 μg of total RNA with the Cells-to-cDNA kit (Ambion, Inc., Austin, TX, USA) at 42°C for 15 min. The primers for ERβ were 5'CGA TGC TTT GGT TTG GGT GAT 3' (forward) and 5'GCC CTC TTT GCT TTT ACT GTC 3' (reverse). The primers for GAPDH were 5'CCA TGG AGA AGG CTG GGG 3' (forward) and 5'CAA AGT TGT CAT GGA TGA CC 3' (reverse). All primers were from Integrated DNA Technologies, Inc. (Coralville, IA, USA). cDNA (1 μl) was amplified in duplicates in Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, USA) by using Brilliant SYBR Green QPCR Master Mix from Stratagene (La Jolla, CA, USA). The reaction was carried out at 95°C for 10 min to denature, 40 cycles of 95°C for 30 sec, 55°C for 60 sec, 72°C for 60 sec. ERβ gene was quantified and normalized with external standard GAPDH. Western blot analysis Cells were treated with retinoids in the presence or absence of E/P for 72 hours. Cell lysates were obtained by incubating cells for 30 minutes at 4°C in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40, 100 mM NaF, 10 mM MgCl 2 , and protease inhibitor cocktail (Sigma, St. Louis, MO, USA), followed by centrifuged at 12,000 rpm for 20 min. Protein content was determined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Cell lysates (~30 μg protein) were separated on 10% polyacrylamide gels in the presence of 0.1% SDS with prestained low-range molecular-weight standards (Biorad, Richmond, CA, USA). After transfer, the membranes were blocked and then probed with antibodies against interested proteins as suggested by manufactures, followed by incubation with a peroxidase-conjugated secondary antibody. Immunoreactive bands were developed with an ECL reagent from Amersham (Arlington Heights, Il, USA). All blots were probed with anti-actin to normalize for loading differences. Quantification of gels was carried out using ImageJ software (NIH, Bethesda, Maryland, USA). The p53 (DO-1), p21 (F-5) and RARγ (G-1) mouse monoclonal antibodies, and RXRα (D-20) polyclonal antibody were from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Anti-ERβ (Ab-2), ER (Ab-1), PR (Ab-1), RARα and RARβ mouse monoclonal antibodies, and Bax (Ab-1) polyclonal antibody were from Calbiochem (San Diego, CA, USA). Monoclonal anti-actin, RXRβ and RXRγ were from Sigma (St Louis, MO, USA). Statistical Analysis Statistical differences were analyzed by T-test. Levels of statistical significance were set at p < 0.05. List of abbreviations 9-cis RA: 9-cis-retinoic acid; ATRA: all-trans-retinoic acid; E/P: ?β-estradiol and progesterone; ERβ: estrogen receptor beta; HPR: N-(4-hydroxyphenyl) retinamide; PR: progesterone receptors; RARs: retinoic acid receptors; RXRs: retinoid X receptors. Competing interests The author(s) declare that they have no competing interests. Authors' contributions JZ designed and carried out most of the assays and drafted the manuscript. YT carried out the real-time PCR experiments. SS conceived of the study and participated in its coordination. All authors read and approved the final manuscript.

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545945

Prognostic significance of metallothionein expression in renal cell carcinoma

Background Metallothionein (MT) protein expression deficiency has been implicated in carcinogenesis while MT over expression in tumors is indicative of tumor resistance to anti-cancer treatment. The purpose of the study was to examine the expression of MT expression in human renal cell carcinoma (RCC) and to correlate MT positivity, the pattern and extent of MT expression with tumor histologic cell type and nuclear grade, pathologic stage and patients' survival. Patients and methods The immunohistochemical expression of MT was determined in 43 formalin-fixed and paraffin-embedded RCC specimens, using a mouse monoclonal antibody that reacts with both human MT-I and MT-II. Correlation was sought between immunohistochemical (MT positivity, intensity and extension of staining) and clinico-pathological data (histological cell type, tumor nuclear grade, pathologic stage and patients' survival). Results Positive MT staining was present in 21 cases (49%), being mild/moderate and intense in 8 and 13 cases, respectively. The pattern was cytoplasmic in 7 cases and was both cytoplasmic and nuclear in 14 cases. MT expression in a percentage of up to 25% of tumor cells (negative MT staining included) was observed in 31 cases, in a percentage 25–50% of tumor cells in 7 cases, and in a percentage of 50–75% of tumor cells in 5 cases. There was no significant correlation of MT intensity of staining to histological type, stage and patients' survival, while it was inversely correlated to higher tumor nuclear grade. MT extent of staining did not correlate with histological type, nuclear grade, and pathologic stage while a statistically significant association was found with patients' survival. Conclusions The inverse correlation between MT staining intensity and tumor nuclear grade in RCC suggests a role of MT in tumor differentiation process. Since extent of MT expression is inversely correlated with survival it may be possibly used as a clinical prognostic parameter.

Background Metallothioneins (MTs) were firstly discovered by Margoses and Valle in 1957 [ 1 ] as cadmium (Cd) binding proteins. Later, Piscator [ 2 ] documented a marked increase of MT in Cd exposed rabbits, as a metal detoxification mechanism. MTs are a family of heavy metal binding proteins with a large degree of sequence homology that have been described in most vertebrate and invertebrate species. They are single-chain proteins, with molecular weight of approximately 6000 Da, characterized by a very high proportion of cysteine residues (30%), resulting in several high affinity Cd and/or zinc (Zn) binding sites [ 3 ]. There are two major isoforms, referred to as MT-I and MT-II [ 4 ], resolvable through ion exchange chromatography, that have closely related but distinct amino acid sequences and are distributed in most adult mammalian tissues. Recently, a further charge-separable MT isoform (MT-0) [ 4 ], and genes for two MT isoforms with restricted tissue distribution MT-III (brain neurons) [ 5 , 6 ] and MT-IV (stratified epithelia) [ 7 ] have been described. The potential for wider tissue distribution of MT-III was suggested by recent studies demonstrating the presence of MT-III mRNA and protein in the adult and developing human kidney [ 8 , 9 ]. MT-I and MT-II isoforms are usually expressed in low levels, but are inducible by a variety of metal ions, hormones, inflammatory cytokines and xenobiotics [ 10 - 12 ]. Induction of MTs is important in detoxification and metal ion homeostasis [ 9 ], in protection against reactive oxygen species [ 10 ] and in tissue regeneration [ 13 - 15 ]. MT expression deficiency implicated in carcinogenesis [ 16 ] and possible relation of MT over expression and resistance of tumors to anti-cancer therapy [ 17 ] has provided evidence of the importance of MT expression in cancer. MT over expression, detected immunohistochemically, has been described in a variety of human tumors, in relation to different stages of tumor development and progression [ 18 ]. The involvement of MT and Zn, in processes such as p53 gene activation and protein structure has been referred [ 16 , 19 ]. There is evidence that some human tumors contain high levels of MT, nevertheless, the importance of MT expression in carcinogenic evolution and in patients' survival is not yet fully understood. In organs such as kidney, colon and liver, normally implicated in metal ions homeostasis, MT protein is apparently expressed. It would be of great scientific importance to elucidate the pattern of MT expression in tumors developed from these organs, since it could not only delineate the role of MT in carcinogenic transformation but could also provide prognostic information for patients' outcome. The aim of the present study was to examine the expression of MT in human renal cell carcinoma (RCC) and to correlate the MT positivity, the pattern and extent of MT expression with tumor histological cell type and nuclear grade, pathologic stage and patients' survival. Patients and methods Forty three consecutive patients, 31 men and 12 women, who underwent nephrectomy for RCC comprised the group of our study. Their age ranged from 33 to 85 years (mean age 59.6 ± 11.1). Tumors were histologically classified as clear cell type in 32 cases, papillary type in 2 cases, chromophobe cell type in 4 cases and sarcomatoid type in 5 cases. The 8, 2, 16, 6 and 9 tumors were pathologically staged as T1 or T2N0M0, T2N+M0, T3N0M0, T3N+M0 and T4N+M+, respectively as per the TNM classification [ 20 ]. The grade of nuclear atypia according to Fuhrman Grading system [ 21 ] was: grade I in 11 cases, grade II in 15 cases, grade III in 6 cases, and grade IV in 11 cases. The patients were followed up from 2 up to 144 months (5 lost in follow up), mean 65.4 months, median 39 months. Immunohistochemistry Sections of 5 μm thickness were deparaffinized in xylene and rehydrated in graded alcohol series. To remove the endogenous peroxidase activity, sections were treated with freshly prepared 0.3% (v/v) hydrogen peroxide in methanol in dark, for 30 min, at room temperature. Non-specific antibody binding was then blocked using normal rabbit serum (Dakopatts, Glostrup, Denmark) diluted 1:5 in phosphate buffered saline (PBS), for 20 minute. A mouse (IgG1k) monoclonal antibody that reacts with both human MT-I and -II isoforms (Zymed, San Francisco, California, USA) was used in this study. The sections were then incubated for 1 hour, at room temperature, with the primary antibody diluted 1:50 in PBS. After three washes with PBS, sections were incubated for 30 minute at room temperature with rabbit, peroxidase conjugated, anti-mouse serum (Dakopatts) diluted 1:200 in PBS and rinsed three times with PBS. Sections were then incubated with swine, peroxidase conjugated, anti-rabbit serum (Dakopatts) diluted 1:100 in PBS and rinsed three more times with PBS. The resultant immune peroxidase complexes were developed in 0.5% (v/v) 3,3'-diaminobenzidine hydrochloride (DAB; Sigma, Saint Louis, MO, USA) in PBS containing 0.03% (v/v) hydrogen peroxide. Sections were counterstained with Harris' hematoxylin and mounted in gelatin (Sigma). Control slides included in MT immunostaining procedure consisted of specific tissues previously shown to express MT (lung cancer) as positive controls, whereas the primary antibody was replaced by PBS in the case of negative controls. Scoring system The stained sections were independently assessed by the pathologists (A.K., S.T., E.M.) without prior knowledge of the clinical data as previously described [ 22 ]. Specimens were considered as "positive" for MT when more than 5% of tumor cells within the section were positively stained. The intensity of staining was graded as mild (+), moderate (++), and intense (+++). To further evaluate the importance of staining extent, cases were stratified into 3 groups according to the percentage of cells staining positive for MT: group A, 0–25%; group B, 26–50%; and group C, >50% of MT positive cells. The pattern of MT staining was also characterized as cytoplasmic only, nuclear only, and both cytoplasmic and nuclear. Statistical analysis Correlation between immunohistochemical data (MT intensity and extent of staining) and clinicopathological data (histologic cell type, nuclear grade, pathologic stage) was assessed using the Chi-square test. The association of intensity and extent of MT immunohistochemical staining with survival was determined by comparing Kaplan-Meier survival curves constructed for different patient groups, and were compared using the log-rank test. Results Tubular cells but not glomeruli and interstitial cells of normal autologous renal tissue stained positive (both in the nucleus and the cytoplasm) for MT, although the intensity and extent varied significantly. Positive MT expression was prominent in 21 out of 43 cases (49%), while 22 out of 43 ones (51%) were MT negative. As far as the intensity of staining is concerned, low/moderate intensity was observed in 8 cases, while intense staining was evident in 13 cases. The pattern of positive MT immunostaining observed was either cytoplasmic (7 out of 21 cases, 33.3%) or cytoplasmic and nuclear (14 out of 21 cases, 66.6%). In certain cases of clear cell RCC membranous staining was also observed. Nuclear pattern of staining only, was not observed in any of the RCC cases examined (Figure 1 ). The extent of MT expression in a percentage of up to 25% of tumor cells (negative MT staining included) was observed in 31 out of 43 cases, in a percentage 25 up to 50% of tumor cells in 7 cases, and in a percentage of 50–75% of tumor cells in 5 cases. Figure 1 Detection of MT expression by immunohistochemistry. Intense membranous, cytoplasmic and nuclear staining in a case of clear cell type RCC (x330). There was no significant difference between MT intensity of staining and the histological types of RCC cases examined (χ 2 = 5.61, p = 0.46). No statistically significant differences were also observed between MT intensity of staining and stage (χ 2 = 9.24, p = 0.32), or patients' survival (log rank test: 4.75, p = 0.09) in the cases of RCC examined (Figure 2 ). Statistically significant correlation was found between MT intensity of staining and histological grade, where higher intensity of staining was found in cases presenting high histological grade (χ 2 = 13.63, p = 0.03). However, no such correlation was found in the case of clear cell RCC (χ 2 = 4.75, p = 0.57). Figure 2 Cancer-specific survival of patients according to intensity of staining for MT (no staining, green line; mild/moderate staining, blue line; intense staining, red line). No statistically significant difference was detected (p = 0.09). Non statistical correlation was found among MT extent of staining and histological types (χ 2 = 2.54, p = 0.86), stage (χ 2 = 7.12, p = 0.52) and grade (χ 2 = 6.24, p = 0.39) of RCC cases examined. Statistically significant inverse correlation was found between MT extent of staining and patients' survival (log rank test: 6.59, p = 0.037) (Figure 3 ). Again, this was not observed in case all other histological types but clear cell RCC were excluded (log rank test 3.36, p = 0.186). Figure 3 Survival curves of patients according to MT extent of staining (0–25% of cells, green line; 26–50% of cells, blue line; >50% of cells, red line). Statistically significant inverse correlation was found between extent of staining and patients' survival (p = 0.037). Discussion MT expression has been observed in different types of human tumors [ 18 , 22 ], including neoplasias of the urogenital tract [ 23 - 28 ]. However, the biological mechanisms underlying MT over expression in tumors, or the consequences of this over expression are not currently well understood. MT is highly expressed in fetal life [ 10 ]; the reappearance of this fetal characteristic in tumors suggests its participation in cellular growth and differentiation [ 16 , 18 , 19 ]. Developing and adult kidneys consistently express MT-I and MT-II mRNA [ 29 ] and the corresponding protein [ 28 ], while the MT-III isoform is also expressed in developing renal tissue, adult proximal tubule and renal cell carcinoma cell lines [ 8 , 9 ]. The MT-0 isoform is absent in adult kidneys but it can be found in nonneoplastic tissue from renal and transitional cell carcinoma [ 30 ]. The isoform specific expression of MT in RCC has not been so far investigated. Using a monoclonal antibody that reacts to both MT-I and MT-II, we demonstrated positive immunoreaction in 49% of our RCC cases, a percentage close to the 55.7% reported by Tüzel et al [ 24 ], using the same antibody. Zhang and Takenaka [ 24 ] used another commercially available monoclonal antibody with unspecified specificity towards different MT isoforms and found positive immunoreaction for the MT protein in 33% of their cases, while such report is not included in the study of Izawa et al [ 23 ] who used a polyclonal antibody prepared by their own laboratory. The cytoplasm and nucleus of normal and malignant cells may both express MT but there is no conclusive data on the functional significance of their subcellular distribution. The majority of the cases in our study expressed MT in both cytoplasm and nucleus, the expression being cytoplasmic only in one third of the cases. Analogous pattern has been reported in other studies [ 23 - 25 ] while the membranous staining observed in some of our cases as well as in two previous series [ 24 , 25 ] could be explained by the nature of clear cell carcinoma. Differential subcellular expression of MT may be related to either cell proliferation or the induction of apoptosis [ 31 , 32 ]. Recently, Kondo et al [ 33 ] pointed out the importance of subcellular distribution of MT in drug resistant-prostatic cancer cells, in which the nuclear MT expression rather the cytoplasmic counterpart seems to predominantly confer resistance to cisplatin. On the other hand, nuclear pattern of MT immunostaining has already been correlated with cellular response to stress stimuli [ 34 ]. The function of MT to protect the cell from apoptosis could be an explanation for MT over expression observed in high grade cases of RCC [ 24 ], as MT is not etiologically correlated with the apoptotic process. In certain tumors MT over expression has been associated with unfavorable prognostic characteristics such as advanced stage and poor differentiation [ 18 ]. In our study, MT intensity of staining did not correlate with stage while it showed an inverse correlation with histological grade. Our results are in accordance to those of other investigators [ 23 , 25 ], who also found an inverse relationship between MT immunoreactivity and tumor grade. In contrast, Zhang and Takenaka [ 24 ] reported positive associations between MT expression and tumor grade. The inverse relationship between MT immunohistochemical expression and tumor grade may suggest a role of MT in cellular growth and differentiation, and reflect alterations of intracellular processes leading to a gradual decline of Zn storage and to the subsequent decrease in MT expression. Although no association was found between MT staining intensity and survival, the reduced extent of MT expression significantly correlated with prolonged survival as reported elsewhere [ 25 ]. The extent of MT expression may offer an additional, prognostic factor in patients suffering from RCC. MT concentrations might also prove useful in predicting the efficacy of a particular cancer treatment protocols. Several types of transformed cells enriched for MT have been shown to exhibit greater resistance to chemotherapeutic agents [ 17 ]. In this context, the thiolate sulfur of the cysteine residues are thought to act as sacrificial scavengers for radicals and alkylyating agents [ 10 ]. Thus, MT might serve as a Cu and Zn reservoir, where their supply may negatively affect the growth of tumor cells. MT could also participate in tumorigenesis by sequestering and then donating these essential bivalent cations to proteins of tumor cells in order to meet metabolic requirements [ 35 ]. Conclusions The current data on the expression of MT in RCC cases examined emphasize the necessity to investigate larger numbers of patients with RCC comparing the staining profile of different MT isoforms with other clinico-pathological parameters and survival status of patients. Currently, it is unknown whether the presence of MT in renal carcinoma cells is related to the induction or inhibition of apoptosis or plays an active role in cell proliferation. Since cytokines may also induce MT expression and immunotherapy is the only, albeit with limited efficacy, available treatment for RCC, the intensity and extent of MT immunostaining should be studied in correlation to the immune status of RCC patients before and after immunotherapy. Thus, it will be possible to elucidate the potential role of MT in renal cell carcinogenesis, as well as its clinical usefulness as a tumor marker and as a tool for selecting patients for adjuvant immunotherapy.

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548508

Mutagenicity testing with transgenic mice. Part II: Comparison with the mouse spot test

The mouse spot test, an in vivo mutation assay, has been used to assess a number of chemicals. It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of in vivo testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the lacI model (commercially available as the Big Blue ® mouse), and the lacZ model (commercially available as the Muta™ Mouse). There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo .

Background This is the second presentation from a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in toxicity testing and mechanistic research. Part I, preceeding this article, discussed comparison of effects of chemicals using certain transgenic assays with results using the bone marrow micronucleus test. The assessment of the potential genotoxicity of chemicals in vivo is important for both the verification and confirmation of intrinsic mutagenicity and for establishing the mode of action of chemical carcinogens. Although the present trend is to reduce animal testing, in vitro data must be confirmed by testing in in vivo conditions which take into account whole animal processes like absorption, tissue distribution, metabolism and excretion of the chemical and its metabolites, and overall toxicity [ 1 ]. In the mid 1980s, the mouse spot test [ 2 ] was suggested as a complementary in vivo test to the bacterial mutagenicity assay for detection of mutagenic substances and as a confirmatory test for the identification of carcinogens [ 3 ]. The mouse spot test has been used to assess a number of chemicals (see e.g. Additional file 1 , see separate file). It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484 [ 4 ]). However to achieve an acceptable sensitivity, a large number of animals are necessary and it is therefore an expensive type of test and seldom used. More recently assays using transgenic animals have been developed for testing in vivo gene mutagenicity. The two transgenic mouse models with the best data base available are the lacI model (commercially available as the Big Blue ® mouse), and the lacZ model (commercially available as the Muta™ Mouse). The present study compares the results of in vivo testing of a number of chemicals using the mouse spot test and compares it with results from these two transgenic mouse models. Descriptions of test systems Mouse spot test In the spot test, mouse embryos which are heterozygous for different recessive coat colour genes, are treated in utero at gestation day 9–11 with the test substance. The exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. The in utero exposure may result in an alteration or loss of a specific wild-type allele in a pigment precursor cell resulting in a colour spot in the coat of the adult animal. The frequency of spots is compared with the frequency in sham-exposed controls [ 2 , 4 ]. In the mouse spot test there are 4 possible mechanisms by which the recessive coat-colour alleles can be expressed: 1) gene mutation in the wild-type allele, 2) deficiency (large or small) of a chromosomal segment involving the wild-type allele, 3) nondisjunctional (or other) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) [ 5 ]. Gene mutagenic but also clastogenic effects are detected by this test system. Transgenic mouse models The transgenic mutation test systems the lacI model (Big Blue ® mouse), and the lacZ model (Muta™ Mouse) are described in detail in the preceding article: Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test Methods Data presented in this documentation are the results of an extensive literature research. Concerning data on transgenic mouse assays only primary literature was used. Data on the mouse spot test were extracted from reliable reviews on this item or from primary literature. For all other data informations from secondary literature or data banks were used. Results and Discussion Comparison of the mouse spot test with transgenic mouse model systems In the literature search chemicals have been identified that had been tested using the spot test and the Muta™ mouse assay (n = 20) or the Big Blue ® mouse assay (n = 9) or both transgenic mutation assays (n = 8). The results (including references) are given in Additional file 1 . The results on 15 out of 20 substances (2-acetylaminofluorene, acrylamide, benzo[ a ]pyrene, 1,3-butadiene, cyclophosphamide, ethylmethanesulfonate, N -ethyl- N -nitrosourea, N -methyl- N '-nitro- N -nitrosoguanidine, N -methyl- N -nitrosourea, 4-nitroquino-line-1-oxide, N -nitrosodiethylamine, N -nitrosodimethylamine, procarbazine, 4-acetylaminofluorene and N -propyl- N -nitrosourea) showed agreement between the Muta™ mouse and the mouse spot test. No agreement was seen with 5 out of 20 substances (4-acetylaminofluorene, 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), hydrazine, mitomycin C, trichloroethylene). The positive results obtained with the Big Blue ® mouse assay agreed with results in the mouse spot test for 7 out of 9 substances (2-acetylaminofluorene, benzo[ a ]pyrene, 1,3-butadiene, cyclophosphamide, N -ethyl- N -nitrosourea, N -methyl- N -nitrosourea, N -nitrosodimethylamine); one (di-(2-ethylhexyl)phthalate) was negative in both test systems and only one (methyl methanesulfonate) showed no agreement between the two test systems. With two exceptions, 4-acetylaminofluorene and N-propyl-N-nitrosourea (discussed later), all of the tested substances showed also clearly positive results in in vitro gene mutation assays (exception of 1,3-butadiene, negative results) and in the majority of in vivo studies on this endpoint. Further they induced carcinogenic effects in long-term studies on mice. Although no data on carcinogenicity on mice is available on N -propyl- N -nitrosourea, this substance might also be included in the category mentioned above, since carcinogenic effects were reported in rats [ 113 ] and in vitro gene mutation assays revealed clearly positive results. The following substances did not show agreement between results in the mouse spot test and transgenic mouse assays or negative results were reported in both test systems (see Additional file 1 ). These are therefore discussed in more detail here; for references see Additional file 1 . Table 1 Characteristics of the Muta™ mouse assay and the Big Blue ® mouse assay for predicting mouse carcinogenicity in comparison with the mouse spot test Term# Calculation* for the mouse spot test Calculation* for Muta™ and/or Big Blue ® mouse combined ** Sensitivity 84% (16/18) 79% (15/18) Specificity 0 (0/0) 0 (0/0) Positive predictability 100% (16/16) 100% (15/15) Negative predictability 0 (0/2) 0 (0/3) Overall accuracy 84% (16/18) 79% (15/18) # Sensitivity = % of carcinogens with a positive result in the specified test system (STS) Specificity = % of noncarcinogens with a negative result in the STS Positive predictivity = % of positive results in the STS that are carcinogens Negative predictivity = % of negative results in the STS that are noncarcinogens Overall accuracy = % of chemicals tested where STS results agree with the carcinogenicity results *: carcinogens with genotoxic and nongenotoxic mechanisms were considered but not substances without data on carcinogenicity; only data on mice were used **: judged as positive in transgenic assays if positive in one of the two test systems For methylmethanesulfonate, the weak positive results were judged as positive. Trichloroethylene was not included in the calculation (inconclusive results in the mouse spot test). 4-Acetylaminofluorene This substance showed mutagenic activity in the Muta™ mouse assay [ 19 ] but negative results in the mouse spot test [ 12 , 13 ]. No data on carcinogenicity are available on 4-acetylaminofluorene. However, data on two in vitro test systems indicated gene mutagenic activity supporting results in the transgenic assay [ 15 - 18 ]. 2-Amino-3-methylimidazo(4,5-f)quinol (IQ) IQ is mutagenic in the Muta™ mouse assay [ 28 ] but negative results were obtained in the mouse spot test [ 29 ]. This negative result in the mouse spot test is in contrast to all other in vivo gene mutation assays on rodents and insects which revealed positive results [ 27 ]. Furthermore, gene mutagenic activity was detected in in vitro test systems and carcinogenic effects were observed in long-term studies on mice [ 27 ]. The results in the Muta™ mouse assay are in accordance with these data. Di-(2-ethylhexyl)phthalate Negative results in the mouse spot test [ 51 ] are in agreement with the negative Big Blue ® assay [ 11 ]. Furthermore no gene mutagenic or questionable activity was reported in in vitro tests and in tests on Drosophila. Carcinogenic effects were obtained in studies on mice but nongenotoxic mechanisms are presumed. Hydrazine This substance induced mutagenic effects in the mouse spot test [ 72 ] but negative results were observed in the Muta™ mouse assay [ 71 ]. Other in vivo as well as in vitro test systems revealed gene mutagenic effects [ 70 ]. Increased tumor incidences were observed in carcinogenicity studies on mice. Overall, the mouse spot test but not the Muta™ mouse assay reflects data on genotoxicity and carcinogenicity. However, a single exposure was used in the Muta™ mouse assay [ 71 ]. Studies on other in vivo genotoxicity endpoints have shown generally negative results after single exposure but genotoxic activity after repeated application, for example the mouse bone marrow micronucleus assay was positive [ 20 ]. It is possible that positive results may be found using another experimental design in the Muta™ mouse assay e.g. repeated exposure. Methyl methanesulfonate Only weak mutagenic effects were observed in the Muta™ mouse [ 19 , 57 , 75 - 77 ] and negative results in the Big Blue ® mouse [ 63 - 65 , 78 ]. In the mouse spot test this carcinogenic substance is mutagenic [ 3 ] as well as in other gene mutation assays in vitro and in vivo [ 73 , 74 ]. However, there is evidence that the chromosome mutagenic activity is detectable at much lower doses than the gene mutagenic activity. Tinwell et al. [ 19 ] have shown in Muta™ mice a weak gene mutagenic effect in the liver but no effect in the bone marrow. The same dose induced in these animals a significant increase in bone marrow micronuclei indicating clear clastogenic activity. However, the transgenic mutation assay is less suitable for detection of these effects [ 1 ]. Mitomycin C No mutagenic activity was observed in the Muta™ mouse assay after single application and ambiguous results after repeated exposure [ 93 ] but positive results were obtained with the mouse spot test [ 2 , 3 ] and other gene mutation assays in vitro and in vivo with this carcinogenic substance [ 90 - 92 ]. The reason for this discrepancy is similar to that presumed for methyl methanesulfonate above. Clastogenicity in bone marrow but no gene mutagenic activity in liver and bone marrow has been shown in the same animals in the Muta™ mouse assay combined with a micronucleus assay [ 93 ]. However, using another experimental design for detection of gene mutations in the Muta™ mouse assay (dose level up to the MTD, repeated exposure) positive results might be obtained. Trichloroethylene Also with this carcinogenic substance, no mutagenicity was detected in the Muta™ mouse assay [ 117 ], the mouse spot test was positive [ 3 ], but this result is possibly related to contaminations with epoxides [ 116 ]. Further in vitro and in vivo assays on gene mutation resulted in weak positive, questionable, or negative effects [ 116 ]. Results in chromosome mutation assays are equivocal. However, a further (simple) reason for this discrepancy between the Muta™ mouse assay and the mouse spot test might be that the MTD was not reached in the Muta™ mouse assay presented by Douglas et al. [ 117 ]. In general, from the studies on genotoxic carcinogens given above, the results do not seem to give a preference for either the spot test or transgenic mouse model system. However, considering the mechanisms of action of specific substances there is some evidence, that the mouse spot test detects gene mutations as well as chromosome mutations whereas the transgenic mouse assays are restricted to gene mutations. Evidence for this hypothesis has been shown with the examples methyl methanesulfonate, mitomycin C, and trichloroethylene. In the mouse spot test, there are four possible mechanisms by which the recessive coat-colour alleles can be expressed (see introduction) including gene and chromosome mutations. Although the chromosome mutations have to survive several mitoses to cause the expression of the recessive allele [ 118 ], there is evidence that also predominantly clastogenic substances might result in a positive mouse spot test. In contrast, the transgenic mutation assays detected point mutations and maximal small deletions and insertions [ 1 ]. Predictivity of the transgenic animal assays and the mouse spot test for carcinogenicity The sensitivity, specificity and predictivity of carcinogenicity for the transgenic mouse model (Muta™ mouse assay and the Big Blue ® mouse assay combined) and the mouse spot test are documented in Table 1 . Data on 18 substances (see Additional file 1 ) are available on carcinogenicity in mice and mutagenic effects in transgenic mice as well as mutagenic effects in the mouse spot test (trichloroethylene not included because of inconclusive results in the mouse spot test). Although the data pool is not sufficient for a comprehensive comparison, there is some indication, that no significant differences were detectable between the two test systems. Advantages and disadvantages of both test systems Sensitivity of the test system In comparison to models using endogenous genes like the target genes in the mouse spot test, the spontaneous mutant frequency in transgenic animals is relatively high. This might be due to the fact that bacterial DNA is the target gene (high methylation rate) and/or the transgene is silent and no transcription related repair occurs as in endogenous genes which are more efficiently repaired [ 1 ]. However, comparing the number of cells and genes at risk at the time of exposure, the mouse spot test is numerical inferior to the transgenic mouse mutation assays. In the mouse spot test, the exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. In the transgenic Big Blue ® mouse, for example, 30–40 copies of the target gene (the constructed λLIZα shuttle vector) are integrated on chromosome 4 of each cell of the animal [ 1 ]. Other factors To achieve an acceptable sensitivity, a large number of animals are necessary in the mouse spot test. Many pregnant dams have to be in one treatment group to get a sufficient number of surviving F1-animals, since the test substance may induce maternal and developmental toxicity. Fahrig [ 2 ] suggested that 30–40 pregnant mice are needed per treatment group for evaluation of spots in the progeny. At least 150 F1-mice are recommended for the concurrent vehicle control [ 5 ] and at least two dose groups are used (OECD guideline 484 [ 4 ]). Therefore, the mouse spot test is an expensive type of in vivo test. In contrast, in transgenic mutation assays ca. 20 animals (3 dose groups and 1 concurrent vehicle control group in laboratories which already established this test system) are recommended per species and gender [ 119 - 121 ]. In the mouse spot test the discrimination between spots of mutagenic and non-mutagenic origin may be problematic [ 2 ]. A comparison of both test systems is presented in Table 2 . Table 2 Advantages and Disadvantages of mouse spot test compared to the transgenic Big Blue ® and Muta™ mouse assays Mouse spot test [2-5] Transgenic mouse mutation assay [1, 122] Age restriction Exposure restricted to embryos at gestation day 9–11 Usually less than 3 months Toxicokinetics and metabolism Restrictions in toxicokinetics: test substance reaches the fetal melanoblasts after administration to the dams and absorption of the test substance itself or the toxic metabolites via the placenta No further barrier like the placenta after absorption and distribution Target tissue Restricted to melanoblasts No tissue restriction; analysis of mutagenic potency in different organs Type of mutation Detects 1) gene mutation, 2) large or small deletions, 3) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) Detects 1) gene mutation, 2) small deletions or insertions Dependency of effects on application route Only systemic effects can be detected; no application route specific effects For different routes systemic as well as local mutagenic effects can be detected Target gene/cell 4 genes per cell in ca. 200 melanocytes Ca. 40 (Big Blue) or ca. 80 (Muta™ mouse) copies of the transgene per nucleus of each cell of the organismk Number of animals Animal consuming test system Not more than 5 animals per gender per dose necessary Specificity of test system Discrimination between spots of mutagenic and non-mutagenic origin may be problematically Identifying and isolating mutated genes with a high specificity Characterisation of mutations by molecular methods Less suitable for identification of mutations in DNA analysis due to size of the genes detection of the "molecular signature" of a particular mutagen by DNA sequence analysis with standardized methods Possibility of parallel investigation of several genetic endpoints No combination with other genotoxic endpoints possible The transgenic mouse assay can be combined with other in vivo genotoxic endpoints in the same animal: e.g. micronuclei, chromosomal aberration, unsheduled DNA synthesis, sister chromatid exchange Endogenous versus foreign target gene The mouse spot test shows an in situ end point (expression of the target genes) Target genes are integrated parts of foreign DNA and consequently no "normal" mutational target Costs Expensive type of in vivo test Less expensive Conclusions Although the mouse spot test is a standard genotoxicity test system according to the OECD guidelines, this system has seldom been used for detection of somatic mutations in vivo in the last decades. This is partly due to considerations of cost effectiveness and number of animals needed for testing but also for toxicological considerations. The usefulness of the mouse spot test in toxicology is limited by restrictions in toxicokinetics, sensitivity, target cell/organ, and molecular genetics. From the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo . Author's contributions UW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript. Supplementary Material Additional File 1 Results in the transgenic mouse assay versus mouse spot test Click here for file

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526206

Patterns of use, dosing, and economic impact of biologic agent use in patients with rheumatoid arthritis: a retrospective cohort study

Background Variability in dosing and costs of biologics among patients with rheumatoid arthritis (RA) is of interest to healthcare descision-makers. We examined dosing and costs among RA patients newly treated with infliximab or etanercept under conditions of typical clinical practice. Methods Integrated pharmacy and medical claims data were obtained from 61 U.S. health plans. RA patients newly treated with infliximab or etanercept between July 1999–June 2002 were selected. A maintenance number of infliximab vials was determined after the "loading period" (2–3 infusions); those with ≥ 2 occurrences of an increase in vials or an interval between infusions of <49 days were considered to have had escalated. For etanercept patients, escalation was based on ≥ 2 instances of increased average daily dose. Multiple logistic regression analyses were conducted to assess variables associated with dose escalation. RA-related costs at one year post-initiation also were examined; comparisons were made using generalized linear models. Results A total of 1,548 patients were identified (n = 598 and 950 for infliximab and etanercept respectively). Infliximab recipients were somewhat older (50.5 vs. 46.6 years for etanercept). Nearly 60% of infliximab patients increased their dose at one year, compared to 18% for etanercept. Infliximab patients who escalated dose incurred a 25% increase in mean one-year costs ($20,915 vs. $16,713 for no increase; p < 0.0001). Costs among etanercept patients did not substantially differ based on dose escalation ($14,482 vs. $13,866 respectively). Conclusions Infliximab is associated with higher rates of dose escalation relative to etanercept, which contributes to substantially higher one-year medical costs.

Background Rheumatoid arthritis is a costly and debilitating autoimmune disorder that is characterized by joint pain, stiffness, and impaired functionality. Symptoms arise from the inflammation and degradation of the synovial membrane, causing progressive disability in joint function [ 1 ]. As the disease progresses, patients require more frequent invasive procedures (e.g., joint injections, synovectomy) as well as the eventual replacement of affected joints. Consequently, the economic costs of RA are considerable, as the estimated direct and indirect costs of related care in the US totals $19 billion annually [ 2 ]. Because there is no known cure for RA, the goal of therapy is to treat the disease's symptomatology while attempting to slow or halt its overall progression. Pharmacotherapy is the cornerstone of treatment where symptoms may be treated with various combinations of nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and narcotic analgesics. In addition, disease-modifying antirheumatic drugs (DMARDs) are sometimes administered in an effort to alter the disease's progression. The effectiveness of DMARDs, however, is offset by the high levels of toxicity experienced by some patients taking these medications, and is problematic for long-term therapy [ 3 ]. In addition to the use of various DMARDs either alone or in combination with other therapies, several new DMARDs have recently been introduced for the treatment of RA. These include two biologic agents, etanercept (Enbrel ® , Amgen, Inc./Wyeth, Inc., Thousand Oaks, CA and St. David's, PA) and infliximab (Remicade ® , Centocor Inc., Malvern, PA) which provide anti-rheumatic activity by inhibiting tumor necrosis factor (TNF), another important mediator of an inflammatory response. The use of such agents in combination with the DMARD methotrexate has been shown to be clinically superior to methotrexate alone in controlled clinical trials [ 4 - 12 ]. The results of recent observational studies examining the effectiveness of infliximab indicate that increased or more frequent dosing (i.e., beyond what is mentioned in the product labeling) may provide additional benefits for patients with rheumatoid arthritis [ 13 , 14 ]. However, the costs associated with use of biologic agents is already far greater than other DMARDs. Therefore, economic considerations may impact physicians' willingness to prescribe as well as commercial insurers' placement of biologic agents in the sequence of care. Given the wide disparity in costs between therapy alternatives and the potential impact of dose escalation on these costs, it is essential to examine the total costs of RA-related care associated with each form of therapy from a perspective of typical U.S. clinical practice. In this study, the direct costs of RA-related care were estimated on an annual basis after initial treatment with anti-TNF biologics. Resource utilization and cost estimates were also stratified by dosing status (increase in dose during follow-up vs. no increase), using retrospective claims data from commercial insurers in the US. Methods Data source Medical and pharmaceutical service claims were obtained from the PharMetrics Patient-Centric Database, and spanned the period from January 1999 to June 2002. At the time of this study, the database contained fully adjudicated service claims from 61 health plans across the US. Inpatient and outpatient diagnoses (ICD-9-CM format) and procedures (CPT-4 and HCPCS formats), as well as standard and mail order prescription records, are included in the data set. Reimbursed payments and charged amounts are available for all services rendered, as well as dates of service for all claims. Additional data elements include demographic variables (e.g., age, gender, geographic region), product type (e.g., HMO, PPO), payor type (e.g., commercial, self-pay), provider specialty, and start and stop dates for plan enrollment. All patients who met the sample selection criteria specified below were included in the analyses. Sample selection Patients with a diagnosis of rheumatoid arthritis (ICD-9-CM 714.XX) who newly started on infliximab or etanercept between July 1999 and June 2001 were initially selected for inclusion in the study sample. A hierarchical procedure was then implemented to stratify patients into treatment cohorts according to their first utilization of a particular medication at a specific point in time. For example, a patient initially receiving infliximab and later receiving etanercept would be classified as an "infliximab" patient for the duration of the study period. An index date for each therapy was established based on the first occurrence of a claim. Patients with no claims activity for the index therapy for six months prior to the index date were deemed "newly started". Those patients not continuously enrolled during the six-month pretreatment and 12-month follow-up periods were excluded from all analyses. Additionally, patients must have had at least five infusions or prescriptions for their index medication. Patients 65 and older who were not enrolled in a Medicare "risk" plan (i.e., a commercial plan that agrees to undertake full financial risk for a Medicare beneficiary) were excluded from each of the analyses, as such patients may not have had fully visible utilization and cost values due to coordination of medical benefits. All medical and pharmaceutical claims spanning the period January 1, 1999 to June 30, 2002 were then extracted for eligible patients in the data set. Measures The primary measures of interest in this evaluation were the frequency and economic impact of an escalation in biologic dose. Dose escalation was assessed for patients new to infliximab and etanercept during the study period described above. Infliximab and etanercept doses reported at the third infusion/prescription respectively were considered to be the maintenance dose levels. Subsequent utilization was then examined to determine dose escalation. Dose escalation for patients initiating infliximab was based on at the presence of least two occurrences of an increase in the number of vials reported on the infusion claim. The standard period (as indicated on the prescribing information for infliximab) between infusions following the third infusion is eight weeks. Therefore, patients with two infusions within seven weeks on two or more occasions were also considered to have an increase in dose. Dose escalation for patients initiating etanercept therapy was determined according to a change in the average daily dose (expressed in terms of mg per day); average daily dose was calculated based on data from pharmacy claims, using the following formula: metric strength(25 mg)*quantity dispensed (in vials)/days supplied Patients having two or more prescriptions with a higher average daily dose than that reported on their maintenance dose were considered to have an increase in dose. Dose escalation results were reported on an overall basis and stratified by age (<18, 18–34, 35–44, 45–54, 55–64, and 65 years and over respectively), geographic region (East, South, Midwest, West), calendar year of biologic therapy initiation (1999, 2000, or 2001), and quartile of pre-index RA-related costs. In addition to dose escalation, the demographic and clinical characteristics of the sample also were assessed and stratified among patients who did and did not escalate their dose. Characteristics of interest included age, gender, health plan type, geographic region, physician specialty as of the index date, presence of selected pre-index medications, procedures, and comorbid diagnoses, co-diagnosis of Crohn's disease, and pre-index total (i.e., RA-related and unrelated) healthcare costs. During follow-up, the numbers of prescriptions or infusions of biologic therapy were tracked, as were the costs of all appropriate medical interventions, inpatient, outpatient, and pharmacy services. Costs were tallied for both RA-related and unrelated services and medications. Costs were deemed to be RA-related based on the presence of a relevant diagnosis, medication claim, or procedure (See Appendix ' Additional file 1 ' for ICD-9-CM, CPT-4, and GPI drug codes). Analyses Based on the sample described above, a series of analyses were conducted as follows: 1. Dose Escalation – compared the rate of dose escalation at one year among patients initiating etanercept or infliximab therapies; 2. Predictive Model for Dose Escalation – identified RA patients most likely to experience an increase in dose at one year, isolating specific medical, pharmaceutical and demographic characteristics that served as predictors for patients increasing drug utilization; and 3. Comparison of Annual Costs – compared costs between etanercept and infliximab stratified by whether or not the patient escalated their dose. The proportion of patients escalating dose was compared between patients receiving etanercept and infliximab using a chi-square test or Fisher's Exact Test (for cell sizes less than five). In addition, a multiple logistic regression model was applied to identify characteristics that were most predictive of patients experiencing an escalation in dose (including the index biologic therapy). The selection of predictors for inclusion in the models began with a univariate analysis of each variable to determine the frequency of the observations associated with each treatment cohort. An initial model run was performed using the following variables: age group, gender, region, biologic therapy group, plan type, prescribing specialty, RA-related costs during the six month pre-index period, non-RA related costs during the pre-index period, maintenance dose, dummy variables (1 = present, 0 = absent) for the receipt of other RA related medications during the pre-index period, and selected comorbidities. A stepwise method was employed to produce the final model specification using an "entry" level of α = 0.15 and a "stay" level of α = 0.05. The amount of costs (reimbursed amounts paid by health plans) for all services previously described were calculated on an annual per patient basis for each cohort. Total RA-related, unrelated, and overall costs during the one-year follow-up period were compared controlling for differences in age, gender, pre-index RA related costs and other appropriate variables between the cohorts. A generalized linear model using a gamma distribution was used to control for these differences. Results Patient demographics and clinical characteristics Clinical and demographic characteristics of the study sample (N = 1,548) are presented in Table 1 . Overall, approximately one-third of patients (31%) were aged 55 or older. However, infliximab use was more concentrated among older patients, as 37% of patients on this therapy were 55 or older; compared to 27% of etanercept patients. Not surprisingly, females had a greater representation than males in the study sample, accounting for nearly three quarters of the study population (74%). Rates were similar for the infliximab and etanercept groups respectively (76.4% and 72.1%). Most patients in the sample were members of an HMO or PPO product; however, the use of infliximab was much lower in the HMO group compared to etanercept (30% vs. 45% respectively) and substantially higher among PPO patients (49% vs. 35% respectively). The use of other RA-related medications prior to biologic use differed numerically by treatment group. NSAIDs were used more frequently by etanercept users relative to infliximab (42% vs. 26% respectively), as were Cox-II inhibitors (35% vs. 27% respectively) and leflunomide (26% vs. 19% respectively). The rate of pretreatment methotrexate use was similar among the etanercept and infliximab groups (55% and 56%). Utilization of joint aspiration procedures was numerically higher during the pre-index period for patients in the infliximab group relative to the etanercept sample (35% and 28% respectively). Additionally, pre-index RA related costs also were somewhat higher among infliximab patients ($3,916 vs. $3,585). Dose escalation Patients who initiated infliximab therapy experienced significantly higher rates of dose escalation during the first year of follow-up relative to patients who were initiated on etanercept (58% vs. 18%; p < 0.001) (Table 2 ). When stratified by pre-index costs, patients initiating infliximab therapy had consistently higher rates of escalation relative to patients on etanercept therapy, although the rate of dose escalation generally increased with pre-index costs; the rate of escalation at one year for patients with the lowest pre-index costs was 50% for the infliximab cohort compared to 17% for patients initiating etanercept (p < 0.001); corresponding rates were 62% and 21% in the highest cost group (p < 0.001). When stratified by year of therapy initiation, age, and geographic region, rates of escalation were significantly higher among patients initiating infliximab therapy relative to the etanercept cohort across all groups. The rate of escalation increased by calendar year for patients receiving infliximab, but declined among etanercept users. Interestingly, while the rate of dose escalation increased with increasing age in the etanercept group, this rate declined in the infliximab group as age increased (beyond age 35); for example, 66.4% of those aged 35–44 in the infliximab group increased their dose, versus 39.3% in patients aged 65 and older. Finally, rates of dose escalation varied considerably by region, with the highest rates observed in the South and Midwest. Predictive model for dose escalation Modeled dose escalation results for patients who initiated infliximab or etanercept therapy are presented in Table 3 . The type of biologic therapy was by far the most significant predictor of dose escalation, as patients starting infliximab were over 6 times more likely to increase dose than patients starting on etanercept (Transformed Odds Ratio [OR] = 6.38; p < 0.0001). Patients who were members of an HMO were less likely to have an increase in dose than those who did not. RA patients with a listed comorbid diagnosis of Crohn's disease were less likely to escalate dose (OR = 0.48; p = 0.0477) than those without, while patients utilizing Cox II therapy were significantly more likely to experience an increase in dose over the course of the year (OR = 1.36; p = 0.0175). Patients in the West were much less likely to experience an increase in dose. Patients in the Northeast were 1.92 times more likely to increase dose (p = 0.0215), while patients in the South and Midwest were 1.87 and 1.89 times more likely to dose escalate (p = 0.0102 and 0.0063 respectively). Age and gender were not significant predictors of dose escalation, although the likelihood of dose escalation increased by 4% with every additional $1,000 of RA-related pretreatment costs. In an effort to better understand the factors associated with infliximab dose escalation, an additional model was conducted among patients initiating infliximab only. Results are presented in Table 4 . In this model, patients who belonged to an HMO were significantly less likely to have an increase in dose at one year relative to patients with other coverage (OR = 0.68; p = 0.0372). Patients utilizing methotrexate during pretreatment were more likely to escalate dose than those without (OR = 1.48; p = 0.0216). There was a trend towards significance in terms of an age effect, as infliximab users between the ages of 35–44 were more likely to escalate dose relative to younger patients (OR = 1.94; p = 0.0682). Comparison of annual costs Overall, costs for patients initiating infliximab therapy were numerically higher than for patients in the etanercept group ($19,144 vs. $13,977). Much of the cost difference was due to the difference in drug costs ($13,470 vs. $10,159 respectively). In addition, infliximab patients had higher costs for physician management visits ($691 vs. $381), ancillary services ($1,511 vs. $866), and hospitalizations ($2,277 vs. $1,322). Use of alternative biologic therapy (i.e., use of etanercept in a patient starting on infliximab and vice versa) was minimal, as illustrated by extremely low average annual costs for these alternative strategies. Average annual RA-related, unrelated, and total costs are also stratified by whether patients underwent dose escalation in Table 5 . Patients in the infliximab group who experienced an increase in dose had significantly higher RA-related costs relative to those who remained at maintenance levels ($20,915 vs. $16,713; p < 0.0001). This difference was primarily manifested in lower pharmacy costs for patients not escalating dose; for example, annual infliximab costs were 60% higher for patients escalating dose ($15,998 vs. $10,000; p < 0.001). Ancillary costs were also higher for patients with an increase in dose ($1,601 vs. $1,387). However, RA-related hospitalization costs were lower for patients who had an increase in dose ($1,516 vs. $3,323). Discussion In an effort to better understand the differences in dosing patterns and costs among RA patients on biologic therapy, a retrospective analysis of pharmacy and medical claims for patients new to biologic therapy was undertaken. Dosing frequency and quantity was examined, as were RA-related costs at one year after therapy initiation. Dosing guidelines suggest that etanercept patients receive two 25 mg vials a week; the use of higher doses has not been studied. The recommended dosing for infliximab is 3 mg/kg of body weight for the first dose, and then at two and six weeks and every eight weeks thereafter. Patients experiencing an inadequate response may increase dose to 10 mg/kg; or they may receive treatment as frequently as every four weeks [ 15 ]. The flexibility in these guidelines appears to be necessary, as infliximab patients in our study were much more likely to experience a dose escalation than patients on etanercept. There also appeared to be a strong relationship between the utilization of Cox-II inhibitors as well as pretreatment RA-related costs and dose escalation, indicating that disease severity may play a role in the decision to increase dose. Recent evidence suggests, however, that the relationship between dose escalation and disease activity is nonlinear. In a recent examination of infliximab and etanercept use in Sweden, improvement in disease activity levels following infliximab dose escalation was similar to that observed among infliximab patients not escalating dose as well as etanercept recipients [ 16 ]. Lastly and most importantly, differences in RA-related cost among patients new to infliximab and etanercept therapy ($19,144 vs. $13,977) were manifested mainly in the treatment costs ($13,470 vs. $10,159). Management and ancillary services accounted for most of the remaining difference. The difference in treatment costs may be attributed to the higher rate of dose escalation among the infliximab group. These patients had treatment expenses that were ~60% higher than patients who did not dose escalate ($15,998 vs. $10,000), while infliximab patients who did not dose escalate had costs similar to patients in the etanercept group. There was little difference in drug therapy costs among etanercept patients who experienced an increase in dose and those with no change ($10,427 vs. $10,100). These findings highlight the differences in treatment patterns and associated costs among patients new to etanercept and infliximab. Our study was subject to some important limitations. First, as this was a retrospective analysis of claims data, results were based on amounts billed to health plans. As a result, the unit of measurement for infliximab is billed whole vials. For example, if 1.2 vials were administered to a patient, 2 vials would be billed to the health plan. Therefore, these results may not reflect the true amount of infliximab utilized and may in fact under- or overstate the rate of dose escalation – for example, a patient who increases from 1.2 to 1.7 vials will be shown to have utilized 2 vials in both instances; in contrast, a patient moving from 1.9 to 2.1 vials will appear as having moved from 2 to 3 vials. In addition, information regarding body mass and/or patient weight was not available. As stated above, infliximab dosing levels may range from 3 mg/kg to 10 mg/kg. Dosing changes resulting from weight changes alone were therefore undetectable. Also, one method of estimation of dose change for infliximab was based on two infusions within seven weeks on two or more occasions. It is possible that some patients may have had these non-standard queuing times simply as a result of scheduling availability, and not as a result of dose escalation. Clear estimates of dose increase due to increased frequency may only be obtained through a more controlled observational study. Furthermore, no information is available in this administrative database regarding the reason for dose escalation – lack of efficacy, increase in symptoms, other reasons. Our major focus for this study was therefore to simply document that standard dosing assumptions regarding infliximab may lead to erroneous conclusions regarding its cost, given the high level of escalation seen in this and other studies. In addition, the database lacks clinical detail on levels of disease severity as well as other potentially important variables (e.g., working status) for consideration of the full clinical and economic impact of dose escalation. As with all retrospective study, we cannot rule out the possibility that differences in disease progression and/or severity between patients who do and do not escalate dose may have influenced our findings. Nevertheless, our results remained statistically significant even after controlling for observable differences between groups, indicating that any selection bias would likely only affect the magnitude, not the direction, of our findings. Finally, while the data used represent final, adjudicated claims in a health plan setting, it is possible that the data elements used are subject to coding or misclassification error. Nevertheless, if such an error rate exists, it is likely not a systematic phenomenon – that is, there is no reason to expect that coding errors would disproportionately affect the infliximab or etanercept samples in our study. Conclusions Despite the limitations noted above, we believe our study has important implications. While, the results of recent observational studies suggest that both infliximab and etanercept are highly effective in clinical practice [ 17 ], our findings suggest that patients with rheumatoid arthritis who initiate infliximab therapy are much more likely to experience an increase in dose over the course of one year relative to patients who initiate etanercept. This increase in dose leads to significantly higher pharmacy costs, as well as increases in many other RA-related medical costs. While there may be clinical factors in the decision to pick one route of therapy administration over another, public and private payers alike should carefully consider the economic implications of coverage decisions when targeting appropriate candidates for anti-TNF therapy. Competing interests All authors were employed by PharMetrics, Inc. at the time of this analysis, which was conducted based on an unrestricted research grant from Abbott Laboratories, Inc. No other competing interests are declared by any author, including stocks or other holdings, other financial interests, or non-financial interests. Authors' contributions TG was involved in the conception and design of the study, oversaw and provided quality assurance on all study output, and drafted the methods and results sections of the manuscript. DS was responsible for the design and conduct of all descriptive and statistical analyses. DO was responsible for the conception and design of the study, drafting of the background, discussion, and conclusions sections of the manuscript, and all formal correspondence regarding the study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 File consists of ICD-9-CM codes, CPT-4 codes, GPI drug codes to describe the various diagnoses, type of drugs and procedures. Click here for file

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Patterns of lung cancer mortality in 23 countries: Application of the Age-Period-Cohort model

Background Smoking habits do not seem to be the main explanation of the epidemiological characteristics of female lung cancer mortality in Asian countries. However, Asian countries are often excluded from studies of geographical differences in trends for lung cancer mortality. We thus examined lung cancer trends from 1971 to 1995 among men and women for 23 countries, including four in Asia. Methods International and national data were used to analyze lung cancer mortality from 1971 to 1995 in both sexes. Age-standardized mortality rates (ASMR) were analyzed in five consecutive five-year periods and for each five-year age group in the age range 30 to 79. The age-period-cohort (APC) model was used to estimate the period effect (adjusted for age and cohort effects) for mortality from lung cancer. Results The sex ratio of the ASMR for lung cancer was lower in Asian countries, while the sex ratio of smoking prevalence was higher in Asian countries. The mean values of the sex ratio of the ASMR from lung cancer in Taiwan, Hong Kong, Singapore, and Japan for the five 5-year period were 2.10, 2.39, 3.07, and 3.55, respectively. These values not only remained quite constant over each five-year period, but were also lower than seen in the western countries. The period effect, for lung cancer mortality as derived for the 23 countries from the APC model, could be classified into seven patterns. Conclusion Period effects for both men and women in 23 countries, as derived using the APC model, could be classified into seven patterns. Four Asian countries have a relatively low sex ratio in lung cancer mortality and a relatively high sex ratio in smoking prevalence. Factors other than smoking might be important, especially for women in Asian countries.

Background Worldwide, over one million people die of lung cancer each year [ 1 ]. In the US, lung cancer is the most common cause of cancer deaths in both sexes [ 2 ], and mortality rates in women have risen 500% since 1950 [ 3 ]. In the European Union countries, although age-standardized mortality rates have decreased for most cancer sites, lung cancer mortality rates have significantly risen in women [ 4 ]. A rising death rate from lung cancer has also been observed in Taiwan. Between 1971 and 2001, age-standardized lung cancer mortality rates per 100 000 per year in Taiwan have increased sharply, from 12.66 to 32.93 among men and from 7.83 to 14.94 among women [ 5 ]. Today, in Taiwan, lung cancer is the leading cause of cancer death in women and the second leading cause in men [ 5 ]. Epidemiological studies have shown that cigarette smoking is the major cause of lung cancer in both sexes [ 6 - 8 ]. However, smoking habits do not seem to be the main explanation of the epidemiological characteristics of female lung cancer mortality in Asian countries [ 9 - 13 ], where the prevalence of smoking is relatively low but lung cancer mortality rates are relatively high. Factors other than smoking habits might contribute to the variability in lung cancer mortality. Long-term geographical trends in cancer mortality can provide useful information to assist etiological research. However, Asian countries are often excluded from studies of geographical differences in trends in lung cancer mortality. In order to clarify the changing patterns of lung cancer mortality worldwide, we examined lung cancer trends from 1971 to 1995 among men and women for 23 countries including four from Asia – Taiwan, Japan, Singapore, and Hong Kong. In addition, we plotted the pattern of mortality rate in these countries by using the age-period-cohort (APC) model. Methods We used data from the World Health Organization (WHO) and Taiwan to analyze secular trends from 1971–1995 in lung cancer mortality in both men and women. Mortality data provided by WHO were relatively incomplete in some countries, so we analyzed data from 22 countries and Taiwan. The twenty-two countries – Hong Kong, Singapore, Japan, Portugal, Poland, Italy, Cuba, Spain, Hungary, France, Greece, Finland, United States, England and Wales, Netherlands, Belgium, Canada, Australia, New Zealand, Denmark, Norway and Sweden – are members of WHO. The data for Taiwan came directly from the Office of Statistics, Department of Health in Taiwan. Since rates for the under 30-year age group are often based on few deaths, and rates for the over 80-year group might be affected by competitive death effects, only rates for the age range 30 to 79 were considered, so as to ensure adequate reliability of the estimates. Lung cancer mortality rates between 1971 and 1995 were analyzed in five consecutive five year periods (1971–1975, 1976–1980, 1981–1985, 1986–1990, and 1991–1995) and in five year age groups. Statistical methods Age-standardized mortality rates (ASMR) were calculated using the world population for 1976 as the reference [ 14 ]. Percent changes in the ASMR were calculated as [(ASMR 1991–1995 - ASMR 1971–1975 ) / (ASMR 1971–1975 )] × 100. In order to apply the APC model, the matrix of age-specific death rates was calculated for each 5-year calendar period (from 1971–1975 to 1991–1995) and age group (from 30–34 to 75–79). The effect of period of death in the APC model was evaluated by a log-linear Poisson model with a modified method as described by Osmond and Gardner [ 15 ]. Briefly, the estimate of period effect results from minimizing the weighted sum of the Euclidean distances from the three possible two-factor models (age/period; age/cohort; period/cohort). The weights used in the minimization process were based on the goodness-of-fit measures of each two-factor model. In this study, these were taken as the inverse of the deviance statistics. The sum of period effects were constrained to be zero. These "effects" can be interpreted as logarithms of "relative" risks. These relative risks were estimated separately for men and women. A computer program written in the SAS/IML language [ 16 ] was developed to perform the above calculations. Results Age-standardized mortality rates from lung cancer per 100 000 population per year in 23 countries for 1971 to 1995 are listed for men in Table 1 and for women in Table 2 . Trends in the ASMR varied by sex. From 1971 to 1995, in men, the rates progressively increased in nine countries (Portugal, Hungary, Taiwan, Spain, Poland, Japan, Norway, France and Greece), progressively decreased in two countries (England and Wales and Finland) and increased then declined in the others. In women, rates increased between 1971–1975 and 1991–1995 in 23 countries except for Hong Kong, Cuba, and Spain, with the highest increasing rate observed in the Netherlands (223.46%). Table 1 Age-standardized mortality rate (per100 000 person years) from lung cancer in males in 23 countries, 1971–1995 ASMR Country 1971–1975 1976–1980 1981–1985 1986–1990 1991–1995 Percent increase* Rank $ Portugal 31.88 40.51 45.96 54.40 59.48 86.57 1 Hungary 97.10 116.05 138.49 161.34 180.91 86.31 2 Taiwan 32.08 39.80 49.95 54.24 58.44 82.17 3 Spain 56.92 68.84 80.68 93.80 101.49 78.30 4 Poland 93.91 113.65 135.11 152.23 158.34 68.60 5 Japan 38.88 46.55 53.40 56.96 59.44 52.87 6 Norway 45.20 53.24 62.61 65.43 67.36 49.03 7 France 74.81 87.45 93.16 99.32 100.08 33.77 8 Greece 79.55 93.63 99.23 103.77 105.44 32.54 9 Italy 94.59 110.47 123.02 125.63 117.10 23.80 10 Hong Kong 94.96 118.63 116.24 116.89 110.13 15.97 11 Canada 96.68 108.38 116.23 118.13 107.71 11.42 12 USA 109.60 117.87 121.16 120.33 115.23 5.13 13 Denmark 101.45 109.36 116.83 113.21 103.74 2.26 14 Sweden 48.34 52.24 50.10 48.56 48.21 -0.27 15 Singapore 92.34 114.24 115.29 102.90 91.47 -0.95 16 Belgium 146.33 163.41 166.59 155.70 144.87 -1.00 17 Cuba 75.86 76.35 76.59 76.16 72.90 -3.90 18 Netherlands 151.10 162.14 160.49 148.73 129.40 -14.36 19 Australia 99.14 100.74 100.11 90.81 80.86 -18.44 20 New Zealand 99.81 104.55 100.52 93.07 79.31 -20.54 21 England and Wales 159.59 152.45 136.82 120.40 101.21 -36.58 22 Finland 142.74 142.51 126.97 105.58 90.20 -36.81 23 *percent increase (%) = 100 × (ASMR 1991–1995 - ASMR 1971–1975 ) / (ASMR 1971–1975 ) $ Rank by percent increase Table 2 Age-standardized mortality rate (per100 000 person years) from lung cancer in females in 23 countries, 1971–1995 ASMR Country 1971–1975 1976–1980 1981–1985 1986–1990 1991–1995 percent increase* Rank $ Netherlands 8.42 11.06 15.46 20.80 27.24 223.46 1 Norway 8.86 11.02 15.61 21.21 26.42 198.24 2 Denmark 21.25 29.28 40.86 49.78 58.41 174.91 3 Canada 17.94 25.44 34.93 43.53 49.13 173.81 4 USA 24.96 33.85 43.41 51.50 56.51 126.46 5 Hungary 16.75 19.34 22.99 29.11 36.83 119.89 6 Sweden 11.36 13.47 16.96 20.68 24.50 115.72 7 Poland 11.40 13.66 16.41 19.68 22.90 100.83 8 Australia 14.91 19.70 23.61 26.47 28.47 90.99 9 Belgium 10.74 12.31 13.79 16.45 19.86 84.90 10 New Zealand 22.58 26.88 30.83 36.53 38.00 68.29 11 France 7.07 7.44 8.42 9.97 11.89 68.19 12 Finland 8.71 11.35 12.39 13.48 14.54 67.02 13 Taiwan 16.03 19.55 23.13 25.69 26.43 64.94 14 Italy 10.69 12.12 13.51 15.09 16.23 51.86 15 England and Wales 30.45 35.75 40.06 43.52 42.84 40.68 16 Portugal 6.74 7.14 7.97 8.56 9.43 39.97 17 Japan 11.90 13.60 14.97 15.35 15.73 32.25 18 Greece 12.64 13.57 13.21 13.86 14.28 12.97 19 Singapore 29.66 34.34 37.07 36.01 31.25 5.36 20 Hong Kong 44.18 48.99 48.01 48.32 43.14 -2.34 21 Cuba 27.87 26.63 26.90 27.77 27.03 -3.02 22 Spain 8.54 7.99 7.32 7.01 7.58 -11.27 23 * percent increase(%) = 100 × (ASMR 1991–1995 - ASMR 1971–1975 ) / (ASMR 1971–1975 ) $ Rank by percent increase Table 3 shows the sex ratio (male:female) of the ASMR for lung cancer for five consecutive five- year periods in 23 countries. The sex ratio was greater than one in each five-year period, indicating that the ASMR from lung cancer was higher in men than in women. Among the 23 countries, the trend in the sex ratio gradually decreased for the whole period in most countries except for Spain, France, Italy, Poland, Greece, Portugal, Hungary, Cuba and the Asian countries. For example, in 1971–1975, the highest sex mortality ratio was seen in the Netherlands with a sex ratio of 17.95, the ratio then gradually decreasing to a value of 4.75 by 1991–1995. The change in the sex ratio of mortality in the Netherlands might be due to the increase and then decrease in male lung cancer mortality and simultaneously to the increase in female lung cancer mortality. On the other hand, the ratio gradually increased in Spain. This might be due to Spain having the the fourth highest increase in male lung cancer mortality accompanied by a progressive decrease in female lung cancer mortality from 1971–1975 through 1986–1990, followed by a slight increase. Table 3 Sex ratio of the age-standardized mortality rate from lung cancer in 23 countries, 1971–1995 Ratio Country 1971–1975 1976–1980 1981–1985 1986–1990 1991–1995 Mean Range Rank* Belgium 13.62 13.27 12.08 9.46 7.29 11.15 6.33 1 Netherlands 17.95 14.67 10.38 7.15 4.75 10.98 13.20 2 Finland 16.40 12.56 10.25 7.83 6.20 10.65 10.20 3 Spain 6.66 8.62 11.02 13.39 13.39 10.62 6.73 4 France 10.58 11.76 11.06 9.97 8.42 10.36 3.34 5 Italy 8.85 9.12 9.11 8.33 7.21 8.52 1.91 6 Poland 8.24 8.32 8.23 7.73 6.91 7.89 1.41 7 Greece 6.30 6.90 7.51 7.49 7.39 7.12 1.21 8 Portugal 4.73 5.67 5.77 6.36 6.31 5.77 1.63 9 Hungary 5.80 6.00 6.03 5.54 4.91 5.66 1.12 10 Australia 6.65 5.11 4.24 3.43 2.84 4.45 3.81 11 Norway 5.10 4.83 4.01 3.09 2.55 3.92 2.55 12 England and Wales 5.24 4.26 3.42 2.77 2.36 3.61 2.88 13 Canada 5.39 4.26 3.33 2.71 2.19 3.58 3.20 14 Japan 3.27 3.42 3.57 3.71 3.78 3.55 0.51 15 New Zealand 4.42 3.89 3.26 2.55 2.09 3.24 2.33 16 Denmark 4.77 3.74 2.86 2.27 1.78 3.08 2.99 17 Sweden 4.26 3.88 2.95 2.35 1.97 3.08 2.29 18 Singapore 3.11 3.33 3.11 2.86 2.93 3.07 0.47 19 USA 4.39 3.48 2.79 2.34 2.04 3.01 2.35 20 Cuba 2.72 2.87 2.85 2.74 2.70 2.78 0.17 21 Hong Kong 2.15 2.42 2.42 2.42 2.55 2.39 0.40 22 Taiwan 2.00 2.04 2.16 2.11 2.21 2.10 0.21 23 *Rank by mean The mean values of the sex ratio of the ASMR from lung cancer in Taiwan, Hong Kong, Cuba, Singapore, and Japan were 2.10, 2.39, 2.78, 3.07, and 3.55, respectively, with a range of 0.17 to 0.51 over the five-year periods. These values were not only relatively constant over time, but were also lower than seen in the western countries. For example, the lowest sex ratio of 2.10 was seen in Taiwan, with the sex ratio remaining a relatively constant value over the entire period. Only age is adjusted when the ASMR is calculated. However, both age and cohort effect are adjusted in the APC model. The period effects for males and females from the APC model applied to the data from the 23 countries could be classified into seven patterns: 1) an increasing trend in both sexes, seen in Taiwan (Figure 1 ), Norway, Japan, Hungary, and Portugal; 2) a sharply increasing trend in women, with little change seen in men seen in USA (Figure 1 ), Sweden, Poland, Italy, Canada, Belgium, Denmark and France; 3) a sharply increasing trend in women, and a sharply decreasing trend in men, seen in New Zealand (Figure 1 ), Finland, Australia and Netherlands; 4) a more gradual increasing trend in women, but a sharply declining trend in men, seen only in England and Wales (Figure 1 ); 5) a decreasing trend in both sexes, seen in Singapore (Figure 1 ) and Hong Kong; 6) a decreasing and then a gradually increasing trend in women, but a sharply increasing trend in men, seen in Spain (Figure 1 ) and Greece; and 7) a relatively steady trend in both sexes, seen only in Cuba (Figure 1 ). In most countries, the long-term trend in the period effect as derived from the APC model was similar to the trend of ASMR. It is worth noting that the trend in the ASMR for female lung cancer increased and then declined in Singapore and Hong Kong. After adjusting for the cohort effect, however, a decreasing trend in the period effect was observed in Singapore and Hong Kong. Figure 1 Secular trend in the relative risk (RR) of dying from male and female lung cancer, 1971–1995, based on analyses using the age-period-cohort model in Taiwan, England and Wales, New Zealand, USA, Singapore, Spain and Cuba Discussion We found that the sex ratio in lung cancer mortality varied over time and geographically. After adjusting for age and cohort effects, seven patterns could be identified using the APC model, indicating that some countries had a similar trend in lung cancer mortality. Koo and Ho [ 17 ] indicated that smoking was a strong risk factor in the west and worldwide where there were high rates of smoking in men. We appreciate that lung cancer mortality rates for a given year depend on smoking habits over a period before that year; however, it is not possible to get data on smoking prevalence before 1975 from WHO. Therefore, based on the smoking prevalence obtained from World Health Organization (Table 4 ), the first, second, third, and fifth highest sex ratios (male:female) of smoking prevalence among the 23 countries were in Taiwan, Hong Kong, Singapore, and Japan, respectively. However, the sex ratios of ASMR from lung cancer in the four Asian countries were significantly lower than in the western countries. That is, the four Asian countries have a relatively low sex ratio in lung cancer mortality and a relatively high sex ratio in smoking prevalence. This fact is of particular interest. Table 4 Smoking prevalence in males and females, and their sex ratio, in 23 countries Country Prevalence Male Female Male: female ratio Rank* Data source $ Taiwan 55.1 3.3 16.7 1 Adult (18 years & older), 1996 Hong Kong 27.1 2.9 9.3 2 Adult (15 years & older), 1998 Singapore 26.9 3.1 8.7 3 Adult (18–64 year olds), 1998 Portugal 30.2 7.1 4.3 4 Adult (15 years & older), 1995–1996 Japan 52.8 13.4 3.9 5 Adult (15 years & older), 1998 Poland 39.0 19.0 2.1 6 Adult, 1998 Italy 32.2 17.3 1.9 7 Adult (14 years & older), 1998 Cuba 48.0 26.3 1.8 8 Adult (15 years & older), 1995 Spain 42.1 24.7 1.7 9 Adult (16 years & older), 1997 Greece 46.0 28.0 1.6 10 Adult, 1994–1998 Hungary 44.0 27.0 1.6 11 Adult (18 years & older), 1998–1999 France 39.0 27.0 1.4 12 Adult (18 years & older), 1997 Finland 27.0 20.0 1.4 13 Adult (15–64 year olds), 1999 USA 27.6 22.1 1.3 14 Adult (18 years & older), 1997 Netherlands 37.0 30.0 1.2 15 Adult (15 years & older), 1998 Belgium 31.0 26.0 1.2 16 Adult (15 years & older), 1999 Canada 27.0 23.0 1.2 17 Adult (15 years & older), 1999 Australia 27.1 23.2 1.2 18 Adult (16 years & older), 1995 New Zealand 26.0 24.0 1.1 19 Adult (15 years & older), 1998 Denmark 32.0 30.0 1.1 20 Adult (14 years & older), 1998 England and Wales 29.0 28.0 1.0 21 Adult (16 years & older), 1996 Norway 33.7 32.3 1.0 22 Adult (16–74 year olds), 1998 Sweden 17.1 22.3 0.8 23 Adult (16–84 year olds), 1998 *Rank by Male: female ratio $ Data were obtained from the World Health Organization Dietary fat consumption has been found to be positively related to lung cancer mortality [ 18 - 20 ]. Our data from Japan, Taiwan and Cuba women (on Tables 2 and 5 ) also indicated that the percent increase of fat consumption was positively related to the percent increase of ASMR by using the Spearman's rank correlation coefficient. Further study of factors other than smoking, like fat intake, on lung cancer mortality seems warranted, especially for women in Asian countries (Japan and Taiwan). Table 5 Changes in annual per caput fat consumption in 23 countries Fat Consumption* Country 1970 1990 Percent increase Rank & Taiwan 38.0 136.8 260.0% 1 Hong Kong 71.4 $ 135.6 # 89.9% 2 Spain 88.9 137.0 54.1% 3 Portugal 78.6 120.6 53.4% 4 Japan 54.6 79.3 45.2% 5 Greece 101.9 138.4 35.8% 6 Hungary 115.3 153.5 33.1% 7 Italy 114.4 151.0 32.0% 8 France 126.4 161.3 27.7% 9 Cuba 67.6 85.1 25.9% 10 New Zealand 115.4 134.7 16.7% 11 USA 119.6 138.8 16.1% 12 Canada 113.7 127.1 11.8% 13 Australia 117.8 130.6 10.9% 14 Netherlands 132.0 140.9 6.7% 15 Poland 103.9 110.3 6.2% 16 Sweden 116.8 122.6 5.0% 17 Finland 123.6 124.2 0.5% 18 Norway 131.6 127.7 -3.0% 19 England & Wales 141.7 135.8 -4.2% 20 Denmark 140.7 132.6 -5.8% 21 Belgium - - - - Singapore - - - - *Data were obtained from FAO & Rank by percent increase $ :1961, # : 1995 Conclusion Period effects for both men and women in 23 countries, as derived using the APC model, could be classified into seven patterns. The four Asian countries have a relatively low sex ratio in lung cancer mortality and a relatively high sex ratio in smoking prevalence. Factors other than smoking might be important, especially for women in Asian countries. Competing interests The author(s) declare that they have no competing interests. Authors' contributions YPL was responsible for the development of intellectual content and the study design, collected and analyzed the data, interpretation of the results, manuscript drafting and the critical revisions of manuscript. YCH was responsible for the development of intellectual content, interpretation of the results and manuscript drafting. GWL was responsible for data coding and entry and statistical analyses. Pre-publication history The pre-publication history for this paper can be accessed here:

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555565.xml

548520

Synaptogenesis and outer segment formation are perturbed in the neural retina of Crx mutant mice

Background In Leber's congenital amaurosis (LCA), affected individuals are blind, or nearly so, from birth. This early onset suggests abnormal development of the neural retina. Mutations in genes that affect the development and/or function of photoreceptor cells have been found to be responsible in some families. These examples include mutations in the photoreceptor transcription factor, Crx. Results A Crx mutant strain of mice was created to serve as a model for LCA and to provide more insight into Crx's function. In this study, an ultrastructural analysis of the developing retina in Crx mutant mice was performed. Outer segment morphogenesis was found to be blocked at the elongation stage, leading to a failure in production of the phototransduction apparatus. Further, Crx-/- photoreceptors demonstrated severely abnormal synaptic endings in the outer plexiform layer. Conclusions This is the first report of a synaptogenesis defect in an animal model for LCA. These data confirm the essential role this gene plays in multiple aspects of photoreceptor development and extend our understanding of the basic pathology of LCA.

Background Photoreceptor cells play a primary role in vision by capturing light energy and converting it into neural stimuli. These sensory neurons are a shared element in all organisms capable of sensing light. In humans, genetic diseases of the eye are common and the primary site of disease is most frequently photoreceptors (for review see [ 1 - 3 ]). Photoreceptors have been well studied, particularly with respect to the biochemistry and physiology of phototransduction. Insight into the development of vertebrate photoreceptors, however, has lagged behind our understanding of function. Only recently have the first molecular mechanisms regulating photoreceptor development been identified (for review see, [ 2 , 4 ]). Crx (cone-rod homeobox) is an otx-family homeobox gene expressed predominantly in photoreceptors, from early in their development through to the adult ages [ 5 - 7 ]. Crx gene expression is critically dependent upon Otx2, another member of the same homeobox family which is expressed in early photoreceptor cells [ 8 ]. In rod photoreceptors, Crx appears to work in concert with Nrl, a leucine zipper protein that is also restricted in its expression in the retina to rod photoreceptors [ 9 ]. Many photoreceptor-specific genes have putative Crx-binding elements in their regulatory regions [ 10 ], including rhodopsin [ 11 ] and arrestin [ 12 ]. Mutations in Crx have been associated with several human diseases that lead to blindness, including cone-rod dystrophy 2 [ 6 , 13 , 14 ], retinitis pigmentosa [ 14 ], and LCA [ 14 - 16 ]. Based on these data, Crx was hypothesized to play a critical role in the differentiation and maintainance of photoreceptor cells [ 5 , 7 ]. LCA is the most severe genetic disease of photoreceptors (see [ 17 ], for recent review). Affected infants exhibit a complete or near complete absence of vision from birth. Mutations in retinal specific genes, such as Crx, have been associated with LCA [ 14 , 15 ], as well as GUCY2D [ 18 ], RPE65 [ 19 ], AIPL-1 [ 20 ], CRB-1 [ 21 ], and RPGRIP-1 [ 22 ]. There also may be as many as three additional genetically linked loci where genes have not been identified [ 23 ]. Crx mutations in LCA are varied, and include a putative dominant mutation that is proposed to encode a dominant-negative form of Crx [ 14 , 15 ]. Recessive mutations also have been reported and at least one allele encodes a protein with decreased DNA-binding activity [ 16 ]. Histopathological and ultrastructural studies of LCA should enable a better understanding of the disease process, and the design of suitable therapies. Few such studies exist for human LCA (reviewed in [ 17 ]) and the majority of such studies examine the globes of adults with LCA, after the tissue has undergone secondary changes. Only a single study exists where the developing eye of an infant was examined [ 24 ]. Animal models for LCA have recently been reported and have already served to broaden our understanding of the pathology of this disease [ 25 - 28 ]. Since LCA is a clinically and genetically heterogeneous disorder, additional mouse models are in order to allow a full understanding of the many ways in which photoreceptor development can go awry. In addition to their importance as a locus of disease, photoreceptor cells serve as an excellent model for studies in neuronal differentiation. Photoreceptor cells are highly polarized. At their apex, these neurons have a membranous outer segment, which contains proteins involved in the phototransduction cascade. Loss of function mutations in rhodopsin [ 29 ], or the structural protein, peripherin [ 30 ], result in an inability to form outer segments. At the other extremity, photoreceptors terminate with synaptic endings that make contact with the processes of horizontal and bipolar cells [ 31 , 32 ]. Rod spherules establish an invaginating synapse with rod bipolar dendrites and axonal endings of horizontal cells. This synapse is characterized by the presence of a ribbon in the presynaptic cytoplasm. Cone pedicles make invaginating synapses with the dendrites of on-cone bipolar cells and horizontal cells and basal junctions with the dendrites of off-cone bipolar cells. The factors regulating the formation of the photoreceptor synapses are completely unknown. At least one photoreceptor synaptic protein, HRG4, contains a potential Crx target sequence in its transcriptional regulatory sequence [ 33 ]. Few studies of LCA animal models have extended their examination of retinal pathology to the ultrastructural level. Certain features of neuronal differentiation, such as synapse formation, can be detected definitively at this level of analysis. With the hope of understanding the neuropathology of LCA in greater detail, we have analyzed the differentiation of the outer retina in Crx-/- mice at the ultrastructural level. These retinas exhibit several prominent defects. Crx-/- photoreceptors demonstrate a complete block in outer segment formation at the elongation stage. Further, these cells exhibit abnormal synaptic morphology, thereby broadening the function of Crx to photoreceptor synaptogenesis. This represents the first report strongly implicating the process of synapse formation in LCA. Results Multiple pathologies in the outer segment layer in Crx-/- mice A standard knock-out protocol was used to generate mice in which the homeodomain of Crx-/- was targeted and deleted. Generation of these Crx-/- mice has been reported elsewhere [ 34 ]. In this study, in order to characterize further the role of Crx in photoreceptor morphogenesis, the outer retinae from Crx-/- mice were examined using transmission electron microscopy. At postnatal day 21 (P21), when Crx+/+ photoreceptors exhibited robust outer segments (Figure 1A , os), Crx-/- retinas were without a recognizable outer segment layer (Figure 1B ). Crx-/- photoreceptors had inner segments, demonstrating at least limited photoreceptor polarization in the Crx mutant, but the inner segments were extremely short (Figure 2 ). Furthermore, the majority of inner segments showed some morphological differentiation, having approximately as many mitochondria as the control (Figure 1 and 2 ). Occasionally, an inner segment undergoing lysis was noted, appearing swollen or with vacuoles and swollen mitochondria (data not shown). Figure 1 Transmission electron microscopy of the outer retina at P21 in (A) Crx+/+ and (B) Crx-/- retinas. pe, pigmented epithelium. os, outer segments. is, inner segments. onl, outer nuclear layer with photoreceptor nuclei. Scale bar = 5 μm for A and B. Figure 2 Transmission electron micrograph of the outer segment layer of Crx-/- retina at P21. Inner segments of Crx-/- photoreceptors exhibit numerous mitochondria (m indicated by arrow) as in Crx+/+ (Figure 1A). pe, pigmented epithelium. is, inner segments. onl, outer nuclear layer. Scale bar = 2 μm. Photoreceptor inner segments and outer segments are joined by a non-motile connecting cilium that exhibits a characteristic 9 + 0 arrangement of microtubule doublets when viewed in cross-section. At P21, in Crx-/- retinas, numerous cross sections of connecting cilia were noted (Figure 3A and 3B ). Sporadically, connecting cilia contained other than the typical complement of microtubule doublets. For example, in Figure 3A , the connecting cilium labelled by arrowhead 1, shows 7 + 0 doublets. The majority exhibited the characteristic 9 + 0 doublets (arrowhead 2 and 3 in Figure 3A and Figure 3B ). These observations indicate that in addition to inner segment formation, ciliogenesis is also largely intact in Crx-/- photoreceptors. Further, in Crx-/- retinas the retinal pigmented epithelium (PE) appeared normal, at least up to P21 (data not shown), the oldest age examined. Figure 3 Transmission electron micrograph of Crx-/- retina at P21 (A and B), and scanning electron micrograph of Crx-/- at P10 (C) of outer segment layer. (A) Evidence of ciliogenesis in the photoreceptor layer of Crx-/- retina. Nonmotile connecting cilia were observed in cross section (arrowheads 1,2, and 3, for examples). Connecting cilium 1 (arrowhead 1) demonstrated seven microtuble doublets, while cilium 2 and cilium 3 exhibited nine. In A, a displaced cell nucleus (n) appearing pyknotic and abnormal deposition of matrix (mx) material of unknown identity were seen, along with large amounts of membranous vesicles (arrow) which filled the photoreceptor space and appeared to be released from inner segments. Scale bar = 3.7 μm. (B) Nonmotile connecting cilium in cross section, from a Crx-/- photoreceptor, demonstrating characteristic 9+0 radial array of microtubule doublets. Scale bar = 88 nm. (C) Scanning electron micrograph (SEM) of membranous vesicles (arrow shows one example) shed from inner segments of Crx-/- photoreceptors at P10. Figure shows inner segments viewed from the scleral side with the pigmented epithelium removed. Scale bar = 1 μm. In addition to the complete absence of outer segments, Crx-/- retinas exhibited three other notable pathologies in the outer segment layer. First, an abnormal deposition of matrix of unknown identity was noted (Figure 3A , mx). Second, sporadically displaced nuclei were found residing in the space abutting the PE. Occasionally, these nuclei appeared pyknotic (Figure 3A , n); but, more frequently exhibited the heterochromatin pattern typical of photoreceptors (data not shown), strongly suggesting that they belonged to ectopic photoreceptors. The third pathological entity noted in the outer segment layer were numerous small vesicles (Figure 3A arrow) 100 to 200 nm in diameter. They appeared to be emerging from the inner segments, as scanning electron microscopic images showed spherical structures budding from the inner segments (Figure 3C , arrow). In order to characterize further the morphogenesis of Crx-/- photoreceptors, the developing outer segment layer was viewed by scanning electron microscopy at P7, P14 and P21 (Figure 4 ). In Crx+/+ retinas, photoreceptor inner segments, connecting cilia, and the first rudimentary outer segment structures were noted at P7. In the Crx-/- retina, only an occassional connecting cilium was noted emerging from inner segments at this stage (Figure 4A and 4B ). This observation was confirmed by comparison with transmission electron micrographs (Figure 5 ). These differences are the earliest noted differences between Crx+/+ and Crx-/- photoreceptors. At P14, elongating outer segments were noted on Crx+/+ photoreceptors, occasionally demonstrating a paddle-like structure at their apex (Figure 4C , os). In Crx-/- retinae, the vast majority of photoreceptors at this stage demonstrated connecting cilia without outer segments (Figure 4D , cc). Sporadically, Crx-/- photoreceptors would exhibit an irregular structure extending from a connecting cilium (Figure 4D , cc*) perhaps representing a malformed outer segment. Such structures were also observed at P21 (Figure 4F , cc*). These putative, abnormal outer segments were only rarely noted in Crx+/+ at P14, and never at P21 (Figure 4C and 4E , cc*). Further, in Crx-/- photoreceptors, unusually long connecting cilia were noted (Figure 4F , cc). Serial examination of Crx-/- photoreceptors at P7, P10, P14, and P21 by TEM, demonstrated a distinctive lack of any structure interpretable as orderly stacks of discs or forming discs. These data demonstrate a complete absence of normal outer segment formation in the Crx mutant mouse, and the arrest of development of the photoreceptor appendage at the elongation stage of outer segment formation. Figure 4 Outer segment morphogenesis in Crx-/- photoreceptors. Scanning electron microscopy of developing photoreceptors viewed from the scleral side with the pigmented epithelium removed at P7, P14, and P21 for Crx+/+ (A, C, and E) and Crx-/- (B, D, F) littermates. In Crx+/+ retina, numerous connecting cilia (A, cc) were evident at P7 with rudimentary outer segments. After P7, in Crx+/+ outer segment elongation occurs. Initially, outer segments have a paddle-like structure (C, os) which is later shed (E, os). In Crx-/- photoreceptors, few connecting cilia were observed at P7 (B, cc). After P7, connecting cilia were more numerous and occasionally a malformed outer segment was noted extending from a connecting cilium (D and F, cc*). These were rarely observed in Crx+/+ and only at P14 (C, cc*). At P21, abnormally long connecting cilia are noted in Crx-/- (F, cc). Scale bars = 10 μm Figure 5 Transmission electron micrographs of Crx-/- photoreceptors in the photoreceptor layer at P7. (A) Photoreceptor layer of Crx+/+ retina demonstrating nascent outer segment structures (arrow) emerging from photoreceptor connecting cilia (cc). (B) Crx-/- photoreceptors exhibited connecting cilia (cc) at this early stage, however, nascent outer segment structures were not observed. Scale Bar = 1 μm. Finally, the morphology of the malformed Crx-/- photoreceptors was compared to rhodopsin-/- and peripherin-/- photoreceptors. Rhodopsin and peripherin are two photoreceptor-specific genes whose expression is significantly downregulated in the Crx-/- retinae [ 10 , 34 , 35 ]. Loss of function mutations in each of these genes separately have been reported to result in a failure to elaborate outer segments [ 29 , 30 ]. Photoreceptors from these two mutant mice examined by SEM from the scleral side appeared highly similar to Crx-/- photoreceptors (compare Figure 4F to Figure 6A and 6B ). Figure 6 Scanning electron microscopy of peripherin-/- (A) and rhodopsin-/- (B) photoreceptors at P21, viewed from the scleral side with the pigmented epithelium removed. cc, connecting cilium. is, inner segment. Scale bar = 10 μm. Crx is necessary for the formation of photoreceptor terminals In a previous study, we demonstrated that forced expression of a dominant-negative allele of Crx in developing rods blocked formation of both rod spherules in the outer plexiform layer (OPL) and outer segments [ 7 ]. To expand on these studies, the ultrastructure of photoreceptor synapses was examined in Crx-/- retinas. In Crx+/+ retinas at P21, newly mature rod spherules were abundant (Figure 7A ). The sperules were blunt or club-shaped structures with a single ribbon associated with a single invaginating synapse (Figure 7A , arrow indicates one example; Figure 8A and 8B ). Two processes from horizontal cells were situated on either side of the synaptic ridge (Figure 8B , labelled H) and one or more dendrites of rod bipolar cells occupied a central position (Figure 8B , bipolar labelled B). Cone terminals are large, flat pedicles that exhibit many invaginating synapses containing separate sets of horizontal and bipolar cell processes. Each pedicle contains numerous ribbons. These terminals were much less common than spherules in Crx+/+ retinas at P21 (Figure 7 , box). In the OPL of Crx-/- retinas, photoreceptor terminals were highly disorganized at P21 (Figure 7B , arrows). Processes containing synaptic vesicles and ribbon-like structures were apparent, suggesting at least limited generation of synapse components. However, well formed spherules and pedicles were not observed. In addition, many terminals appeared to contain multiple ribbons (Figure 8C and 8D , r) not tethered to the plasma membrane and occasionally in perinuclear positions (Figure 8D ). Figure 7 Transmission electron micrographs of the outer plexiform layer in Crx-/- retinas. (A) In Crx+/+ retina at P21, newly formed rod spherules were abundant (arrow demonstrates one example). The spherules were club-shaped and contained a single invaginating synapse with one ribbon complex. Cone terminals form large, flat pedicles that contain many invaginating synapses with separate ribbon structures. These terminals were more scarce, but apparent in Crx+/+ retinas at P21 (one example in box). (B) In the outer plexiform layer (OPL) of Crx-/- retinas, photoreceptor terminals appeared highly disorganized at P21 (arrows). Well-formed pedicles and spherules were not evident. Putative terminals containing ribbon-like structures were apparent, suggesting at least limited generation of synapse components. Many terminals appeared to contain multiple ribbon-like structures, instead of a singule ribbon. For example, terminal 1 and 2 contained two ribbons each, whereas terminal 3 appeared to contain only one. opl, outer plexiform layer. Scale bar = 2 μm. Figure 8 Transmission electron micrographs of the outer plexiform layer in Crx-/- retinas at P21. (A) Crx+/+ rod spherules contained a single invaginating synapse with one ribbon complex. The spherule was a blunt or club-shaped structure. (B) Crx+/+ rod terminals contained a single ribbon structure (r). Two processes, from horizontal cells (h), contacted the rod laterally. An additional process, the postsynaptic bipolar dendrite (b), was situated more centrally. Terminals were filled with synaptic vesicles. One coated vesicle originatinf from the cell membrane was observed (arrow). (C) In the OPL of Crx-/- retinas, photoreceptor terminals appeared highly disorganized. Putative terminals containing synaptic vesicles and ribbon-like structures were apparent (arrows), suggesting at least limited generation of synapse components. However, well formed spherules and pedicles were not observed. Further, many terminals appeared to contain multiple ribbon-like structures (r). The majority of these ribbons were not associated with the synaptic membrane, but instead were found free floating and, in some instances, were perinuclear (D, arrow). H, horizontal cell; B, bipolar cell; N, nucleus; r, ribbon. (A) Scale bar = 500 nm, (B) Scale bar = 250 nm, (C and D) Scale bar = 500 nm. Discussion In this study, an ultrastructural analysis of Crx-/- photoreceptors was carried out. As Crx mutations have been associated with Leber's congenital amaurosis, the findings in this study broaden our understanding of the pathology of this disease. Two prominent pathologies were characterized in the Crx-/- retina: (1) An absolute block in outer segment morphogenesis was noted, with the block occuring at the elongation stage of outer segment formation; (2) Crx-/- photoreceptors exhibited a severe perturbation in synapse formation. This represents the first report of a synaptogenesis defect in an animal model of LCA. Crx-/- photoreceptors cannot complete outer segment morphogenesis Mutations in Crx represent one of a collection of gene mutations that lead to an outer segment formation defect. Homozygous null mutations in the peripherin/RDS gene [ 36 ] or in rhodopsin [ 29 ] lead to a failure of outer segment formation. The deficits in peripherin-/- and Crx-/- photoreceptor morphogenesis were found to be very similar. Vesicular structures in Crx-/- photoreceptors were observed that were similar to those previously noted in the rds/peripherin-/- mouse. It was initially proposed that these vesicles were due to the breakdown of outer segment membranes that were not properly recruited to the outer segments in the absence of peripherin, or were from the result of the breakdown of the microvilli of Müller cells [ 30 ]. Strong support in favor of the former explanation was provided by Nir and colleagues who demonstrated the presence of rhodopsin protein in these vesicles using immunoelectron microscopy against a rhodopsin epitope [ 37 ]. Further, as shown here, the vesicles appear to bud from the inner segments themselves. In developing photoreceptors, an extraordinary growth process occurs whereby the outer segment is generated from the nascent connecting cilium (see [ 38 ] and references therein). Peripherin/RDS and ROM-1 proteins (localized in disc rims) and the opsin proteins (localized throughout the discs) have important roles in the structural integrity of mature outer segments (see [ 39 , 29 ]). ROM-1-/- mice produce disorganized outer segments with large disks [ 40 ]. Crx, by virtue of being a transcription factor, presumably controls genes that are responsible for the building and perhaps maintenance of the outer segment structure, including rhodopsin and peripherin. Using northern blots [ 34 ], microarrays [ 10 ], and serial analysis of gene expression (SAGE) [ 35 ], we have defined a large number of genes that are altered in their expression level in Crx-/- mice. We found that rhodopsin expression was severely diminished in Crx-/- animals, and peripherin mRNA was reduced by approximately 30%. Transgenic mice with variable levels of expression of wild type rhodopsin exhibit rod degeneration [ 41 ], indicating the importance of the level of rhodopsin expression. In addition, the timing of rhodopsin expression may be very important, as indicated by studies in Drosophila. In Drosophila , rhodopsin ( ninaE ) is expressed in photoreceptors R1–R6. In ninaE null mutants, the rhabdomere, a structure analogous to vertebrate outer segments, fails to develop in R1–R6 photoreceptors [ 42 ], reminiscent of the situation in rhodopsin-/- mice [ 29 ]. An intriguing experiment by Kumar et al. [ 43 ] demonstrated a temporal requirement for rhodopsin expression during rhabdomere development. In ninaE null flies, a ninaE transgene under the control of a heat shock promoter was subjected to various temperature shifts during development. Heat shock during the normal time of rhodopsin onset resulted in substantial and long-lasting rescue of photoreceptor structure and transient rescue of photoreceptor physiology. However, expression shortly before or after this critical period failed to rescue, suggesting that rhodopsin expression during a discrete window of time in development is essential for proper rhabdomere morphogenesis. This result is consistent with observations in the rat wherein rhodopsin onset occurs with strict timing in the developmental history of most rods in vivo [ 44 ]. Thus, through its regulation of rhodopsin levels, or perhaps through control of the kinetics of the up-regulation of rhodopsin beginning at about P6, Crx may be regulating outer segment morphogenesis. The similarty of the two cases may extend further. At present, the closest Crx relative in Drosophila is Otd, the founding member of the class of homeobox genes to which Crx belongs. Interestingly, in one hypomorphic allele of Drosophila otd , otd uvi , photoreceptor morphogenesis is also disrupted [ 45 ]. We found that there are many other genes that are dependent upon Crx. Those that are expressed at a lower level in the Crx-/- retina, such as rhodopsin and peripherin, comprise many that are either enriched or specific to photoreceptors in their expression [ 35 ]. They include enzymes that are important in lipid metabolism, protein folding and transport, as well as in other processes that one might envision would be important in building a structure such as the outer segment. In situ hybridization using probes from this collection of genes has revealed that some of them have their RNA localized to the inner segment, a finding typical for proteins targeted to the outer segment. Future analyses of the function of these genes might reveal their role in outer segment biogenesis. Finally, polarization of photoreceptors was found to be largely intact, as was ciliogenesis. Another LCA gene, CRB1, and a related gene CRB3, have been implicated in ciliogenesis in in vitro models [ 46 ]. The Drosophila homologue of CRB1, Crumbs, has been implicated in photoreceptor morphogenesis [ 47 ]. However, the spontaneously occurring mouse mutant in CRB1, the Rd8 mouse, develops shortened outer segments that subsequently degenerate [ 48 ], suggesting that photoreceptor polarization and synaptogenesis are intact in this mutant. While CRB1 and Crx have been both linked to LCA, further work is necessary to determine if their function is linked in retinal development. Synaptogenesis is perturbed in Crx-/- photoreceptors The Crx-/- mouse demonstrates the most severe abnormality of photoreceptor synapses reported to date. The peripherin-/- mouse develops a normal complement of photoreceptor terminals which then degenerate as the photoreceptors are lost [ 30 ]. Also, similarly in rhodopsin (Rho) and cyclic nucleotide-gated channel, alpha-3 (CNGA3) double knockout mice (Rho-/-, CNGA3-/-), synapses are reported to form normally [ 49 ]. These observations demonstrate that photoreceptor synaptogenesis can occur in the absence of outer segment formation. In keeping with this observation is the fact that some electroretinogram activity is present in peripherin-/- mice, suggesting that minimal phototransduction is present in these mice, enough to drive activity at the photoreceptor synapse. In vitro studies wherein synapse elements are formed in the absence of proper outer segment development and, therefore, possible absence of light-dependent photoreceptor activity, have indicated the independence of phototransduction and synapse formation, at least for the initial stages [ 50 , 51 ]. These data then suggest that the fact that the Crx-/- photoreceptors do not have proper synaptic endings is not due to a lack of outer segment formation. A more likely explanation is that Crx plays a role in photoreceptor synapse formation, perhaps by regulating directly, or indirectly, important genes in this process. Using immunohistochemistry, we examined the expression of common pre-synaptic terminal proteins, including KIF3a, SV2, and synaptophysin, and were unable to observe qualitative differences between Crx-/- and control tissue at P14 (data not shown). Examination of their RNA levels by SAGE showed no significant difference for all 3 genes, though very few tags were recovered from these genes and thus the analysis of RNA levels may not be significant [ 35 ]. However, since other genes expressed in photoreceptors were significantly altered in their expression level in the Crx-/- mouse, there are many candidates that could be important for photoreceptor morphogenesis. Tags from three genes from proteins expressed in photoreceptor terminals were found to be decreased in a statistically significant fashion, namely the HGF-regulated tyrosine kinase substrate, the CRIPT protein, and synaptotagmin 1 (Blackshaw and Cepko, unpublished data). An example of a gene that was increased in the Crx-/- retina is HRG4 (a homologue of the C. elegans Unc119 gene) (Blackshaw and Cepko, unpublished data) which encodes a component of the ribbon synapse [ 33 ]. The fact that it is upregulated might indicate a response to the lack of proper terminal structures. Several other genes encoding putative cytoskeletal elements also were increased (e.g. microtubule associated protein 4) or decreased (e.g. cofilin 1) in the Crx-/- retina, with P values of <.005. It is not known whether any of these genes are involved in building or regulating synaptic structures, but they are now genes that might lead to a better understanding of the construction and function of the relatively unique structure of the ribbon synapse. Abnormal photoreceptor terminal formation was noted in a study that examined retinal development in the laminin beta2 chain-deficient mouse [ 52 ]. Several pathologies were noted in these mice. First, laminin beta2 chain-deficient mice displayed abnormal outer segment elongation, but a more mild phenotype than that of the Crx-/- mice; the outer segments were reduced by 50% in length. Also photoreceptor terminals were perturbed in laminin beta2 mutants, but again the phenotype was more subtle then that of Crx-/- mice. The outer plexiform layer of the beta2-deficient retinas demonstrated only 7% normal invaginating synapses, while the remainder had various pathologies, including floating synaptic ribbons, as seen here. The mechanistic relationship of these two molecules, if any, in photoreceptor morphogenesis is unknown to date. The mRNA for laminin beta2 was not detected in the SAGE study of the relative RNA levels in Crx-/- and Crx+/+ and thus we cannot comment on whether the levels of RNA for laminin beta2 were altered. Crx-/- mice are a model for LCA Crx has been implicated in three photoreceptor diseases that result in human blindness, cone-rod dystrophy2, Leber's congenital amaurosis, and retinitis pigmentosa (for review, see [ 53 ]). The cone-rod dystrophies (CRDs) are characterized by loss of cone-mediated vision in the first decade of life or later, with concomitant or subsequent loss of rod-mediated vision [ 54 ]. Conversely, RP is notable for initial loss of rod function, followed by loss of cone-mediated vision [ 55 ]. The majority of known genes responsible for human genetic blindness, encode proteins expressed almost exclusively, or exclusively, in photoreceptors, particularly in the outer segment [ 35 ]. Many of these proteins are required for phototransduction or outer segment structure. The mechanisms whereby mutations in rod-specific genes, such as rhodopsin, lead eventually to cone degeneration in RP remain obscure. Mutations in Crx were the first, and still one of a very few examples of a transcription factor mutation leading to photoreceptor disease. LCA is a disease in which there is little or no photoreceptor function in infancy; thereby, likely developmental in etiology ([ 17 , 56 ] for review). The Crx-/- mouse may be an excellent model for studying the pathology of this disorder, particularly the subtype of the disorder where Crx mutations are involved. The vast majority of histopathological studies of LCA in human tissue have been derived from adult patients with LCA where secondary changes are likely to be present. Indeed in animal models of LCA, secondary reactive and/or degenerative changes occur early after the abnormal formation of retinal tissue [ 57 ]. The only study in human tissue derived from a human 33-week retina with proposed RPE65 mutations was reported to have abnormal retinae at this early stage [ 24 ]. These authors report cell loss, including thinning of the photoreceptor layer. In addition, they claim in the text to have seen aberrant synaptic and inner retinal organization, although their examination of photoreceptor synapses unfortunately are not presented in the data section of the paper. Given the scarcity of available human tissue, the characterization of the primary pathology of LCA will require animal models. In the current study, we present data that argue that, in addition to outer segment morphogenesis, synaptogenesis may also be critically impaired in at least a subset of LCA. Methods Mice Crx-/- mice were generated as reported elsewhere [ 34 ]. Rhodosin-null mice [ 29 ] were obtained from Paul Sieving (University of Michigan). Rds mice were acquired from Jackson Laboratory. Transmission electron microscopy Littermate Crx-/- and wildtype pups were perfused in 1% formaldehyde and 0.5% glutaraldehyde at various postnatal stages. The eyes were then enucleated, and the cornea and lens were removed. The eye cup was immersed in fixative (1% formaldehyde and 2.5% glutaraldehyde) for 3 to 4 hours at 4°C. The sclera was then partially removed and the retinas were sliced into small pieces and fixed (1% paraformaldehyde and 2.5% glutaraldehyde) overnight at 4°C. These procedures were found optimal for maintaining the structural integrity of the photoreceptor outer segments. After fixing, the tissue was washed 2X in PBS for thirty minutes per wash. The tissue was then postfixed in a 1% osmium tetroxide/1.5% potassium ferrocyanide mixture for 2 hours at 4°C. Staining was carried out for 30 minutes in 1% uranyl acetate in maleate buffer (pH = 6.0) at room temperature followed by 1% tannic acid in 0.1 M cacodylate buffer (pH = 7.4) for thirty minutes. The specimens were then dehydrated and embedded in Epon/Araldite. Thin sections were stained with uranyl acetate and lead citrate, and examined in a Jeol JEM-1200EX electron microscope. Scanning electron microscopy Specimens used for SEM required removal of the retinal pigment epithelium (PE), enabling visualization of the outer surface of the neural retina. Retinae from Crx-/-, rhodopsin-/-, RDS, or wildtype eyes were dissected free from PE in a dispase solution and fixed in 1.25% glutaraldehyde and 1.0% formaldehyde overnight at 4°C. Tissue was then washed 5X in cacodylate buffer and dehydrated in ascending grades of ethanol. Tissues were subsequently critical point dried in carbon dioxide. All specimens were mounted and coated with sublimated gold-palladium by the sputtering technique. Micrographs were obtained with a Jeol JSM-35CF scanning electron microscope. Authors' contributions EM and ER conducted transmission electron microscopy. EM performed scanning electron microscopy. TF and EM generated, characterized and maintained the Crx-/- mouse line. CC participated in the design and coordination of the study and all data analysis. EM and CC drafted the manuscript. All authors read and approved the final manuscript.

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514539

Tests for finding complex patterns of differential expression in cancers: towards individualized medicine

Background Microarray studies in cancer compare expression levels between two or more sample groups on thousands of genes. Data analysis follows a population-level approach (e.g., comparison of sample means) to identify differentially expressed genes. This leads to the discovery of 'population-level' markers, i.e., genes with the expression patterns A > B and B > A. We introduce the PPST test that identifies genes where a significantly large subset of cases exhibit expression values beyond upper and lower thresholds observed in the control samples. Results Interestingly, the test identifies A > B and B < A pattern genes that are missed by population-level approaches, such as the t-test, and many genes that exhibit both significant overexpression and significant underexpression in statistically significantly large subsets of cancer patients (ABA pattern genes). These patterns tend to show distributions that are unique to individual genes, and are aptly visualized in a 'gene expression pattern grid'. The low degree of among-gene correlations in these genes suggests unique underlying genomic pathologies and high degree of unique tumor-specific differential expression. We compare the PPST and the ABA test to the parametric and non-parametric t-test by analyzing two independently published data sets from studies of progression in astrocytoma. Conclusions The PPST test resulted findings similar to the nonparametric t-test with higher self-consistency. These tests and the gene expression pattern grid may be useful for the identification of therapeutic targets and diagnostic or prognostic markers that are present only in subsets of cancer patients, and provide a more complete portrait of differential expression in cancer.

Background Studies of differential expression of individual genes often find genes that are up-regulated in some tumors, and down-regulated in others. Microarray studies typically seek to identify differentially expressed genes using use fold-change [ 1 ], t-tests [ 2 ], and models [ 3 - 6 ]. Studies of global gene expression patterns in cancer have focused largely on the identification of novel cancer subtypes via classification [ 7 - 13 ] or the identification of differentially expressed genes [ 14 - 18 ]. Such studies typically use fold-change [ 1 ], t-tests [ 2 ], and models [ 3 - 6 ]. The methods of analysis for identifying differentially expressed genes in data from microarray experiments vary widely [ 20 - 45 ], but all are focused on the question of whether genes are over-expressed or under-expressed in samples in group A (e.g., tumor, or treatment, or metastastic, or responder) compared to samples in group B (e.g., normal, or control, or quiescient, or nonresponder). These patterns can efficiently be referred to as AB (overexpressed in A) and BA (underexpressed in A) patterns. Typically, researchers use study designs that favor biological replication to maximize the ability to detect reproducibly genes that are differentially expressed in a patient population, at a sacrifice of the ability to detect individual-specific patterns of differential expression with technical replication. Most cancers are diseases with heterogeneous etiologies; moreover, the development of every primary tumor in different individuals is a unique biological event. Thus, the expression levels of genes in the individual patient are also important; some important gene dysregulation may be highly specific to each individual. Statistical methods that average gene expression may hide important expressotypes (expression phenotypes). Current tests that compare mean group expression intensities are not likely to find genes that are in fact significantly dysregulated in only a proportion of the individuals in the case population, unless the magnitude of differential expression is very high in the subset of individuals. Unsupervised clustering can be used to attempt to identify unknown partitions, or subgroups within patients, but clustering is not a well-defined method for finding differentially expressed genes, and, upon discovery of novel groups, researchers are restricted to comparing group means, and cannot identify genes that may be dysregulated in subsets of patients where the combined patterns of dysregulation patterns do not suggest coherent subgroups. Results A remarkable pattern emerges when the PPST test is applied to published cancer data sets, including breast cancer [ 7 ], ovarian cancer [ 16 ], colon cancer (epithelial-rich normals only [ 17 , 47 ]), lymphoma [ 18 ], and lung cancer [ 19 ] at the 99th percentile. We find an abundance of AB and BA pattern genes, with roughly the same number of genes called significant under the parametric t-test. We also find large numbers of genes with significant ABA test scores, and some with 'BAB' pattern genes (Table 1 ). There is a marked tendency in most data sets for more ABA (cancer-normal-cancer) type genes than BAB pattern genes. These patterns are also reflected in 'expression pattern grids' of gene with significant s3 (ABA) statistics (Fig. 1 ). These patterns are reproducible at more stringent levels of α (Table 1 ). The capability of the PPST test to identify genes that are in fact differentially expressed in only a subset of patients is made evident by a comparison of genes that are found to be significant under the PPST test, but missed by, for example, the t-test (even without Bonferroni-type adjustments). These are listed in Table 2 , for the lymphoma data18, and notably include B-cell growth factor 1 (IL4; ABA pattern). Furthermore, 'classic' oncogenes such as cyclin D1 are found by the PPST test in the lung cancer data set [ 19 ] are not reported to be significant by the t-test. Cyclin D1 ranks 1009th among significant genes in the colon cancer data set under the t-test but ranks 90th under the PPST test (AB/BA pattern results only). Discussion Our initial results are compelling in that they suggest that we can expect biomarkers of high clinical significance for subsets of patients to be important for distinct subsets of patients. This also suggests that clinical validation of the utility of biomarkers should examine panels of expression biomarkers, not individual biomarkers. Disruption of genomic function via these patterns cannot be studied in the population level biomarker framework for the simple reason that methods that compare, say, group means, will find no difference between the sample groups if the number of case samples found in the two tails are even approximately equal. This is a sensible approach even from within the framework of population-based hypothesis testing, because the PPST test can be expected to be more robust to one or two outliers that might mislead simple parametric tests. Note that a number of genes are 'nearly significant' under the t-test but are strongly significant under the PPST test for the AB/BA patterns (e.g., Table 2 ). Our re-analysis of two independently generated data sets on astrocytoma progression demonstrates the utility of extending analysis to include a search for genes that are differentially expressed in a subset of patients. Of the tests examined, the parametric t-test showed the least internal consistency, while the PPST exhibited the highest internal consistency in identifying progression markers. Comparison to the non-parametric t-tests demonstrates that PPST is most similar to the nonparameteric t-test, but is more self-consistent. While the ABA test showed the least internal consistency across populations, it also exhibited low overlap with any other test, so the genes reported are unique and tend not to be found by others tests, matching expectations. Our results are consistent with Knudsen's 'two-hit' hypothesis on the genomic etiologies of cancer [ 49 ] with some insight into the diversity of genomic pathologies (functional 'hits') that may be relevant in patient populations. Studies of differential gene expression – and its role in the etiology of cancer and its responses to treatment – should seek these types of genes in addition to population-wide biomarkers, because they represent a subset of the genes that are expressed differentially in a significant subset of cancer patients. We recommend a major shift in perspective on the study on gene expression dysregulation away from the study of 'tumor populations' – which do not exist – toward the study of genomic pathologies in individual patients. For example, tumor subtypes are typically characterized by morphological characters, and these classifications may conflict with important expressotype subtypes that do not follow classical morphological tumor classes. Imposition of these subtypes on a study design may interfere with identifying expressotypes that provide high diagnostic, prognostic and theranostic value to the individual – and sets of individuals with similar expressotypes. This view is also consistent with the Hanahan-Weinberg model of oncogenesis [ 50 ], which envisions multiple possible mechanistic strategies to the acquisition of characteristics and capabilities of cancers including self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion & metastasis, limitless replicative potential, sustained angiogenesis and evasion of apoptosis. We also expect that individual cancers in different patients will be found to have evolved unique sets of solutions to each of these problems. Current prevailing methods for finding differentially expressed genes such as fold-change and t-tests do not allow for such complexities. Our comparison of the methods (Table 3 ) highlights the uniqueness of the ABA test. It is an extension of the PPST test; it specifically focuses on genes that are differentially expressed in subsets of patients. This ability is extremely important in search of genes with expression patterns that correlate with drug response. The ABA and the two-tailed t-test are not comparable because the ABA test allows us to find genes that the t-test specifically cannot (genes that are simultaneously overexpressed in some patients while underexpressed in others). Such test will have high variance (leading to a low t-test score) and low mean difference, and will thus not be significant. The PPST and the ABA tests extend our abilities beyond the t-test. Other improvements or even superior alternatives to these tests may be possible. The performance of these tests and all tests described to date for the AB type patterns and now for ABA patterns should be compared using extensive numerical simulations and cross-validation. Developments are needed to determine how best to select a threshold to allow deliberate control of the false positive and false negative error rates. Conclusions The two major conclusions these results suggest are (1) that the most commonly applied tests for identifying differentially expressed genes will miss important genes that are dysregulated in only a fraction of patients, and (2) that important aspects of differential expression may be, to a degree, highly individualistic in most cancers. Some potentially important genes with this form of unusual differential expression (ABA; Table 2 ) would be missed by methods that compare group means, because the means of the two sample groups would be approximately identical, and the variance in tumors would be high, leading to a large error term. The high internal consistency of PPST compared to the non-parametric t-test and our observation that the PPST test exhibited high consistency with the nonparametric t-test suggests that the PPST test may be of interest to researchers interested in identifying both population-level biomarkers and biomarkers important to a subset of patients. An online implementation of this test, it source code (Java), and that for many other methods of analysis, are accessible online in the Cancer Gene Expression Data Analysis tool . It is hoped that the development and application of more approaches like this will lead to a more complete representation of differential expression, leading to more meaningful and specific hypotheses of dysregulation, and thus a better comprehension of how diverse genomic pathologies contribute to the etiologies of cancers, and thereby facilitate the identification of targets that may lead to individual-specific therapies. Methods We have developed a test we call the Permutation Percentile Separability Test (PPST), which attempts to refute a null hypothesis that is slightly different from A = B, but which is capable of detecting AB, BA, ABA and BAB patterns. Under this test, we are interested in the question "are there are statistically significant number of samples in group A (e.g., tumor) that exhibit expression intensities beyond a particular percentile of the observed expression intensities in group B (e.g., normal)?" and vice versa. By 'statistically significant' we mean that the number of samples that exhibit apparent overexpression (or underexpression) exceeds that expected under the null distribution. To test these hypotheses, we count the number of samples in both groups that are found beyond the n th percentile of the samples in the opposite group. This provides two scores, s 1 , and s 2 , for each gene (PPST scores). s 1 is the number of samples in group A that are beyond the upper percentile (say, 95 th ) of group B plus the number of samples in group B that are below the lower 95 th percentile of group A. This measure will tend to be large when all samples in both groups are significantly distinct from the alternate group in the same way (comparisons consistent with A > B). It can also be significant when a surprising number of samples in only one group varies from the expression levels in the alternate group. s 2 is the number of samples with correspondingly opposite pattern (comparisons consistent with B > A). Sample class label permutations are used to generate an arbitrarily large number of permuted data sets. These scores s 1 and s 2 are calculated in each permuted data set to produce unique null distributions for each gene. For the sake of convenience of interpretation, we use - s 2 when reporting s 2 to denote underexpression. Genes with values of s 1 beyond the specified acceptable Type 1 error risk (e.g., α = 5%) are determined to be significantly overexpressed in sample group A relative to B. Individuals in sample group A with expression intensity values over the 95 th percentile of sample group B for a given gene may be considered overexpressed. Similarly, genes with values of s 2 beyond the specified Type 1 risk for s 2 are deemed underexpressed in sample group B relative to A. Varying the percentile threshold allows direct control over the false discovery rate. Test for ABA patterns (ABA Test) Genes that exhibit both significant s 1 and s 2 scores in this comparison may be considered 'ABA pattern genes' (Fig. 1 ); however, for stronger inference, permutation tests are also used to calculate s 3 , to determine, for a given gene , the number of samples from one group (A) that can expected to be distributed both in the upper and lower n th percentile tails of the intensity distribution of that gene in the other group (B); i.e., in the ABA ( s 3 ) or BAB ( s 4 ) pattern. These scores are not redundant to but rather allow for exploration of distribution-wise (upper and lower) false discovery rates. The application of the PPST test to find ABA patterns is called the 'ABA' test. Under the ABA test, differential expression of a gene may be deemed to be significant in both directions at once, i.e., simultaneously significantly over-expressed and under-expressed in a surprising number of patients in the case population. Both the PPST test and the ABA test will perform optimally when the variation in expression intensities in the normal sample population is well characterized. A collection of published microarray data sets we have placed 'on-tap' in the caGEDA (Gene Expression Data Analysis) web application [ 51 ] were subjected to the PPST test and the ABA test. To avoid idiosyncracies that can result from the study of extreme values, we ran the tests at a fairly relaxed Type 1 error risk (α = 0.05 in both tails, or α = 0.10 overall). To compare the self-consistency of the parametric t-test, the nonparametric t-test, the PPST test and the ABA test, we re-analyzed two published data sets from independent astrocytoma progression studies [ 52 , 53 ]. Details of these studies are available in the original papers. In brief, Khatua et al. [ 52 ] studied global gene expression profiles from 6 early stage and 7 late-stage astrocytoma patients, while van den boom et al. [ 53 ] studied global gene expression profiles from 8 early stage and 8 late-stage astrocytoma patients. We calculated the overlap in the gene lists using our online Overlap tool . Abbreviations PPST: permutation percentile separability test. ABA test: a test that can detect genes with both A > B (gene is overexpressed in sample A compared to sample group B) and B < A (gene is overexpressed in sample B compared to sample group A) patterns. s1, s2, s3, s4: measures of the number of samples that exhibit expression intensities beyond a specified percentile in an alternate group; used as scores in the determination of significance under the PPST and ABA tests. Author contributions JLW conceived of the PPST and ABA tests and executed the analyses, SP encoded the methods in caGEDA, TG provided direction, input and scientific motivation for pursuing a test with the capabilities of the PPST and ABA tests, MJB provided, direction, input and coordination of the research. All authors read and approved the final manuscript.

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516448

Stress, burnout and doctors' attitudes to work are determined by personality and learning style: A twelve year longitudinal study of UK medical graduates

Background The study investigated the extent to which approaches to work, workplace climate, stress, burnout and satisfaction with medicine as a career in doctors aged about thirty are predicted by measures of learning style and personality measured five to twelve years earlier when the doctors were applicants to medical school or were medical students. Methods Prospective study of a large cohort of doctors. The participants were first studied when they applied to any of five UK medical schools in 1990. Postal questionnaires were sent to all doctors with a traceable address on the current or a previous Medical Register . The current questionnaire included measures of Approaches to Work, Workplace Climate, stress (General Health Questionnaire), burnout (Maslach Burnout Inventory), and satisfaction with medicine as a career and personality (Big Five). Previous questionnaires had included measures of learning style (Study Process Questionnaire) and personality. Results Doctors' approaches to work were predicted by study habits and learning styles, both at application to medical school and in the final year. How doctors perceive their workplace climate and workload is predicted both by approaches to work and by measures of stress, burnout and satisfaction with medicine. These characteristics are partially predicted by trait measures of personality taken five years earlier. Stress, burnout and satisfaction also correlate with trait measures of personality taken five years earlier. Conclusions Differences in approach to work and perceived workplace climate seem mainly to reflect stable, long-term individual differences in doctors themselves, reflected in measures of personality and learning style.

Background Sir William Osler (1849–1919), one of the most distinguished physicians of the nineteenth and early twentieth century, recognised that only some doctors are happy in their professional lives: "To each one of you the practice of medicine will be very much as you make it – to one a worry, a care, a perpetual annoyance; to another, a daily joy and a life of as much happiness and usefulness as can well fall to the lot of man."[ 1 ] The modern medical workplace is a complex environment, and doctors respond differently to it, some finding it stimulating and exciting, whereas others become stressed and burned out from the heavy workload. The medical workplace also provides an environment where new skills are continually being learned, both as a result of medical knowledge evolving and because a doctor's work changes, in part due to career development and progression through different jobs. In an important study, Delva et al [ 2 ] used earlier research [ 3 , 4 ] to develop two separate instruments for studying how doctors work, the Approach to Work Questionnaire (AWQ) and the Workplace Climate Questionnaire (WCQ). In Canadian physicians [ 2 , 5 ] the AWQ showed three separate factors, which were called Surface-Rational , Surface-Disorganised , and Deep (see table 1 ). These approaches related to different methods and motivations for continuing medical education. Those with a deep approach preferred independent and problem-based learning and motivation was internal. Surface-rational and surface-disorganised approaches were primarily driven by external motivation, with the preferred mode of continuing education learning being independent for the surface-rational, and in consultations for the surface-disorganised. The WCQ showed three dimensions, called Choice-Independence, Supportive-Receptive , and Workload (see table 1 ), which correlated with the AWQ. Doctors reporting Choice-Independence and Supportive-Receptive work environments had a Deeper approach, whereas those describing an environment dominated by Workload tended to be more Surface-Disorganised. Some doctors are unhappy with their work, which can manifest as stress (usually assessed by the General Health Questionnaire) or burnout, which has three separate components of emotional exhaustion, depersonalisation and reduced personal accomplishment (see table 2 ). Greater stress and burnout in doctors are related to the personality trait of neuroticism or 'negative affectivity' [ 6 ]. The AWQ and WCQ provide a snapshot of a doctor's learning environment and approach to work at one particular time, as also do measures of stress and burnout. A key question, as Deary et al recognised [ 6 ] when considering stress, is the extent to which different approaches to work and the climate of the workplace are consequences of the workplace or of the doctor . At first sight it might seem that the workplace itself has to be the primary force driving both workplace learning and workplace climate. However, it is also possible that approaches to learning and work mainly depend upon pre-existing differences among doctors, differences that may already have manifested earlier in the doctors' careers. The AWQ bears a strong formal similarity to the surface, deep and strategic study habits and learning styles identified by the Study Process Questionnaire (SPQ), which assesses the motivations and approaches used by students in higher education (see table 3 ). The similarity is not accidental since the AWQ was developed by adapting items from Entwistle and Ramsden's Approaches to Study Inventory [ 7 ], which has a similar factor structure to that of the Study Process Questionnaire [ 8 ]. It is therefore expected that there may be significant continuities across approaches to study and approaches to work. In this paper we describe a large cohort of UK doctors, typically aged 29 or 30 at the time of the study, who have been qualified for five or six years, who are practising as SHOs or SpRs in hospital or are in general practice, and who previously had been studied when aged 17 or 18 at application to medical school in autumn 1990 [ 9 ], in their final year at medical school [ 10 ] and as PRHOs [ 11 ]. The main interest here will be in the extent to which a doctor's present approaches to work and their workplace climate, as well as their stress and burnout, relate to earlier measures of study habits and personality at application to medical school and subsequently. Method Participants In the autumn of 1990 a questionnaire was sent to all individuals with European Community postal addresses who had applied to any of the five UK medical schools taking part in the study [ 9 ]; they represented about 70% of all applicants and acceptances for medical school in that year. The response rate was 93%. Students who were accepted for entry in 1991, 1992 or 1993 were followed up in their final year at medical school (1995–1998), when the response rate was 56%, and at the end of their PRHO year (1996–1999), when the response rate was 58%. In 2002 a tracing exercise searched the Medical Register and Medical Directory from 1995 to 2002 to find the addresses of as many doctors as possible who were in the original survey, and who were known not to have died, left medical school during basic medical sciences, or otherwise to be no longer in the survey. For study design see figure 1 . Questionnaire Questionnaires were sent to all individuals with current or recent GMC addresses. The questionnaire consisted of a single folded A3 sheet of paper (4 A4 sides). Included in the present questionnaire (described in the results as '2002') were the 12-item General Health Questionnaire (GHQ) [ 12 ]; an abbreviated version of the Maslach Burnout Inventory (aMBI), which has three sub-scales, Emotional Exhaustion, Depersonalisation, and Personal Accomplishment [ 13 , 14 ]; a three-item scale modelled on the aMBI, which assesses Happiness with a Medical Career [ 15 ]; an abbreviated version of the Study Process Questionnaire, which has three sub-scales of Surface, Strategic and Deep learning [ 16 , 17 ]; an abbreviated questionnaire assessing the 'Big Five' personality dimensions of Neuroticism, Extraversion, Openness to Experience, Agreeableness and Conscientiousness [ 15 , 18 ]; and abbreviated versions of the Approach to Work Questionnaire (aAWQ) and the Workplace Climate Questionnaire (aWCQ) [ 19 ], each of which has three sub-scales, and for which a detailed description is provided in the Supplementary Information (see Additional file: 1 ) [ 2 ]. The GHQ, aMBI and personality questionnaire had also been administered previously in the PRHO survey, and the SPQ had been administered in the Applicant and Final year surveys. Procedure Questionnaires, along with a postage-paid return envelope, were posted at the beginning of December 2002. Two reminders were sent to non-respondents. Although the official closing date was 25 th March 2003, a few questionnaires were returned up until the end of August 2003. Statistical analysis used SPSS version 10.5, and structural equation modelling used LISREL 8.52. Results The tracing exercise looked for 2,912 individuals thought to have completed basic medical sciences and entered a clinical course. Eighty-nine had never been on the UK Medical Register, and either had failed finals, had never registered, or had emigrated. Of 2,823 individuals who were traced, 2,754 doctors were on the 2002 Register, 7 returned to the Register during 2002, and 64 were on an earlier Register. Of 2,823 questionnaires sent, 176 were returned by the Post Office as undeliverable, 10 doctors were travelling and hence uncontactable, and2 had died. Of the remaining 2,635 doctors, 1,668 returned questionnaires, giving a response rate of 63.3%. There was no evidence of response bias (see Supplementary Information see Additional file: 1 ). Respondents The mean age of respondents on 1 st December 2002 was 30.4 years (SD 1.86, range 28.3 – 49.2). There was substantial variation in the scores on the aAWQ and the aWCQ, and the factor structures of the aAWQ and aWCQ were similar to those reported elsewhere [ 19 ] (see Supplementary Information see Additional file: 1 ). There was also substantial variation on the measures of stress, burnout and satisfaction with medicine as a career, with 21.3% of doctors (345/1617) reporting GHQ scores of 4 or more, the conventional level of 'caseness'. Approaches to work and learning were correlated with climate in the workplace, and as in the Delva et al study, the highest correlations were for a surface-disorganised approach correlating with high workload, and a deep approach correlating with a supportive-receptive environment and with choice-independence (table 4 ). Approaches to work Table 4 shows correlation of the stress measures with approaches to work and study habits. The largest correlations were of a surface-rational approach with a strategic learning style, and a deep approach to work with a deep learning style. In each case the correlations were not only highly significant when study habits were measured in the final year at medical school, six or seven years earlier, but were also very significantly correlated with study habits measured at selection, twelve years earlier. Correlations of approaches to work and stress, burnout and satisfaction with medicine were generally small, and generally were only with measures taken in 2002, and not with measures taken as a PRHO, five or six years earlier. The sole exception was that a surface-disorganised approach correlated with high stress as measured by the GHQ, both in 2002 and with stress when the doctors were PRHOs. Workplace climate Table 5 shows correlations between the workplace climate and study habits, stress, burnout and satisfaction with medicine. In contrast to the associations with approaches to work, the workplace climate showed only small correlations with study habits, but showed strong correlations with stress, burnout and satisfaction with medicine. In particular, high stress in the PRHO year showed very significant correlations with measures in 2002 of a perceived high workload, a less supportive-receptive environment, and less choice-independence. In addition, emotional exhaustion both in 2002 and in the PRHO year were related to a high perceived workload in 2002. Personality Table 6 shows the correlations of approaches to work and workplace climate with the 'Big Five' measures of personality, measured both in 2002 and also measured five to six years previously when the doctors were PRHOs. The surface-disordered approach to work is associated with high neuroticism and low conscientiousness, the PRHO correlations also being highly significant in each case. Neuroticism, both in 2002 and as a PRHO, is also associated with a perceived high workload (although in contrast to its prediction of a surface-disordered approach, conscientiousness is not a significant correlate of workload). The deep approach to work and learning is associated with being extravert and with greater openness to experience, and again the measures taken six years earlier are predictive. Finally a supportive-receptive work climate is associated with greater reported agreeableness, both in 2002 and six years earlier as a PRHO. There were no substantial correlations between personality and the surface-rational approach to work or choice-independence in work climate. Multiple regressions Tables 4 to 6 show a large number of correlations, which are not always straightforward to interpret, both because they are numerous and because many variables are themselves inter-correlated. Multiple regression was used to clarify the relationships (for technical details see Supplementary Information see Additional file: 1 ). Each individual measure of the aAWQ and aWCQ was regressed on the measures of study habits at application (n = 3) and in the final year (n = 3), of stress and burnout during the PRHO year (n = 4) and in 2002 (n = 4), and of personality in the PRHO year (n = 5) and in 2002 (n = 5). Alpha for entry was set at p < 0.0001 in view of the large sample size and the number of independent variables. The variables that were significant are shown in tables 4 , 5 and 6 and 3 in italics. Of particular interest are variables that show not only show significant contemporaneous correlations but also significant correlations when measured five or more years previously. A surface-disorganised approach to work is predicted by surface learning in medical school and by higher neuroticism scores and lower conscientiousness (see tables 4 and 6 ). The surface-rational approach to work is predicted by strategic learning in medical school, and by less openness to experience and higher conscientiousness. The deep approach to work is predicted by a deep approach to learning at medical school, by greater extraversion, by greater openness to experience, and by lower emotional exhaustion. A workplace climate dominated by a high workload is predicted by higher stress and emotional exhaustion measures five years earlier, and by lower openness to experience (see tables 5 and 6 ). A supportive-receptive workplace is predicted by lower stress and depersonalisation, and a higher sense of personal accomplishment when measured previously, and by a more agreeable personality. Choice-independence in the work environment is predicted only by lower previous measures of stress. Stress, burnout and satisfaction with medicine Although in the previous analyses, stress and burnout have been used as predictors of approaches to work and workplace climate, they are also important outcome measures in their own right. Table 7 shows the correlations of the five 'stress-related measures' (GHQ, the three burnout measures and satisfaction) with measures of learning style and personality, in each case measured on two separate occasions. Personality correlates with each of the measures, as do study habits. Because of the complex inter-correlations between the dependent variables, multiple regression was used, as before, to find the most important relationships (for technical details see Supplementary Information see Additional file: 1 ). Doctors who are most stressed showed higher levels of neuroticism, both currently and previously, and those reporting most emotional exhaustion also had higher neuroticism levels, as well as being more introvert. High levels of depersonalisation related to lower levels of agreeableness. A greater sense of personal accomplishment related to previous deep approaches to study and learning, as well as to being more extravert. Overall satisfaction with medicine as a career related to lower levels of neuroticism. Path analysis The complex relationships described by the various correlations are best analysed and described by means of path analysis or causal modelling [ 20 ], which analyses the entire set of correlations between variables, using plausible assumptions about causality and removing non-significant paths. The path diagram, which was analysed using LISREL 8.52 [ 21 ], is shown in figure 2 . Measures to the left can causally influence measures to their right. Based on the time-lagged correlations reported previously, we assumed that stress causes different approaches to work, and we also assumed that approaches to work cause differences in workplace climate rather than vice-versa. (Nevertheless, we acknowledge that the causation may well be reciprocal, as suggested by the originators of the scale [ 2 , 19 ]; further longitudinal data will be required to test that hypothesis). Study habits are temporally and causally prior to stress, approaches to work and workplace climate. Personality, being a trait, was prior to all other measures. For technical details see the Supplementary Information (see Additional file: 1 ). Although several of our variables are measured at different time points, we have chosen not to present a model in which each variable has been included on each occasion that it is measured, as the resulting diagram becomes unmanageably complex. Although the path diagram in figure 2 is complex at first sight, the paths are readily interpretable. The diagram divides into two broad sections, with the measures of learning style and approach to work at the bottom, and stress at the top. Here we have simplified the model by omitting the closely correlated measures of burnout, and only including paths with t-values greater than 3.6. Estimates of all the paths are available in the Supplementary Information (see Additional file: 1 ). Stress in our model is caused by personality differences, being greatest in those having high neuroticism scores, low extraversion scores, and low conscientiousness scores. It is unrelated to learning style. Learning styles at medical school relate to different personality measures, in particular showing no relationship to neuroticism. Deep learning is highest in extraverts who are open to experience, whereas strategic learning is highest in highly conscientious individuals with low openness to experience. Surface learning style is higher in introverts who are low in openness to experience. These findings are similar to those of others [ 22 ]. Approaches to work are mainly but not entirely driven by learning styles. A deep approach to work occurs in extraverts who are open to experience and have a deep learning style. The surface-rational and surface-disorganised approaches to work are both greater in those with a surface learning style. However, a surface-disorganised approach occurs in individuals with higher neuroticism scores, in those with lower conscientiousness scores, and in those who have been stressed, whereas the surface-rational approach to work occurs in strategic learners and in those who are low in openness to experience. Workplace climate has a range of influences. High perceived workload occurs in those with a surface-disorganised approach to work, who have been stressed and are more neurotic. In contrast, choice-independence and a supportive-receptive environment both occur in individuals who have not previously been stressed, the choice-independence approach occurring in those with a deep approach to work, whereas the supportive-receptive approach occurs in those who have higher scores on the personality trait of agreeableness. Discussion Many doctors at the age of 30 are unhappy in their jobs, and a fifth of our sample reached the conventional GHQ criterion of psychiatric 'caseness'. In contrast, many doctors reported high levels of personal accomplishment, choice and independence in their work environment, satisfaction with medicine as a career, and intellectual and emotional satisfaction from their work. That is not new; Sir William Osler in 1905 contrasted doctors "whose stability of character and devotion to duty make one proud of our profession" with those who find it difficult to keep "the flame alive, smothered as it is apt to be by the dust and ashes of the daily routine" [ 1 ]. In 2001, Richard Smith asked "Why are doctors so unhappy?" and concluded that "The most obvious cause of doctors' unhappiness is that they feel overworked and undersupported" [ 23 ]. Certainly many doctors in our study report a high workload and a work climate that is neither supportive nor receptive, and those doctors also report more stress, burnout and dissatisfaction with medicine as a career. It is tempting therefore to conclude, as did an article in a special edition of BMJ Careers devoted to "Doctors' Wellbeing", that excessive workload and absence of support are directly caused by poor working conditions: "the way in which the NHS is run generates stress for members of the workforce every day" [ 24 ]. However, such an interpretation is not straightforward in general [ 25 ]. It is particularly difficult for the doctors in our study because the study is longitudinal, and workload and lack of support correlate with stress and burnout reported five or six years earlier , when the doctors were PRHOs and carrying out entirely different jobs. High perceived workload and poor support are therefore determined as much by doctors themselves as by specific working conditions. That view was expressed in another article in the special edition of BMJ Careers : "A critical element contributing to the stress that many conscientious doctors experience is internal..." [ 26 ]. A similar conclusion was reached in a previous study of ours when these doctors were PRHOs, and multi-level modelling showed that stress is not a characteristic of jobs but of doctors, different doctors working in the same job being no more similar in their stress and burnout than different doctors in different jobs [ 11 ]. If differences in reported workload are partly explained by differences among doctors, what in turn explains those differences? Doctors reporting a high workload also have what Delva et al [ 2 ] describe as a surface-disorganised approach to work, which in turn is correlated with being a surface learner at application to medical school, a dozen years previously. Surface-disorganised doctors are also high on the personality trait of neuroticism and low on the trait of conscientiousness; and again those correlations are with measures taken six years earlier when the doctors were PRHOs. Doctors reporting a work climate low in support were lower on the personality scale of agreeableness in the measures collected when they were PRHOs. Some doctors may be stressed and burned out, but what predicts those others who are happy in their work? Doctors reporting high satisfaction with medicine as a career have a deep approach to work, and that approach is more common in those who also had a deep learning style when they applied to medical school. Satisfaction with medicine also relates directly to the personality traits of greater extraversion and lower neuroticism, and the deep approach to work correlates with greater extraversion and more openness to experience. Doctors who describe their colleagues as receptive and supportive score more highly on the personality trait of agreeableness; and as in many other correlations reported here, that correlation is stable across time – those who are more agreeable at the age of 24 have a more receptive and supportive work environment when aged 30. An overview of our findings is that approaches to work are predicted by earlier measures of study habits and learning styles, whereas perceived work climate, and its pathologies such as stress and burnout, are predicted mainly by personality. Although unfortunately our study did not measure personality during selection, the high stability of the Big Five measures across the life-span [ 27 - 29 ] (and across our two measures six years apart), as well as their heritable component [ 30 ], means that we have little doubt that personality at selection would also have been predictive, particularly given that a similar pattern of correlations was found in a different cohort of doctors in mid-career [ 15 ]. Other studies on very different groups of students have also found, like us, that both strategic and deep learning correlate with conscientiousness, and that deep learning also correlates with extraversion and openness to experience [ 22 , 31 ]. Our study has, for various reasons, not looked at academic performance in relation to study habits, learning styles and personality, although previous work of ours has found clear correlations between learning styles and examination performance [ 32 ]. In contrast we have not found any correlation of undergraduate or postgraduate academic achievement with personality [ 15 ], and although some studies have found correlations of conscientiousness with academic achievement [ 33 ], this does seem to vary according to the learning context [ 34 , 35 ]. Although we will be looking at this question again in more detail in a further analysis, it does seem probable that personality mostly has an indirect effect upon academic achievement via approaches to learning [ 31 , 36 ]. If, as William Wordsworth said, "the child is father to the man", then the seeds of subsequent job satisfaction and dissatisfaction in doctors may be visible in the personality, motivations and learning styles of medical school applicants. This argument may provide some justification for using such measures in selection, particularly given the general association of job performance and satisfaction with personality [ 37 ] and motivation [ 38 ], and learning styles with personality [ 22 ] . However, just as genes are not destiny, so neither personality nor learning style is destiny. Nurture interacts with nature [ 39 ], the environment building upon the genes, and the genes using what is provided by the environment; the poetic complement to William Wordsworth is therefore Alexander Pope, who said, "This education forms the common mind: Just as the twig is bent, the tree's inclined." Extreme introverts can, with sufficient insight, preparation and appropriate training become effective public speakers, less conscientious individuals can learn to be more organised and efficient, and those who are more neurotic can transcend their anxieties (and indeed neuroticism may be beneficial if sublimated into a professional concern for detail in critical situations, rather than merely being undifferentiated personal anxiety). . Formal education, particularly effective formal education [ 40 ], can also alter study habits and learning styles, which are less fixed and 'trait-like' than personality measures [ 17 ]. Intercalated degrees increase deep and strategic learning and decrease surface learning at medical school [ 41 ], making it likely that they also encourage surface-rational and deep approaches to work. Deep and strategic learning also relate to the clinical experience gained by medical students [ 32 ], making it possible that greater patient involvement during undergraduate clinical training, rather than mere reliance on textbook learning to pass exams, a characteristic of surface learners, will also reduce surface-disorganised approaches to work. Conclusions Longitudinal data suggest that personality and learning style are not merely correlates of approaches to work, workplace climate, stress, burnout and satisfaction with a medical career, but are causes , events later in time being predicted by events earlier in time [ 35 ]. Doctors with greater stress and emotional exhaustion, who were less satisfied with medicine as a career, had higher neuroticism scores and were more likely to be surface-disorganised. Lower conscientiousness on the personality measure also predicted greater stress. Extraverts reported more personal accomplishment and were more satisfied with medicine. The personality measure of agreeableness predicted a more supportive-receptive work environment. These results imply that differences in approach to work and workplace climate in our study result from differences among doctors themselves, as much as they do from differences in working conditions. Competing interests None declared. Authors' contributions The cohort study was designed by ICMcM. The present follow-up was designed by ICMcM and EP, who also prepared the questionnaire. AK was responsible for day-to-day running of the study, and for data entry and cleaning. ICMcM was primarily responsible for data analysis and for writing the first draft of the paper. ICMcM, EP and AK were all involved in preparing the final draft of the paper. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 Click here for file

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Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

Background Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA. Methods The subunit cDNAs were cloned by a combination of reverse transcriptase polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA ends (RACE) RT-PCR from adult rhesus monkey testis RNA. Results Both the inhibin alpha and betaB subunit nucleotide and predicted protein sequences are highly conserved with other mammalian species, particularly with humans. During the course of these investigations, a novel inhibin alpha mRNA isoform was also identified. This form, referred to as rhesus monkey inhibin alpha-variant 2, appears to derive from both alternative transcription initiation as well as alternative splicing. rmInhibin alpha-variant 2 is comprised of a novel 5' exon (exon 0), which is spliced in-frame with exon 2 of the conventional inhibin alpha isoforms (variant 1). Exon 1 is skipped in its entirety such that the pro-alpha and part of the alpha N regions are not included in the predicted protein. rmInhibin alpha -variant 2 is of relatively low abundance and its biological function has not yet been ascertained. Conclusion The data show that the predicted inhibin B protein is very similar between monkeys and humans. Therefore, studies in monkeys using recombinant human inhibins are likely to reflect actions of the homologous ligands. In addition, we have observed the first inhibin alpha subunit mRNA variant. It is possible that variants will be observed in other species as well and this may lead to novel insights into inhibin action.

Background The inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion [ 1 , 2 ]. Inhibins are comprised of an α subunit (inhibin α) and one of two inhibin β subunits (inhibin βA or inhibin βB). In adult male mammals, inhibin B (α-βB dimer) appears to be the primary circulating form of the hormone, whereas females produce both inhibin A and B and do so in discordant fashion across the reproductive cycle [ 3 - 15 ]. One exception to this general pattern is in rams, where inhibin A appears to be the primary circulating form [ 16 ]. Historically, investigations of inhibin action have relied principally upon recombinant preparations of inhibin A because inhibin B has not been available in sufficient quantities to permit in vivo studies of its role in the negative feedback regulation of gonadotropin secretion [ 17 , 18 ]. Because inhibin B is the biologically relevant ligand in male primates, this has placed some constraints on our understanding of inhibin action in these animals. For this reason, we cloned the inhibin B subunit cDNAs from adult monkey testis as a requisite first step to producing recombinant monkey inhibin B. In the course of cloning the monkey inhibin α subunit, we identified a novel transcript, which has not been observed in other species. In this paper, we describe the new transcript called rhesus monkey inhibin α-variant 2. Methods RNA extraction Total RNA was extracted from frozen testis samples of two adult male rhesus monkeys ( Macaca mulatta ) (#1861 and #2333) using Trizol following the manufacturer's instructions (Invitrogen, Carlsbad, CA). RNA was dissolved in diethyl pyrocarbonate-treated H 2 O and quantified by spectrophotometry. Animals were treated in accordance with institutional and federal guidelines. Reverse transcriptase polymerase chain reaction (RT-PCR) Contaminating genomic DNA was removed from RNA samples using RQ1 DNase (Promega) following standard protocols. Four μg of DNased RNA (from #1861) was reverse transcribed into cDNA using 100 ng random hexamer primers and 100 U MMLV-RT (Promega). Four hundred ng of cDNA was subjected to PCR to amplify part of the αN domain and the entirety of the mature domain (αC) of the inhibin α subunit using the following primer set: 5'-CCYTTCCTGGTGGCCCACACT (forward) and 5'-TTAGATACAAGCACAGTGYTG (reverse) (see primers A and B in Fig. 3 ). Reactions were subjected to 35 cycles of 94C for 30 sec, 55C for 30 sec, and 72C for 30 sec. No amplified products were observed in H 2 O or RT- controls (data not shown). The amplified 465 bp product was ligated into pCR3.1 (Invitrogen) following the manufacturer's instructions. Recombinant clones were screened by colony hybridization using the gel purified PCR product as probe. Plasmids were purified from hybridizing clones and sequenced using DyeTerminator Cycle sequencing (ABI). All hybridizing clones corresponded to inhibin α. Figure 3 Rhesus monkey inhibin α gene structure. Schematic representation of the genomic organization of the inhibin α subunit in rhesus monkey. Boxed regions reflect exons and the intervening straight line is the intron (the 2 kb is an estimate based on the 2051 bp intron in humans). Black boxes reflect 5' and 3' UTRs. White boxes reflect sequences encoding the signal peptide (in inhibin α-variant 1) or a domain of unknown function (inhibin α-variant 2). The other shaded regions correspond to the pro-α, αN, and αC domains of the inhibin α prepro-hormone and are labeled in the figure. Inhibin-α variant 1 is the canonical inhibin α mRNA described previously in other species and is produced through splicing of exons 1 and 2. Inhibin α-variant 2 is produced through splicing of the novel exon 0 and exon 2. In this latter form, the entirety of exon 1 is removed, in addition to intron 1. An A to G transition in the monkey genomic sequence (relative to the human sequence) at the end of exon 0 appears to introduce a GT 5' splice donor site, which is absent in other species. This allows the splicing event observed in inhibin α-variant 2 when an upstream transcription start site is utilized. This variant is not predicted to exist in other species and also appears to be rare in monkey. The approximate positions of the primers used in different RT-PCR analyses are shown. A and B refer to the forward and reverse primers, respectively, designed to amplify the final 465 bp of the open reading frame. C and D are the outer and inner gene specific reverse primers used in the 5' RACE procedure. The full-length monkey inhibin α cDNA was amplified by RT-PCR from monkey testis RNA as described using a primer (5'-ATGGTGCTGCCCCTACTGCT) directed against the putative start of translation as determined by 5' rapid amplification of cDNA ends (RACE) (see below) and the reverse primer described above. Three prominent bands of approximately 1100, 700, and 400 bp were amplified. The top band was of the predicted size and was purified, cloned and sequenced. The identities of the other two bands have not yet been determined. The mature region of the inhibin βB subunit cDNA was amplified from monkey (#1861) testis RNA by RT-PCR as described for the α subunit using the following primer set: 5'-AGCTGGCCGTGGTGCCBGTGTT (forward) and 5'-TCAGGCGCAGCCGCACTCCTC (reverse). The resulting 455 bp product was gel purified and sequenced directly. 5' RACE RT-PCR 5' RACE was performed on monkey (#1861) testis RNA using RNA ligase mediated (RLM)-RACE reagents following the manufacturer's instructions (Ambion, Austin, TX). The primary PCR was performed using the Outer Adapter Primer (Ambion) and the following gene specific primer: 5-GGCAGGTTTGGTGGGATGTGCA (Fig. 3 , primer C). The secondary PCR was performed on 4 μl of a 1:100 dilution of the primary PCR reaction using the Inner Adapter Primer and the following nested gene specific primer: 5'-GGAAGGAGATGTTCAGTGCTAC (Fig. 3 , primer D). For both PCR reactions, the following reaction conditions were used with AmpliTaq (Perkin-Elmer): 35 cycles of 94C for 30 sec, 55C for 30 sec, and 72C for 1.5 min. Three prominent amplicons were observed in the secondary PCR reaction. No bands were observed in the primary PCR or in any of the negative controls (data not shown). A pool of the different RACE products was ligated into pCR2.1 (Invitrogen). Plasmids were isolated from recombinant clones screened by α complementation and were sequenced as described. Northern blot Twenty μg of total RNA prepared from testes of two adult rhesus monkeys were run on a 1% MOPS-formaldehyde agarose gel. RNA was transferred to Hybond N+ charged nylon membrane by capillary action using 20X SSC. The blot was first probed with a 32 P-labeled (Ready-to-go; Amersham Pharmacia) cDNA corresponding to the last 465 bp of the coding sequence of monkey inhibin α. The blot was hybridized overnight at 42C in 50% formamide, 5X SSC, 1X Denhardt's, 20 mM NaPO 4 (pH 6.8), 1% SDS, and 100 μg/ml denatured salmon sperm DNA using a modified sandwich method [ 19 ]. Following washes in 2X SSC/0.1% SDS at RT and 70C, the blot was exposed to X-ray film (Kodak) overnight with an intensifying screen at -85C. The blot was subsequently stripped and re-probed with a 32 P-labeled cDNA corresponding to 256 bp of monkey inhibin α exon 1. Hybridization and washing conditions were as described for the first probe. Results Cloning of the rhesus monkey inhibin α cDNA The inhibin α cDNA was cloned from rhesus monkey testis using a combination of RT-PCR based approaches. First, the cDNA encoding the last 20 amino acids of αN and the entirety of the mature (αC) domain was amplified by RT-PCR using adult monkey testis RNA as starting material. The resulting PCR product was cloned and sequenced. BLASTN of the non-redundant database showed highest sequence identity (97%) to the human inhibin α cDNA. Within the 402 bp encoding the αC domain, sequence identity was also 97% and the predicted amino acid sequence was 98% conserved (Fig. 1 ). Within the 134 amino acid αC domain there are three non-conservative differences between human and rhesus monkey (L4P, S72P, and Y86P; the first letter refers to the amino acid in human and the number denotes the residue in the αC domain); however, all occur at residues that vary between the mammalian inhibin α subunits sequenced thus far (Fig. 1 ). Proline at position 4 of rhesus monkey is also observed in pig, horse, cow and sheep. The proline at position 72 is observed in all mammalian species examined, except human. Finally, the proline at residue 86 is leucine in all non-human mammalian species examined thus far and is tyrosine in human. Figure 1 Inhibin α amino acid sequence alignment. Alignment of the inhibin α mature domain (αC) amino acid sequence in several mammalian species. Differences from the human sequence are bolded and underlined. Differences between the human and monkey sequences are indicated by arrows. To clone the full-length cDNA, we used 5' RACE to amplify the remainder of the αN and the pro-α regions. Three prominent RACE products were amplified. The longest (919–927 bp) and shortest (642 bp) were cloned and sequenced. The intermediate sized amplicon has not yet been definitively characterized. The long product was within the expected size range and was somewhat heterogeneous in that the clones had 5' ends that extended to differing extents. This likely reflects differences in transcription start sites, but all were within 8 bp of each other. The 5' untranslated region (UTR) ranged from 105 to 113 bp, which is slightly shorter than the 144 bp described in humans (GenBank acc.# NM_002191). When combined with the original PCR fragment, the contiguous sequence contained an open reading frame of 1098 bp predicted to encode a 366 amino acid prepro-hormone, consistent with the size of the human prepro-inhibin α. To confirm expression of a transcript containing this uninterrupted open-reading frame, PCR primers were designed against the start and end of the translation and the predicted 1101 bp fragment (including the stop codon) was amplified. DNA sequencing confirmed its identity. A monkey placental EST (CB548960) overlapped with and confirmed the sequence of the final 183 bp of the monkey inhibin α open reading frame and included an additional 188 bp of 3' UTR (not including the poly A+ tail). Thus, the mRNA encoding the inhibin α subunit would be predicted to be approximately 1.4 kb. Northern blot analysis of monkey testis RNA using a probe directed against sequence within αC domain (exon 2 probe) hybridized to an mRNA of approximately 1.7 kb (Fig. 2A ). The slight size discrepancy may result from a long polyA+ tail, the use of alternative polyadenylation sequences (although a consensus AAUAAA sequence appears 21 nt upstream of the polyA+ tail in the EST), and/or alternative transcriptional start sites. An additional transcript of about 4 kb was also detected with this probe (top arrow in Fig. 2A ). The identity of this less abundant transcript has not yet been ascertained. The monkey inhibin α sequence from the end of the longest 5'RACE product through the end of the open reading frame has been deposited in GenBank (GenBank Acc. #AY574369). Figure 2 Inhibin α mRNA expression in monkey testis. Northern blots showing inhibin α mRNA expression in adult monkey testes. Equal amounts of total RNA from two adult males were run on a MOPS-formaldehyde gel. RNA was transferred to a nylon membrane and hybridized consecutively with 32 P-labeled cDNA probes corresponding to 465 bp of exon 2 (A) and 256 bp of exon 1 (B) of monkey inhibin α. Both probes detected transcripts of 1.7 and 4 kb (top two arrows). The exon 1 probe also detected a 0.4 kb transcript (bottom arrow in B). Molecular weight standards (in kb) are shown at the left of each panel. Hybridization patterns were the same in both animals. Novel inhibin α mRNA in monkey testis The short RACE product when cloned and sequenced was determined to correspond to a novel variant of the inhibin α subunit. In humans (and other species), inhibin α has been described as a two exon gene (Fig. 3 ). Exon 1 encodes the 5' UTR, pro-α, and 85 bp of the αN region. The remainder of αN, αC and the 3'UTR are contained within exon 2. The 546 bp at the 3' end of the short RACE product corresponded exactly to sequence within monkey exon 2 (based on the human nomenclature). The 96 bp at the 5' end of the amplicon did not, however, correspond to the exon 1 sequence determined in the long RACE product. Upon BLASTN search, this 96 bp sequence was determined to show highest identity (91–97%) with human (GenBank acc.# AF272341), mouse (GenBank acc.# M95526), pig (GenBank acc.# AF510728), cow (GenBank acc.# S72864), and rat (GenBank acc.# M32754) inhibin α proximal promoter sequences (Fig. 4A ). These data suggested that transcription of the short RACE product was initiated in what is conventionally thought of as inhibin α promoter (5' flanking sequence) in all species described to date. Figure 4 DNA sequence alignment of novel exon 0 in monkey inhibin α. A) Alignment of the novel exon 0 in inhibin α-variant 2 with inhibin α promoter sequences from Human (GenBank acc.# AF272341), mouse (GenBank acc.# M95526), pig (GenBank acc.# AF510728), cow (GenBank acc.# S72864), and rat (GenBank acc.# M32754). Note that the numbering is relative to the 96 bp of the monkey exon 0 and does not reflect the numbering in the GenBank entries. Bolded and underlined bases reflect differences from the monkey sequence and spaces (-) are added where needed to facilitate the alignment. The non-consensus cAMP responsive element (CRE), which is important for basal and FSH stimulated expression of inhibin α, is boxed and is conserved in all species. The arrows denote the start sites of the human (h, GenBank acc.# CB997542), mouse (m1, GenBank acc.# BY303064; m2, GenBank acc.# BI082792), and pig (p, GenBank acc.# BP457576) ESTs referred to in the text. B) Alignment of the end of monkey exon 0 and the start of exon 1 with human inhibin α genomic sequence. The AT dinucleotide in human is GT in monkey, thereby introducing a novel 5' splice donor site. The bold and underlined base reflects a difference from the monkey sequence. We used this 96 bp sequence to screen the expressed sequence tag (EST) database to determine whether or not this portion of the inhibin α gene was included in transcripts in other species. BLASTN showed significant homology to four entries from three species: human placenta (GenBank acc.# CB997542), mouse dpc 14.5 Rathke's pouch (GenBank acc.# BY303064), mouse mammary tumor (GenBank acc.# BI082792), and pig ovary (GenBank acc.# BP457576). None of the ESTs extended as 5' as the monkey short RACE sequence, but one mouse EST (BY303064) started 15 bp 3' of where monkey RACE product began (Fig. 4A ). In all cases, the EST sequences where contiguous with previously described 5' UTRs in exon 1 of the various species. Therefore, these ESTs appear to define alternative transcription initiation sites in the inhibin α gene and ostensibly increase the length of the 5' UTRs, but do not alter the exon-intron structure of the gene nor do they alter the open reading frame of the mRNA. This contrasts with what we observed in monkey. We aligned both the short and long (see above) RACE product sequences to human genomic sequence derived from a BAC clone in GenBank (acc.# AC009955). The 96 bp unique to the short RACE product terminated 12 bp 5' of where the longest RACE product began (Fig. 4B ). We noted that the first two bp of this intervening sequence was AT in human. We hypothesized that if the adenine in the first position in human was guanine in monkey, this would provide a 5' splice donor site (GT; [ 20 ]) and might explain how exon 1 sequence was skipped in its entirety in this transcript. We designed PCR primers corresponding to sequences flanking the intervening region and amplified genomic DNA extracted from monkey testis. Resulting amplicons of the predicted size were gel purified and sequenced directly. The results confirmed that the first two bp of the intervening sequence were GT in monkey, and therefore potentially provided a novel 5' splice site (Fig. 4B ). The same 3' splice acceptor used in the long transcript also appears to be used in the short transcript, such that exon 2 is spliced identically in both cases (Fig. 3 ). We propose to call the unique sequence in the short RACE product exon 0. The resulting transcript reflects the splicing together of exon 0 and exon 2 (Fig. 3 ). Exon 1 and the intron are removed in the process. The long RACE product reflects transcription initiation from a downstream (more common?) site and is produced through splicing together of the canonical exon 1 and exon 2 (Fig. 3 ). We propose to call the transcript identified in the short RACE product rhesus monkey inhibin α-variant 2 (GenBank acc.# AY574370) and the canonical form inhibin α-variant 1. The short RACE product was initiated from a reverse primer directed against sequence within exon 2 (Fig. 3 , primer D). By virtue of its positioning, this primer excluded the last 287 bp of the open reading frame in exon 2. To confirm that inhibin α-variant 2 extended at least as far as the stop codon in exon 2, we used RT-PCR to amplify a contiguous sequence from the 5' end of exon 0 to the end of the ORF in exon 2. A faint band was amplified, but was of insufficient abundance to clone or directly sequence. We therefore performed nested PCR on this amplicon using primers directed against exon 0 (15 bp 3' of the first primer) and exon 2 (170 bp 5' of the first primer; primer C in Fig. 3 ). A band of the predicted size was amplified and directly sequenced following gel purification. The product corresponded to inhibin α-variant 2, indirectly confirming that the entirety of the open reading frame in exon 2 is contained within this transcript. The ORF of inhibin-α variant 2 is 888 bp, potentially encoding a protein of 296 amino acids. A putative AUG start codon is observed 38 bp from the 5' end. However, the surrounding sequence does not conform to the consensus Kozak sequence [ 21 ]. The next AUG is observed 129 bp 3' of the first, within the αN encoding portion of exon 2. This potential start site also fails to conform to the consensus Kozak sequence. Therefore, it is not clear which, if either, of these codons may be used to initiate translation of inhibin α-short. The putative protein contains 19 novel amino acids (encoded by exon 0) at its N-terminus followed in-frame by amino acids 90–366 of inhibin α variant 1. The N-terminal 19 amino acid peptide does not encode a signal sequence nor does it possess significant homology to sequences in the public databases. The amino acids from exon 2 encode the majority of αN and the entirety of αC (see Fig. 3 ). The northern blot in Fig. 2A was probed with a cDNA corresponding to exon 2, which is contained in both inhibin α-variants 1 and 2. Two transcripts were detected. To determine whether the transcripts might encode these two alternative forms, we stripped the blot and re-probed it with an exon 1 specific probe (Fig. 2B ). Both transcripts were again detected, indicating that both contained exon 1 sequence and, by extension, that neither transcript encoded inhibin α-variant 2 (which lacks exon 1). This is perhaps not surprising in light of the difficulty we experienced in amplifying inhibin α-variant 2 by RT-PCR and is consistent with the notion that it is a relatively low abundance mRNA. Surprisingly, a smaller transcript of ~0.4 kb was also detected with the exon 1 probe. These data suggest that yet another inhibin α transcript may be expressed in monkey testis. The identity of this mRNA species is currently unknown; however, it is predicted to be truncated and contain some or all of exon 1 sequence. Cloning of the rhesus monkey inhibin βB cDNA The mature domain of inhibin βB is highly conserved across all species investigated to date. We used RT-PCR to amplify this region of the cDNA in rhesus monkey (GenBank acc.# AY574371). Not surprisingly, the 115 amino acid domain shared 99% sequence identity with several other mammalian species, including human (Fig. 5 ). The one amino acid difference was a non-conservative threonine to alanine substitution at position 75 of the mature domain. This amino acid is also divergent (proline) in rat and mouse. Figure 5 Inhibin βB amino acid sequence alignment. Alignment of the inhibin β B mature domain amino acid sequence in several mammalian species. Differences from the human sequence are bolded and underlined. An arrow indicates the one amino acid difference between the human and monkey sequences. Discussion In this report, we describe the cloning of the inhibin B subunit cDNAs from testis of the adult rhesus monkey. The results indicate that both the inhibin α and inhibin βB subunits are highly conserved with other mammalian species, particularly within the mature domains of the prepro-hormones. For inhibin α, monkey and humans share 131 out of 134 amino acids. The three differences are all non-conservative; however, all occur at residues that vary across mammalian species. The human and monkey βB mature regions share 114 of 115 amino acids. Again, the one difference is non-conservative but occurs in a residue that varies across mammalian species. Collectively, these data suggest that the mature inhibin B is nearly identical in the human and rhesus macaque. As a result, current assays developed to measure human inhibin B are expected to accurately measure native monkey inhibin B. In addition, because rh-inhibin A and rh-inhibin B are equipotent at suppressing FSH secretion by monkey gonadotrophs in primary culture (Winters and Plant, unpublished observations), it seems reasonable to assume that the FSH suppressing potency of rh-inhibin A may be comparable to that of native monkey testicular inhibin B. If this assumption is substantiated when recombinant monkey inhibin B becomes available, then it will allow the physiological significance of previous and future studies using rh-inhibin A administration in male monkeys to be placed into perspective. In the course of cloning the inhibin α subunit, we identified at least one alternative inhibin α mRNA, which we call rhesus monkey inhibin α-variant 2. The canonical inhibin α subunit mRNA, rhesus monkey inhibin α-variant 1, is comprised of two exons. Monkey inhibin α-variant 2 is produced through a combination of alternative transcription initiation and alternative splicing. As a result, a novel exon (exon 0) is used in place of exon 1 observed in inhibin α-variant 1. Both transcripts incorporate exon 2. 5' RACE indicated that transcription of inhibin α-variant 2 initiates about 108 bp 5' to the start of inhibin α-variant 1 (exon 1). As a result, the short variant contains an additional sequence at its 5' end that is conventionally referred to as inhibin α promoter or 5' flanking sequence. In monkey, an A to G transition (relative to the human sequence) in the genomic sequence upstream of the conventional transcription start site (in exon 1) leads to the introduction of a novel 5' splice donor site. As a result, exon 1 and the intron are removed from the pre-mRNA and a new exon (exon 0) is spliced to exon 2. The putative protein encoded by this variant is predicted to contain the majority of the αN and the entirety of the αC domains. At its N-terminus, however, it lacks a signal sequence as well as the pro-α region. Thus, if produced, it is unlikely that the protein would be secreted. We have not yet tried to express the protein to see if it is indeed synthesized in mammalian cells and where it may be trafficked. The putative translation initiation codon does not conform to the consensus sequence so there is some question about the efficiency with which this variant may be translated. In addition, its low abundance (at least at the mRNA level) also calls into question its functional significance. At this point, we do not know to what extent inhibin α-variant 2 may be expressed in other species, nor have we examined its expression in monkey ovaries. However, this isoform may be unique to monkey because of the A to G transition (relative to human) in the genomic sequence. An alignment of the relevant "promoter" sequences in human, mouse, rat, and cow indicates that the splice donor (GT) in monkey is AT in human and GG in the other three species (not shown). Moreover, screening of the EST database with the monkey inhibin α-variant 2 unique sequence (exon 0) identified very few clones and in each case merely extended the 5' UTR in these species. That is, where monkey inhibin α-variant 2 skipped exon 1 entirely, the mouse, human, and pig EST sequences were contiguous with exon 1. Perhaps the most important aspect of the identification of monkey inhibin α-variant 2 is that in this primate and perhaps in other species, transcription can be initiated further 5' than previously considered. As a result, sequences previously characterized as promoter may actually be 5' UTR, at least in some mRNA species. For example, a mouse EST contains sequence in its 5' UTR previously identified as a conserved non-consensus CRE in the inhibin α promoter [ 22 ] (Fig. 4A ). Because this EST contains the CRE sequence, its transcription may not be cAMP dependent. Conclusions Cloning of the inhibin B cDNAs from rhesus monkey testis indicates that the mature inhibin B is highly conserved in monkeys and humans. Therefore, the results in monkeys obtained with recombinant human inhibins may accurately reflect results that would be obtained with recombinant homologous ligands. The characterization of the inhibin subunit cDNAs in monkeys will greatly facilitate the production of macaque inhibins and will permit a direct test of this hypothesis. In addition, a novel inhibin α mRNA isoform was isolated in this investigation. This is the first example of an inhibin α mRNA variant described in any mammalian species. The results of both northern blot (Fig. 2 ) and RT-PCR analyses indicate that additional inhibin α mRNA variants also exist. Future studies will not only more thoroughly characterize these variants, but will examine their expression in other species. Moreover, functional analyses may highlight heretofore-unknown aspects of inhibin biology and function. Authors' contributions DJB participated in the design of the study, performed all of the molecular biological experiments and analyses, and drafted significant portions of the manuscript. TKW participated in the design of the study and critically revised the manuscript. TMP provided the animal tissues, participated in the design of the study, and drafted sections of the manuscript. All authors read and approved the final manuscript.

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509296

Grafting the Way to the Systemic Silencing Signal in Plants

Grafting is a powerful but complex means to study the spread of RNA silencing

Grafting is an ancient technique used by farmers and gardeners to combine desired attributes of the rootstock with those of the donor plant shoot, or scion. Grafting essentially saved European wine making: when the insect Dactylosphera vitifoliae devastated European grapewine varieties over the course of the late 1800s and early 1900s, the varieties were saved by grafting them onto resistant rootstocks from the New World. Since then, these rootstocks have been used to maintain the susceptible Old World cultivars. But grafting is also an excellent tool for scientists studying systemic signals traveling between the rootstock and distal parts of the plants, and vice versa. For example, two important studies ( Palauqui et al. 1997 ; Voinnet et al. 1998 ) used grafting to demonstrate the spreading of RNA silencing in plants. However, it was a subsequent paper ( Crete et al. 2001 ) that followed up on certain inconsistencies in the grafting results that pointed to subtleties important for both experimental design and understanding systemic signaling in plants. RNA silencing (termed posttranscriptional gene silencing in plants, quelling in fungi, and RNA interference in animals) refers to the phenomenon whereby specific gene transcript levels are reduced in the presence of a related RNA. From studies of RNA silencing in several systems, much is now known about the mechanisms involved ( Matzke 2002 ; Mlotshwa et al. 2002 ), but the systemic spreading in plants is still a bit of a mystery. Posttranscriptional gene silencing spreads systemically throughout the individual plants in a very characteristic manner reminiscent of viral spread. This has led to the hypothesis of a systemic silencing signal that is produced in the tissues where silencing is initiated and is then transmitted to the distant parts of the plant where it can initiate silencing in a sequence-specific manner. The sequence specificity of the silencing strongly implies that the signal is a nucleic acid, most likely an RNA, but the identity of the signal remains unknown. Silencing spreads mainly in the direction from carbon source to carbon sink, that is, from tissues such as leaves that export the sugar products of photosynthesis, to tissues such as roots that import these products, and it can take up to several weeks until it is established in the whole plant ( Palauqui et al. 1997 ; Palauqui and Vaucheret 1998 ; Voinnet et al. 1998 ; Sonoda and Nishiguchi 2000 ). As expected, the discovery of this process triggered a quest for the “systemic inducer” of the process: a signal that travels through the plant and is able to initiate silencing in a remote location within the plant. Grafting was an obvious tool to use in the quest for this signal, as it allowed silencing source and sink tissues to be of different origin. Palauqui et al. (1997) were the first to unambiguously demonstrate that silencing spreads from a silenced rootstock to a nonsilenced scion. They used as a stock a transgenic tobacco carrying an additional copy of the endogenous nitrate reductase gene, Nia . Some of the transgenic lines generated always showed higher levels of Nia transcripts than the wild type—as expected from the presence of an additional gene—and were termed class I lines. However, other transgenic lines had undergone silencing for both the endogenous and exogenous Nia genes and those were termed class II lines. Pallaqui et al. (1997) found that silenced class II rootstocks were able to silence class I scions. This was true even in a “sandwich graft,” where a wild-type (nontransgenic) segment was grafted between the silenced stock and the nonsilenced scion. Spreading of silencing was unidirectional from stock to scion. Though not explicitly stated, it was implied that it took more than 3 wk after grafting for systemic silencing to occur in the scion. The reported rate of transmission was 100%. Related experiments by Voinnet et al. (1998) used scions that transgenically expressed green fluorescent protein grafted onto plants with established silencing of the same transgene. There, too, silencing spread through the graft to the nonsilenced scion, even when a wild-type section was grafted between transgenic rootstock and scion. Unlike the Nia transgene, which has an endogenous counterpart in the wildtype plant, green fluorescent protein has no homolog in nontransgenic lines. Therefore, silencing spreading in the wild-type “spacer” in the sandwich grafts could not be assisted by an endogenous sequence. Rather, the systemic signal must have traveled all the way to the scion and induced gene silencing there. The establishment of systemic silencing took 4 wk in the “direct” grafts and 6 wk in the sandwich grafts. However, silencing spread to the scion only in some of the grafts: in ten out of 16 direct grafts and five out of 11 sandwich grafts. I found these papers were very important not only for what they proved—the existence of a systemic signal of silencing—but also because they gave an unequivocal answer to the scientific questions they posed, using relatively simple methodology. Although excited by these successful examples of the transmission of silencing, I kept coming back to two questions: (1) what prevents transmission in some of grafts, and (2) why does it take longer to transmit silencing to the scion than it takes systemic silencing to reach the most remote parts of an intact plant? When we started working on the silencing signal ourselves, we repeated some of the above experiments but found somehow lower efficiencies in the initiation of silencing in the scions. We soon realized that our results were influenced by the developmental stage of our scions. A paper from Jeff Meins's laboratory in Switzerland shed light on some aspects of the grafting puzzle. Researchers there introduced additional chitinase genes using bolistics, in sense or antisense orientation under the control of a strong promoter (35S) into chitinase transformant lines of tobacco that never exhibited spontaneous gene silencing ( Crete et al. 2001 ). In lines bombarded late in plant development, triggering of silencing was rarely observed. However, when the transgene was introduced earlier in development, a large portion of the lines showed a substantial decrease and eventually full suppression of the chitinase mRNA levels. Lines that showed silencing were used as rootstocks and nonsilencing lines were used as scions in three types of grafting experiments. In the first type of grafting experiment, called top grafting, a 5-cm scion cut into a wedge at the bottom was inserted into the vascular ring at the cut surface of a 50-cm-high rootstock ( Figure 1A ). In the second type of experiment, reciprocal transverse grafts of 50-cm-tall plants were exchanged between class I and class II plants ( Figure 1B ). Finally, the third type of experiment involved plug grafts, which were made by exchanging transverse plugs of stems cut with a 5-mm-diameter cork borer from an internode approximately in the middle of a 50-cm plant ( Figure 1C ). Figure 1 Grafting Method May Influence the Spreading Efficiency of the Silencing Signal (According to Crete et al. 2001 ) (A) Top grafting, the most effective in transmitting the silencing signal. (B) Reciprocal transverse graft. (C) Plug graft. Surprisingly, only top grafting resulted in scions that were systemically silenced by a rootstock signal. Furthermore, even transmission after top grafting was less effective than expected; in one stock/scion combination only 27 out of 71 grafts exhibited transmission of the silencing signal. The authors also found that antisense-induced silencing was never transmitted to the scion. These findings do not answer the many questions about the mechanisms underlying systemic silencing, but they point us in certain directions. The individual parts of a whole plant are, in terms of import and export, in an equilibrium that changes with development. When grafting takes place, how this equilibrium is altered depends on the individual “parts” that contribute to the “new” whole plant. In addition, there is now indirect evidence ( Fagard and Vaucheret 2000 ) that what is source tissue and what is sink tissue in terms of sugar transport affects what is source and what is sink in terms of the systemic silencing signal. Taking into account the above findings and choosing the right combination of stock/scion, we have managed to significantly increase the efficiency of graft-transmitted silencing, a prerequisite for continuing the search for the systemic signal. From the grafting experiments to date, it is now evident that the transporting capacity of the vascular tissue bypass that is formed at the graft junction does not fully reach the level of the original vascular tissue. The basis for these restrictions is not known. In a way, the graft interface functions as an unintentional filter. If the specificities of this “filter” were known, it would help us comprehend some transmission inconsistencies. Keeping in mind these limitations, grafting remains an invaluable tool in the search for the systemic silencing signal.

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544396

Participation of African social scientists in malaria control: identifying enabling and constraining factors

Objective To examine the enabling and constraining factors that influence African social scientists involvement in malaria control. Methods Convenience and snowball sampling was used to identify participants. Data collection was conducted in two phases: a mailed survey was followed by in-depth phone interviews with selected individuals chosen from the survey. Findings Most participants did not necessarily seek malaria as a career path. Having a mentor who provided research and training opportunities, and developing strong technical skills in malaria control and grant or proposal writing facilitated career opportunities in malaria. A paucity of jobs and funding and inadequate technical skills in malaria limited the type and number of opportunities available to social scientists in malaria control. Conclusion Understanding the factors that influence job satisfaction, recruitment and retention in malaria control is necessary for better integration of social scientists into malaria control. However, given the wide array of skills that social scientists have and the variety of deadly diseases competing for attention in Sub Saharan Africa, it might be more cost effective to employ social scientists to work broadly on issues common to communicable diseases in general rather than solely on malaria.

Introduction Malaria control remains ineffective in many endemic areas in spite of efficacious interventions, such as combined antimalarial therapies and insecticide-treated materials. Biological, environmental, political, socio-cultural, economic and behavioural factors influence the transmission of malaria, thus requiring a multidisciplinary and integrated approach to effectively control the spread of malaria [ 1 , 2 ]. In recent years international initiatives, such as Roll Back Malaria, have highlighted the contributions that social scientists bring to a multidisciplinary approach for malaria control. Various studies in Africa have illustrated the benefits of collaborative research between social and biomedical scientists [ 3 - 17 ]. However, social science knowledge and practices are still not fully integrated into malaria research and control programmes, especially when compared to other public health areas such as HIV/AIDS prevention. While it is difficult to do a comparison on the actual number of social scientists involved in HIV/AIDS versus malaria control, a review of the literature illustrates the numerous contributions that social and behavioural research has had in HIV/AIDS control and prevention [ 18 - 27 ]. The early recognition and inclusion of socio-behavioural research was part of the HIV/AIDS prevention strategy at the onset of the pandemic [ 27 ]. Significant funding allowed social scientists to work directly with epidemiologists in HIV/AIDS research. Several factors continue to limit the application of social science research in tropical public health in general, and malaria control in particular. These include: confusion among many non-social scientists and social scientists alike about the variations in social science theory and methods and the different types of contributions that each can make to public health and malaria control activities, a perception among many public health and malaria control professionals that the problems of communicable diseases can be solved with technical and clinical interventions, ignoring the social nature of illness, limited communication among social scientists working in applied health and malaria control, insufficient access to current social science and malaria literature, and the lack of trained social scientists with applied experience in the endemic countries [ 28 , 29 ]. Given the poor academic and professional structures found in many African countries, an increasing number of highly skilled citizens emigrate abroad for further studies but fail to return to their respective country of origin, a phenomenon commonly referred to as 'brain drain' [ 30 - 36 ]. As a result, many African countries now have a vast pool of highly skilled professionals who are permanently living abroad, making little to no contributions to their country of origin [ 37 , 38 ]. While the "brain drain" significantly contributes to the lack of social science capacity in malaria control in Africa, it is unclear the extent to which the current pool of trained social scientists that remain in Africa is being integrated into malaria control. Experience has shown that commissioning a well-trained and field-experienced applied social scientist (i.e. one with both an understanding of the theoretical and applied perspectives of the discipline) can be extremely beneficial for informing malaria programme-related decisions, as well as helping in the development of effective intervention programmes [ 39 , 40 ]. This study was designed to better understand the level of involvement of African social scientists in malaria control, in order to identify potential approaches to facilitating collaborative work among social scientists and malaria control programme personnel and stakeholders. Methods A convenience sample was derived from existing social science networks such as, the Partnership for Social Science and Malaria Control (PSSMC) Network, the Social Science and Medicine Africa Network (SOMA-Net), Pan African Anthropological Association (PAAA), as well as the CHANGE Project database of African social scientists, and the United Nations Development Program, World Bank and World Health Organization's Special Programme for Research and Training (WHO/TDR) database of social scientists trained in the last decade (1992–2000). Snowball sampling was used to elicit names of additional social scientists known personally to the participants and investigators. Social science was broadly defined to include disciplines such as anthropology, sociology, health economics, demography and population studies, development studies and public health. Enrolment criteria included specific social science training within these disciplines, and participants had to be of African origin. Enabling factors were defined as those factors that made it easy to work in malaria control, including factors that first attracted social scientists and those that facilitated entrance into the field. Constraining factors were defined as those factors that impeded work in malaria control and factors that made malaria control unattractive to social scientists. Data collection was organized in two phases. In Phase I, consent forms and questionnaires were sent by email and/or fax to potential participants. Participants could respond directly and/or refer other eligible colleagues. Those who were ineligible or uninterested could decline. For those emails that could not be delivered, three attempts were made to resend the survey and consent form. The questionnaire focused on demographic characteristics, work experiences, factors influencing career choices and sources of career development information. The survey instrument was piloted among five participants from different countries. Based on the feedback from the pilot survey, minor adjustments were made to improve use via email. Email communication was preferred but, in the absence of email, other methods of communication were used. If the survey was not returned within three weeks, attempts were made to follow-up. The survey responses in Phase I provided an overview of the involvement of the participants in malaria related activities but gave only a limited understanding of the specific factors affecting career trajectories. In Phase II, we conducted in-depth phone interviews using a purposive sample derived from Phase I that was selected according to country of origin, sex, academic field (social science discipline) and interest in malaria and applied or operational work experience (both in malaria and non-malaria). A deliberate effort was made to ensure that the sample represented those with or without an interest in malaria, those who actively sought employment in malaria and those who did not. The more descriptive data from Phase II clarified Phase I data. For example, with regards to participants' interest in malaria, some participants in Phase I indicated an interest in malaria while in school, but did not include malaria in their thesis research and did not pursue employment in malaria. The in-depth, open-ended phone interviews in Phase II provided greater detail as to other factors that might have influenced participants' career decisions in malaria control. Open-ended questions focused on key events and times in the career trajectory during which critical decisions were made, current and past work experiences, reasons for selecting specific jobs, technical experiences with proposal writing, challenges and rewards of working in malaria, and suggestions for improving the integration of social science in malaria control. Descriptive statistics were generated using EPI-Info. Qualitative data were transcribed and content analysis was used to develop contextual themes. The study was reviewed and approved by the Institutional Review Board of the Centers for Disease Control and Prevention. Verbatim quotes from the participants are used throughout the text to illustrate contextual points of discussion. Results This study was conducted between May 2002–May 2003. Of the 136 surveys sent out, 40 completed surveys were found to fit our enrolment criteria (Fig. 1 ). Eighteen participants were interviewed in Phase II. Figure 1 Response to surveys. Flow chart illustrating response to surveys All 40 participants were nationals of African countries and resided in the countries of Cameroon, Ethiopia, Mali, Ghana, Kenya, Nigeria, Tanzania, South Africa and Uganda. Most participants (75%) were male between the ages of 30–39 years old (Table 1 ). Fifty percent (n = 20) had a masters degree, 42% (n = 17) were either doctoral degree students (n = 3) or had a doctoral degree (n = 14). Almost all participants had an undergraduate degree in the social sciences and chose various social sciences disciplines for specialization during their postgraduate training. Table 1 Age and Sex Distribution (n = 40) Age group Male Female Percentage 20–29 1 0 2.5% 30–39 17 5 55.0% 40–49 9 4 32.5% 50–59 2 1 7.5% 60+ 1 0 2.5% Total 30 10 100.0% Of the 18 participants selected for a phone interview, 55% (n = 10) were between 30–39 years and 22% (n = 4) were female. Of those with postgraduate degrees, 50% (n = 9) had a masters degree, 44% (n = 8) either had a doctoral degree or where doctoral degree students while 5% (n = 1) had a bachelor's degree. When difficulties arose in ascertaining whether the professional discipline linked to the social sciences, participants were contacted for clarification. Eighty-five percent of the participants (34/40) received funding for postgraduate training from their national government, international organizations (such as the WHO or DFID), or other foreign governments. Sources of career development information included the Internet, friends and colleagues, local universities, conferences, journals, professional networks and local newspapers. Enabling factors Interest in malaria When asked if participants were interested in malaria during their academic training, 82% (33/40) of the participants said yes. Only 36% (12/33) of those interested included malaria as a research topic in their thesis or dissertation. Three participants (7.5%) who were not interested in malaria included malaria in their thesis. Post graduation, 35% (14/40) of the participants actively sought employment in malaria control. Three of the 14 (21%) who sought employment in malaria were turned down for jobs due to insufficient technical knowledge of malaria control, while six participants (15%) returned to previous health care positions, some of which were positions in malaria control. Although some participants were not currently working on malaria-related projects, nearly all participants, at some point in their career, had worked as a consultant in malaria control in addition to their full time positions. Participants who specialized in malaria for postgraduate degrees were often funded by a national or international organization, on condition that they would return to their jobs in malaria following the completion of studies. Eleven of the 40 participants said that the primary factor that prevented those with an interest in malaria from seeking further research and employment opportunities in malaria control was the perception that social scientists could not be employed in malaria control. As one participant remarked "Malaria is an endemic disease in my community, so I can't say I was never interested. I wanted to do a thesis on malaria, but I realized that social scientists could not get jobs in malaria so I had to look elsewhere." When followed up during the phone interviews in Phase II, it was clear that some participants turned down jobs in malaria due to competing employment in areas such as HIV/AIDS, while others declined malaria control contracts due to the competing demands of doctoral-level training. Another issue that was revealed in Phase I was the influence of the epidemiology of malaria on participants' interest in malaria. Eleven of the 18 participants during the phone interview explained that they developed an interest in malaria after learning about the complex nature of the disease, its high prevalence and the possibility of access to research funds. "For years I thought the field of malaria was boring and I was not interested in it. I only became interested in malaria when I realized that it was a complex disease with a major public health impact and it appeared that funding for research might be available." Factors influencing employment in malaria All 40 participants were asked to identify factors that attracted social scientists into general employment opportunities, as well as malaria-specific opportunities (Table 2 ). Responses in Phase II provided further clarification on the specific factors affecting career trajectories. Of the 18 participants included for an in-depth interview in Phase II, 11 indicated that a senior lecturer or mentor during the practical training experience had encouraged participants to work with them on their research project. Mentors helped to develop participants' research and proposal writing skills and identify funding and publication opportunities, and in some cases international contracts. These contracts often resulted in participants gaining local and international visibility within the larger malaria community, which led to subsequent employment opportunities. Table 2 Factors Important for Employment (n = 40) Malaria Employment Frequency General Employment Frequency Sufficient funds to complete job 11 Potential for career advancement a 26 Supportive environment that facilitates the translation of research findings into programmatic use 9 Competitive salary a 13 Ability and opportunity to contribute alternative solutions to malaria control from a social science perspective 6 Proximity to family a 12 Type of social science dimension to job 5 Social value of job b 5 Sufficient technical skills to complete jobs 5 Epidemiology of malaria 4 a Indicates factors that were identified as important for both general employment and malaria specific employment. b The social value of a job was defined as the positive impact that a given job made in the community, as well as the professional and community respect awarded to the position. Participants were more likely to seek out employment from organizations with proven track records of using research findings to improve the health of the community. Opportunistic events also shaped career development. For example, the successful completion of a project, fellowship or consultancy often produced a cascade effect by opening up other opportunities in malaria control, resulting in individual professional recognition by local and international collaborators as an expert in the field. Attending workshops sponsored by international organizations, such as the World Bank and WHO, enabled participants to get their name in the organizations' database for possible consultancies. Strong writing skills were identified as essential for job and training opportunities by most of the 18 participants in Phase II. Ten of the 18 participants (55%) in the phone interview noted that while it was initially difficult to identify and develop fundable proposals, receiving constructive feedback from rejected proposals helped improve their writing skills and led to the development of more successful proposals. "Most of the rejected proposals do not give reasons for rejection, which is unfortunate. I have been lucky to have one rejected proposal that was returned with explanations as to why it was rejected. I was told the issue we raised was very broad and vague and it made me realize that I had to be very clear in my framework. After that rejection, I was able to develop projects that had a clear framework and include a multidisciplinary team to strengthen other key areas and the next proposal we developed was accepted." When asked why they chose employment in malaria, one of the participants echoed many others by saying, "In Africa we take what we get. One rarely ends up in a profession of their choice. People take the job that is at hand and learn to deliver as best as they can." This was true for both those interested in malaria originally and those not interested. During the in-depth interviews, we also asked participants who did not initially express an interest in malaria but sought and gained malaria related employment, why they choose to stay in malaria? Of the 18 participants eight agreed with the following view: "I would probably stay in malaria because it is a major problem in my country, and I feel I have gained significant experience in malaria over the years. This has strengthened my confidence and ability to handle current and hopefully future opportunities, and maybe I am too old right now to start learning something new." Constraining factors Malaria employment challenges Participants were also asked to identify factors that constrained employment in malaria. All 40 participants were more likely to refer to employment challenges specific to malaria research, rather than malaria control, as national malaria control programmes employ very few social scientists. Ten of the 18 participants in Phase II noted that social science involvement in malaria control was a recent development in their countries, as biomedical scientists had previously dominated malaria control. Another phone interview participant remarked that "specifically focusing on malaria as the only area for job opportunities narrows ones fields for job opportunities. Although malaria is an endemic disease in my country, it is largely dominated by physicians and there are not sufficient job opportunities for social scientists in my country so it is impossible to focus only on malaria. You need something to fall back on besides malaria." Participants also noted that a paucity of research centres and limited funding for social science research also contributed to the lack of job opportunities. In situations where job opportunities existed for social scientists, they were generally limited to short-term rather than long-term career paths. Individual perceptions of malaria as a 'normal' everyday event limited participants' understanding of the impact of malaria and, thus, diminished their interest in malaria control. As one of the phone interviewed participants remarked, "At first, I did not realize the impact of malaria in my community. Everybody was talking about HIV/AIDS. I think it is because people have lived with malaria for so long that they treat it at home. It was only after doing some training at the hospital (as part of the national training requirement), that I realized how many people, especially children and mothers, die of malaria and I got interested in working in malaria control." Of the 40 participants in Phase I, 36 identified the lack of professional development opportunities as a constraining factor to employment in malaria. The lack of a supportive work environment with good communication and mutual respect was mentioned by those working primarily with biomedical scientists. "I prefer working on maternal and child health issues, as well as HIV/AIDS, because personnel in these areas are able to recognize the role of social science and request social science input. This makes me confident that my discipline is important and I can make a contribution." Insufficient technical skills in proposal writing and professional isolation from other social scientists hampered the development of collaborative relationships, which made it difficult to develop competitive proposals for funding and publication. As a participant observed, "Research is largely uncoordinated. We have no knowledge of who is doing what, there is no database of social scientists and the projects that they're working on from which we can develop collaborative projects. We neither have the infrastructure nor the access to current literature, which makes it difficult to develop manuscripts for publication." In order to meet the challenges of employment, eight of the 18 participants in Phase II stressed the importance of understanding all technical aspects of malaria control. One participant echoed the view of several others in their advice to junior social scientists, "try to learn not only about social science in malaria control, but all other aspects of malaria control as well, such as entomology, vector control and treatment. Having only a social science background without any understanding of the other areas of malaria control might make it difficult to effectively apply the social science aspect within malaria control. Besides you will always be working as part of a multidisciplinary team, therefore, you will need to understand their language in order to be able to effectively communicate the social science dimension to them." Participants also emphasized the importance of linking students to practical training opportunities in endemic areas for fieldwork experience and possible long-term opportunities. As one participant noted, "Encourage donors to develop grants for student researchers working with senior scientists, and identify conferences where students could present research findings." Integrating social science in malaria control Participants in Phase II were asked to describe strategies for improving the integration of social science in malaria control. Of the 18 participants interviewed, 13 (72%) stressed the need for social science advocacy as an important step in malaria research and control. Advocacy efforts were recommended for social scientists and non-social scientists alike, as well as policy makers in health and education and those who fund research. Five participants said their involvement in malaria projects was as a result of specific requests for social scientists in the call for proposals. All 18 participants emphasized the need to develop outreach efforts for junior social scientists in order to enhance their understanding of the potential contributions that social scientists could offer, the type of training needed, and the types of jobs they could perform in malaria control. Three of the 18 participants also suggested the revision of graduate and medical school curricula to include malaria and social science courses, but noted that support had to come from the national level for curricula change to take place. The establishment of a sustainable network of African social scientists working in malaria control was identified as a useful tool for integrating social sciences in malaria control by six of the 18 participants. Possible functions of the network were described as developing a mentoring programme for junior social scientists, a forum to disseminate current local social science research, information relating to employment and training and funding opportunities. To facilitate the application of social science research findings to national malaria control policies, one of the phone-interviewed participants explained how to include policy makers in the process of social science research. "Make the research process more participatory. Involve policy makers and consult with them so that eventually they are the ones who demand the research and in this way, social science research can influence policy. We developed working papers with key findings for distribution to various stakeholders. We published our findings on the Internet and conducted department seminars or short courses during which we discussed our research and presented the information in a very manageable manner. At least, in this way, we were able to get our findings into the hands of policy makers and hopefully begin a dialogue with them." Discussion Results of this study indicate that factors such as having an interest in malaria, a mentor during one's academic and professional career, strong writing skills, and technical experience in malaria made it possible for participants to take advantage of opportunistic events and job opportunities that led to employment in malaria control activities. As well, limited job opportunities, the lack of career development opportunities, and the lack of understanding about social scientists' role in malaria limited the involvement of social scientists in malaria control. This study revealed that many of the enabling and constraining factors for social scientists' involvement in malaria control are not unique to malaria control but are common to African researchers in general. An examination of the intrinsic and extrinsic factors regarding career trajectories of social scientists in this study revealed that the majority of participants did not specifically seek malaria as a career path but, rather, were looking for employment opportunities in general, some of which happened to be in malaria. It is unclear to what extent the career development process in many African countries contributes to the way employment is sought or if this is due to the relatively new role that social science plays in malaria control, which was noted by numerous participants. Career development is a process that ideally begins during one's formal academic training and continues throughout one's professional life. However, if career development is concentrated more towards academic training and less on postgraduate employment opportunities, it is no wonder that post graduation, students are forced to 'take whatever they can get,' as opposed to seeking opportunities that complement their interest and training. The lack of proper career counselling could also limit a student's understanding of what courses and practical experiences are most beneficial for social science and health careers, without which social scientists cannot be hired as part of a health intervention team [ 39 , 41 ]. More initiatives that provide research-training grants, career development fellowships and proposal development workshops, such as those currently established by the UNDP/World Bank/WHO/TDR, are needed to strengthen the social science capacity in endemic countries. However, creative strategies need to be identified to capture those who do not easily have access to such information and opportunities. In order to effectively design and implement malaria control interventions, it is necessary to have social scientists that are knowledgeable about the technical issues that underlie malaria control. This knowledge is acquired over time and needs to be supplemented with direct field experiences. However, given the economic constraints facing national malaria control programmes and Ministries of Health, this study shows that it might not be reasonable to expect that social scientists will be assigned solely to malaria control. Given the limited number of trained social scientists with practical experience and the variety of deadly diseases competing for attention in sub-Saharan Africa, there are few social scientists that are both willing and able to dedicate their expertise only to malaria control. Ministries of Health and Education should work with social scientists to establish employment opportunities in communicable diseases. Given the wide array of skills that social scientists have, it would be cost effective to employ social scientists to work broadly on issues common to communicable diseases, rather than solely on malaria. This "generic" approach would also support the programmatic shift from malaria as a vertical programme to its being integrated within primary health services or the wider arena of communicable diseases. Although this may not be ideal for building strong social science capacity specifically for malaria, this offers a pragmatic approach to utilizing scarce resources. Lessons learned from behavioural interventions in other diseases can also be applied to malaria control, thus strengthening social science contributions to malaria control. The acknowledgement by many participants of their need for a better understanding of both biomedical and technical aspects of malaria control is reinforced by data from other successful projects that are aimed at establishing collaborative multidisciplinary research teams. For example, the International Clinical Epidemiology Network (INCLEN) social science programme seeks to enhance clinicians' understanding of social science by exposure to relevant issues in social science design and measurement and evaluation issues, while teaching principles of clinical epidemiology and biostatistics to established social scientists. As a result, strong collaborative partnerships among individual clinical epidemiologists, social scientists, and biostatisticians have been developed to produce the interdisciplinary solutions required for priority health problems in society [ 42 ]. A key factor identified as important for employment in malaria was the ability to contribute social science solutions to malaria control and the integration of research findings into malaria control programmes and policies. Social scientists are more likely to work directly with members of the community therefore, it is important that strong relationships are developed with local communities and their leaders, as a trust building mechanism and a matter of social prestige for the researcher. This link to the community facilitates the application of research findings on a local level [ 39 , 42 ]. Limitations While findings from this study give us greater insight into factors that affect career trajectories of African social scientists, caution must be used when interpreting the results. The use of convenience sampling and snowball sampling techniques to identify eligible participants appeared to have excluded the younger more recently qualified social scientists who might not have been members of the various networks we used to identify eligible participants for this study. It is possible that some of the junior social scientists do not have much work experience in malaria and are not yet known by the more experienced social scientists that helped with referring other participants for the study. In addition, although telephone interviewing enabled us to reach social scientists from several countries, we were not able to reach those social scientists with no access to the Internet and telephone who might have had other unique challenges not captured in this study. Further research should examine younger or more recent social science graduates as well as those with no access to the Internet. Conclusion Malaria control is at a point where there is good evidence-based data to demonstrate that various approaches can make a difference in malaria-related morbidity and mortality. Prompt and timely treatments with an effective drug, intermittent preventive treatment for malaria in pregnancy, and use of insecticide-treated materials are examples of interventions that have been shown to be effective. However, the success of these approaches depends, to a great extent, on human behaviour. Epidemiologists and clinicians, who work in malaria control, traditionally, do not have the necessary training to incorporate behavioural and social science knowledge into the development, implementation, and evaluation of control interventions. It is time to broaden the perspective of malaria control, which can be best achieved through employing a multidisciplinary team that includes social scientists. To support such collaboration, attention needs to be given to understanding the factors that influence job satisfaction, recruitment and retention of the social scientists. At this point in time, social science capacity is severely limited in most African malarious areas due to insufficient funding, lack of career structure, loss of trained individuals to international postings, and a limited understanding of how social scientists can contribute to malaria control. In order to improve this situation, capacity development is needed as a first step. Joint collaborative efforts should be made to offer technical malaria knowledge to social scientists and social science methodology to epidemiologists and control personnel. Social scientists need forums and networks to exchange information, learn about existing research and educational opportunities and promote collaborative partnerships with national malaria control programmes. However, these mechanisms cannot be realized without adequate levels of funding. In order to attract skilled social scientists to Ministries of Health, a new line of thinking needs to emerge that situates social scientists more broadly within the ministry. Rather than focusing on how to integrate social scientists solely within malaria, the emphasis should shift to thinking creatively about how to best utilize social science expertise across an array of specific programmatic areas. Novel approaches within malaria control interventions are starting to emerge, such as integrating the distribution of insecticide-treated nets with measles immunizations. This same type of non-traditional thinking should be applied when developing career tracks for social scientists within Ministries of Health. However, incentives for employment must rival or exceed those offered in other areas. Pay scales must be equitable and the value of multidisciplinary perspectives must be appreciated and desired by national malaria control programmes and Ministries of Health in order for career structures for long-term employment of social scientists to be established. For contractual employment, the same considerations should be shown. Short-term salaries for malaria-related projects should be competitive with salaries offered for similar work being conducted with other diseases, such as HIV/AIDS. In the last five years, there has been a noticeable increase in the collaboration among social scientists and others engaged in malaria control. The interest in malaria by social scientists is clearly present. What are needed now are structures for training and employment that offer a professional career path over time. Authors' contributions HAW and CJ obtained funding for the project. All authors contributed to the study conception and design. PN coordinated fieldwork, data collection and analysis with ongoing supervision from HAW and CJ. PN wrote the first draft of the article with critical revisions from HAW and CJ. PN, HAW, CJ, IN, SD and FG all read and approved the final manuscript.

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524363

Relationship between CRP and hypofibrinolysis: Is this a possible mechanism to explain the association between CRP and outcome in critically ill patients?

Background- Endothelial cell dysfunction may be implicated in the development of multiple organ failure (MOF) by a number of mechanisms. Among these, altered fibrinolysis promotes fibrin deposition, which may create microvascular alterations during inflammation. Elevated concentrations of C-reactive protein (CRP), especially when these persist over time, are correlated with an increased risk of MOF and death. CRP may inhibit fibrinolysis by inducing plasminogen activator inhibitor-1 (PAI-1) release from human aortic endothelial cells. Moreover, the administration of recombinant CRP in volunteers may increase circulating PAI-1 levels. In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis in intensive care patients with and without sepsis. Methods- We studied the association of inflammation and abnormal fibrinolysis in intensive care unit (ICU) patients with (n = 11) and without (n = 21) sepsis. The inflammatory response was assessed by serum concentration of C-reactive protein (CRP), a marker of the acute phase reaction, which increase rapidly in the inflammatory response, and the plasma fibrinolytic capacity was evaluated by the Euglobulin Clot Lysis Time (ECLT), determined by a new semi-automatic method. Results- ECLT was significantly higher in septic than non-septic patients (1104 ± 439 vs 665 ± 275 min; p = 0.002) and was significantly correlated with CRP concentration (R 2 = 0.45; p < 0.001). In a multivariate analysis, CRP was the strongest predictor of ECLT (R 2 = 0.51, F = 25.6, p < 0.001). In addition, the overall ICU length of stay was significantly correlated with CRP (R 2 = 0.264, p = 0.003) and ECLT (R 2 = 0.259, p = 0.003). Conclusion- In critically ill patients a significant correlation thus exists between plasma fibrinolytic capacity and serum CRP levels. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. This finding is compatible with a link between inflammation and abnormal fibrinolysis, and may explain the negative prognostic value of CRP in critically ill patients.

Background Endothelial cells have a key role in the control of vascular permeability and vessel tone, coagulation and fibrinolysis, and inflammatory response [ 1 ]. There is an increasing body of evidence supporting the critical role of the vascular endothelium in the pathogenesis of multiple organ failure (MOF) in critically ill patients [ 2 ]. Endothelial dysfunction/or activation is associated with an imbalance in hemostatic functions. Endothelial cells are responsible for the release of tissue plasminogen activator (t-PA) and contribute to the release of plasminogen activator inhibitor-1 (PAI-1). Inhibition of the fibrinolytic system amplifies the pathogenic role of fibrin deposition during severe inflammation [ 3 ]. Multiple factors, including lipoproteins, cytokines, and inflammatory proteins can modulate the endothelial cells to produce t-PA and PAI-1 [ 4 ]. In infected patients, elevated concentrations of serum CRP are correlated with a risk of MOF and death [ 5 ], especially when these persist over time [ 5 ]. However, the possible biological involvement of CRP in the development of MOF and death is unknown. CRP can act directly on endothelial cells, inducing, for example, the expression of intercellular adhesion molecule (ICAM)-1 [ 6 ] and the production of inflammatory cytokines such as interleukin- (IL)-6 [ 7 ]. It may also inhibit fibrinolysis by inducing PAI-1 release from human aortic endothelial cells [ 8 ]. Moreover, the administration of recombinant CRP in volunteers may increase circulating PAI-1 levels [ 9 ]. In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis as measured by ECLT in intensive care patients with and without sepsis. Material and methods Subjects After approval by the A. Vésale hospital ethics committee, we studied 32 ICU patients with severe sepsis (n = 11) or other diagnoses (n = 21). Infection definition required isolation of a microorganism from a normally sterile body site, concurrent with accompanying signs and symptoms of sepsis and decision of antibiotic therapy. Criteria for severe sepsis included signs of at least one organ dysfunction attributed to sepsis [ 10 ]. All patients (septic and non-septic) were enrolled in the first day of ICU admission to limit the delay of inflammatory response. The exclusion criteria were: antibiotics treatment in the non-septic group except for surgical prophylaxis, red blood cell transfusion in the last 72 h, active hemorrhage, hematological disorders, recent cytotoxic chemotherapy, burns, cardiogenic shock, cirrhosis, pregnancy. The simplified acute physiologic score (SAPS II score) [ 11 ] was determined in each patient during the first 24 hours after admission. Blood samples Blood samples were obtained during the first 24 hours of sepsis or on the first day of admission for non-septic patients. Serum samples were collected in vacuum tubes without anticoagulant. Plasma samples were harvested in citrated vacuum tubes and put in melting ice. Whole blood was collected on EDTA-treated tubes. CRP was evaluated by antibody-binding and turbidity measurement on SYNCHRON LX ® . Fibrinogen was determined by thrombin time on a STA ® automate (STAGO). Leukocyte and platelet counts were determined on a hemocytometer (CELL-DYN4000 ® , ABBOTT). All tests were performed on blood obtained from the same venipuncture. Plasma fibrinolytic capacity The Euglobulin Clot Lysis Time (ECLT), which is the most common test used to estimate the plasma fibrinolytic capacity, represents the balance between t-PA and PAI-1 activities [ 12 ]. ECLT was measured on fresh plasma the same day as other parameters by a method described elsewhere [ 13 ]. Briefly, we designed a completely computerized, semi-automatic, 8-channel device for measurement and determination of fibrin clot lysis (Lysis Timer, EREM, Belgium). The lysis time is evaluated by a mathematical analysis of the lysis curve and the results are expressed in minutes (range: 5 to 9999). The efficiency scores of the method are <4% in intra-assay and <7% in inter-assay. Statistics We used SigmaStat ® software package (Jandle Scientific). The data are presented as mean ± SD. Correlation between variables was analyzed using a Pearson correlation test. A multivariate analysis was used with stepwise backward selection of the explicative variables. Sepsis was considered as a dichotomous variable while all other data were considered as continuous (CRP, fibrinogen, leukocyte, monocyte and platelet counts). ECLT was the dependent variable. A probability level of p < 0.05 was considered as statistically significant. Results The major cause of severe sepsis (9 patients) or septic shock (2 patients) was pneumonia (8 patients); angiocholitis was the cause in 1 patient, and in 2 patients the cause was not identified. In 8 patients (7 with pneumonia and 1 with angiocholitis), the infection was due to a Gram negative bacteria. Only 1 patient had documented bacteremia. Non-septic patients were admitted for postoperative surveillance (7 patients), intracerebral hemorrhage (3 patients), heart failure (3 patients), drug intoxication (3 patients), or aggravated chronic obstructive pulmonary disease (5 patients). As expected, inflammatory parameters such as white blood cells and CRP levels, the SAPS II score and ECLT were higher in the septic than the non-septic population (Table 1 ). In an univariate analysis (in all patients), the ECLT was strongly correlated with serum CRP concentrations (R 2 = 0.45; p < 0.001) with no perceptible threshold (Fig 1 ). Surprisingly, there was no relationship between the SAPS score and ECLT (R 2 = 0.08; p = 0.15). In multivariate analysis, ECLT was best predicted by the CRP level (R 2 = 0.51; F = 25.6; p < 0.001) and not significantly by sepsis or the fibrinogen concentration. Interestingly, the ICU length of stay was significantly correlated with CRP (R 2 = 0.264, p = 0.003) and ECLT (R 2 = 0.259, p = 0.003) in all patients, and in the survivors (R 2 = 0.13, p = 0.05 and R 2 = 0.3, p = 0.003, respectively). Table 1 Population characteristics Sepsis (n = 11) Non-sepsis (n = 21) p value Age, years 68 ± 19 67 ± 17 0.95 SAPS II 47 ± 11 25 ± 14 0.001 ICU stay, days 11.6 ± 7.8 6.7 ± 6.8 0.07 Death, n (%) 2 (18) 3 (14) 0.57 Leukocytes (×10 3 cells/μl) 10.4 ± 4.5 10.9 ± 3.7 0.74 Monocytes (×10 3 cells/μl) 549 ± 225 711 ± 398 0.24 Platelets (×10 3 cells/μl) 207 ± 157 234 ± 94 0.54 Fibrinogen (mg/dl) 657 ± 123 445 ± 132 <0.001 CRP (mg/dl) 24.2 ± 10.5 7.6 ± 6.1 <0.001 ECLT (min) 1104 ± 439 665 ± 275 0.002 Mean ± SD; p value (t-stest). SAPS : Simplified Acute Physiologic Score; ICU : Intensive Care Unit; CRP : C-Reactive Protein; ECLT : Euglobulin Clot Lysis Test Figure 1 Correlation between serum CRP levels and Euglobulin Clot Lysis Time. Discussion Many studies have demonstrated that sepsis is associated with endothelial cell dysfunction and promotes coagulation activation [ 14 ]. Although levels of t-PA antigen increase in sepsis, fibrinolysis is inhibited by increased levels of PAI-1. ECLT is, therefore, an interesting test, because it represents the balance between t-PA and PAI-1 activities. Previously considered as an imprecise method, we have been able to improve the precision and reproducibility of the test with a new semi-automatic device [ 13 ]. Inhibition of the fibrinolytic system may contribute to MOF, as fibrin can activate endothelial cells leading to a disorganization of the monolayer and the release of inflammatory cytokines [ 4 ]. Increased CRP levels are associated with worse outcomes and MOF in ICU patients [ 3 ]. The role of CRP on fibrinolysis is unknown in vivo . Our data suggest that CRP could itself be involved in the processes leading to endothelium dysfunction. The observed relationship does not prove a direct biological link between increasing CRP and hypofibrinolysis; however, indirect arguments exist in support of the concept. Several in vitro studies have reported the direct effects of CRP on endothelial cells [ 6 - 8 ]. In vivo , Cleland et al. [ 15 ] reported a relationship between serum CRP levels and the forearm blood flow response to N G -monomethyl-L-arginine (L-NMMA), reflecting endothelial dysfunction. Bisoendial et al. reported that the administration of CRP in volunteers impairs the fibrinolytic balance [ 9 ]. In addition, CRP has a strong prognostic value in acute coronary syndromes [ 16 ]. In a non-selected population with no inflammatory syndrome (CRP below 1.5 mg/dl, n = 160), we also observed that ECLT was significantly correlated with serum CRP levels [ 17 ]. CRP could also act indirectly on endothelial cells via the action of monocytes and the release of tumor necrosis factor-α (TNF-α). TNF-α is a strong inducer of PAI-1 production in-vitro and in-vivo . This mechanism seems to be important in sepsis, as high plasma levels of PAI-1 are associated with poor outcome [ 18 ]. Moreover, an association between CRP and TNF-α has also been described [ 19 ]. CRP can induce the monocyte release of cytokines such as IL-1β, IL-6, and also TNF-α through Fc receptors (γRI/CD64, FcγRIIa/CD32) [ 20 ]. CRP also has essential biological functions. No polymorphism of either the gene coding sequence or of the protein itself has been described in humans [ 21 ]. Also, high levels of human CRP protect against lethal infection. Transgenic mice capable of produce human CRP are protected against lethal infection by Gram positive and negative bacteria, Szalai et al [ 22 , 23 ]. This work is a pilot study. We have chosen to include patients in the first day of ICU admission to limit the possible rapid effect of inflammatory response on fibrinolysis, especially in septic patients. Indeed, this particular patient population has inflammatory reaction before signs of severe sepsis and thus before their admission to ICU. In fact, we have studied ECLT test at the onset of the organ dysfunction and the inflammatory reaction. Other studies with serial measurement of ECLT in patients who developed nosocomial ICU infections are needed to study the time course of these events. Moreover, we could not definitively exclude that all patients in the non-septic groups were non infected. For example, some non-septic patients with decompensated COPD may have minor infections, despite the negative microbiology cultures and the absence of antibiotic therapy. Viral infections were also possible in some patients. Moreover, it would be of great interest to determine the interactions between CRP and ECLT with IL-6, TNF-α and endothelium dysfunction markers such as soluble thrombomodulin and soluble von Willebrand factor. Conclusion Despite accumulating evidence that the inflammatory and coagulation systems are activated in sepsis, little is known about the mechanisms that ultimately lead to organ dysfunction and death. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. Prospective studies including the time course of CRP and hypofibrinolysis would provide additional information about this relationship. Authors' contributions KZB: Laboratory analysis, writing of the manuscript and design of the study. MP: patients recruitment and design of the study. DB: coordination and design analysis of the results. MG: design of the study. PC: laboratory analysis. JLV: design of the study and analysis of the results. CR: design of the study and analysis of the results. YB: patients recruitment. MV: statistical analysis and coordination.

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524377

Limited Durability of Viral Control following Treated Acute HIV Infection

Background Early treatment of acute HIV infection with highly active antiretroviral therapy, followed by supervised treatment interruption (STI), has been associated with at least transient control of viremia. However, the durability of such control remains unclear. Here we present longitudinal follow-up of a single-arm, open-label study assessing the impact of STI in the setting of acute HIV-1 infection. Methods and Findings Fourteen patients were treated during acute HIV-1 infection and subsequently subjected to an STI protocol that required retreatment if viral load exceeded 50,000 RNA copies/ml plasma or remained above 5,000 copies/ml for more than three consecutive weeks. Eleven of 14 (79%) patients were able to achieve viral loads of less than 5,000 RNA copies/ml for at least 90 d following one, two, or three interruptions of treatment. However, a gradual increase in viremia and decline in CD4+ T cell counts was observed in most individuals. By an intention-to-treat analysis, eight (57%), six (43%), and three (21%) of 14 patients achieved a maximal period of control of 180, 360, and 720 d, respectively, despite augmentation of HIV-specific CD4+ and CD8+ T cell responses. The magnitude of HIV-1-specific cellular immune responses before treatment interruption did not predict duration of viremia control. The small sample size and lack of concurrent untreated controls preclude assessment of possible clinical benefit despite failure to control viremia by study criteria. Conclusions These data indicate that despite initial control of viremia, durable viral control to less than 5,000 RNA copies/ml plasma in patients following treated acute HIV-1 infection occurs infrequently. Determination of whether early treatment leads to overall clinical benefit will require a larger and randomized clinical trial. These data may be relevant to current efforts to develop an HIV-1 vaccine designed to retard disease progression rather than prevent infection since they indicate that durable maintenance of low-level viremia may be difficult to achieve.

Introduction The use of highly active antiretroviral therapy (HAART) can dramatically prolong the life of individuals infected by human immunodeficiency virus 1 (HIV-1) [ 1 ], but early hopes for virus eradication have not been realized [ 2 ]. The successful use of HAART is limited by drug-related toxicities, high costs, and drug resistance [ 3 ], factors which have led to the development of alternative therapeutic strategies, including the use of supervised, or structured, treatment interruption (STI). This approach, involving recurrent limited exposure to autologous virus, has not been successful in chronic infection [ 4 , 5 ], but has been shown to lead to at least transient containment of viremia after intervention in the acute phase of infection in humans and animals exposed to AIDS-associated retroviruses [ 6 , 7 , 8 , 9 ]. In the present study, we performed a detailed longitudinal assessment of the impact of early treatment followed by STIs in patients treated during acute or early HIV-1 infection. The main hypothesis of the study was that early treatment of acute HIV-1 infection followed by STI would lead to immune boosting and subsequent control of viremia without the need for drugs. The primary endpoint was the time to viral rebound above 50,000 copies/ml once or above 5,000 copies for three determinations separated by a week each. The early results of this trial were previously reported, showing that five of eight patients were able to achieve a plasma viral load of 500 copies/ml or less at a median of 6 mo off therapy [ 6 ]. The current study investigates the frequency and durability of control achieved with this intervention, with follow-up to a median of 5.3 y after infection, and with an increase in size of the cohort to 14 patients. Our results indicate that, although the majority of patients treated in the acute phase of infection go on to control HIV-1 to less than 5,000 RNA copies/ml plasma for at least 6 mo off therapy, the ability to contain viremia below this level over the long term is maintained in a minority of patients. Methods Objective The hypothesis of the study was that early treatment of acute HIV-1 infection would confer immunologic maturation and subsequent control of HIV-1 without the need for ongoing drug therapy. Alternatively, if a breakthrough of virus replication was observed, this would provide a boost in HIV-1-specific immunity after reinstitution of antiviral therapy. The primary endpoint was the time to viral rebound to more than 50,000 copies/ml or viral loads above 5,000 copies/ml for three determinations separated by a week each. The secondary objective was to correlate immunologic and virologic parameters with any observed effects including evolution of HIV-1-specific T helper and cytotoxic T lymphocyte responses. The original study protocol, including the patient consent form and the institutional review board approval, can be found in Protocols S1–S4 . Study Population Fourteen patients presenting with acute or early HIV-1 infection were enrolled in this study between July 1997 and January 2000 ( Table 1 ). Acute HIV-1 infection was defined by the presence of HIV-1 RNA in the plasma, a negative or weakly positive HIV-1 antibody by HIV-1/2 ELISA, and the detection of no more than three bands in an HIV-1 Western blot; early HIV-1 infection was defined by a positive ELISA and confirmation of early infection by either detuned negative ELISA or previously known negative ELISA. All participants in the study had symptoms compatible with the acute retroviral syndrome and were treated with HAART (one protease inhibitor and two nucleoside reverse transcriptase inhibitors) within a median of 19 d (range, 9–33) from the onset of symptoms. Study participants were recruited from the Massachusetts General Hospital, the Brigham and Women's Hospital, and the Fenway Community Heath Care Center in Boston. All individuals gave written informed consent to participate, and the study was approved by the respective institutional review boards and conducted in accordance with the human experimentation guidelines of the Massachusetts General Hospital. Table 1 Characteristics of the Study Participants a All patients were males. All individuals were White Americans except AC-45, who was Hispanic b ELISA and Western blot positive, but infection within 180 d from time therapy was initiated Ind, indetermined; NA, not applicable; neg, negative; pos, positive Six of the 14 individuals were investigated in an interim study [ 10 ]. These patients were AC-02 (AS2 in [ 10 ]), AC-05 (AS5), AC-14 (AS1), AC-15 (AS3), AC-25 (AS6), and AC-46 (AS4). STIs Entry criteria included treatment with HAART before or shortly after HIV-1 seroconversion, viral suppression on HAART to less than 400 RNA copies/ml for 2 mo, HIV-1 viral load at the time of entry into the study of less than 50 RNA copies/ml, and lack of significant mutations conferring drug resistance [ 11 , 12 ]. Lymphocyte proliferative response to recombinant HIV-1 p24 protein had to exceed a stimulation index of ten before treatment discontinuation, and net counts per minute had to be 800 or greater. All antiretroviral drugs were discontinued simultaneously. After a treatment interruption, patients were seen at least once a week for the first 24 wk, and then monthly, with a total of at least 30 visits for the first year after cessation of therapy. In the second year, visits continued monthly. Treatment was restarted if viral load remained above 5,000 RNA copies/ml plasma for greater than three consecutive weeks, or was in excess of 50,000 copies/ml on any single occasion. Human Leukocyte Antigen Typing High- and intermediate-resolution human leukocyte antigen class I typing was performed at a commercial laboratory (Dynal Biotech, Oxford, United Kingdom) by sequence-specific PCR as described [ 13 ]. Detection of GB Virus C RNA GB virus C (GBV-C) RNA was detected using a two-step nested PCR amplification reaction from whole plasma RNA. Briefly, GBV-C RNA was extracted from plasma using the Qiagen Viral RNA Mini Kit (Qiagen, Valencia, California, United States) according to the manufacturer's instructions. Extracted RNA was reverse transcribed using the Qiagen OneStep RT-PCR Kit and amplified by nested PCR; in both steps primers specific for the 5′ UTR of GBV-C were used, as described previously [ 14 ]. Chemokine Receptor Genotyping In order to analyze the chemokine receptor (CCR) 5Δ32 deletion polymorphism, the region spanning the 32-nt deletion was amplified by PCR, and the two alleles were separated by gel electrophoresis [ 15 ]. The CCR2–64I polymorphism was detected by PCR–restriction fragment length polymorphism analysis as described previously [ 16 ]. Synthetic HIV-1 Peptides We synthesized 410 synthetic peptides 15–20 amino acids long at the Massachusetts General Hospital Peptide Core Facility on an automated peptide synthesizer using Fmoc technology, as described [ 17 ]. Peptides overlapped by 10 amino acids and spanned the entire HIV-1 clade B 2001 consensus sequence. ELISPOT Assays ELISPOT assays were carried out as described previously [ 18 ]. Peripheral blood mononuclear cells (PBMCs) were incubated overnight at 50,000 to 100,000 cells/well in 96-well polyvinylidene plates that had been precoated with 0.5 μg/ml anti-human interferon-γ monoclonal antibody (Mabtech, Stockholm, Sweden). The final concentration of the peptide per well was 14 μg/ml. The numbers of spots per well were counted using an automated ELISPOT plate reader (AID EliSpot reader system, Autoimmune Diagnostika, Strassberg, Germany). A response was considered positive if there were more than 50 spot-forming cells (SFCs)/10 6 PBMCs and if the well had at least three times the mean number of SFCs in the three control wells. The dependence of responses on CD8+ T cells was determined by measuring the depletion of CD4+ T cells using the Minimacs cell depletion system (Miltenyi Biotec, Bergisch-Gladbach, Germany). When HIV-1-specific CD8+ T cell responses were detected against adjacent peptides, and therefore might represent targeting of the overlap region, responses to the weaker peptide were excluded for calculations of magnitude and breadth, as previously described [ 19 ]. Proliferation Assays Freshly isolated PBMCs (10 5 cells) were incubated with baculovirus-derived recombinant p24 protein (Protein Sciences, Meriden, Connecticut, United States) at 5 μg/ml for 6 d and then pulsed with 3 H thymidine at 1.0 μCi for 6 h before harvesting as previously described [ 20 ]. A stimulation index of five or greater was considered significant. Statistical Analysis Time to failure during the different treatment interruptions was assessed by Kaplan-Meier analysis. Patients who were still controlling viremia at the time of last visit, who failed because they restarted therapy without meeting the criteria of virologic failure, or who were lost in follow-up were included in the analyses, but the data were censored at the last evaluable time point. Equality of survival distributions for the first and second treatment discontinuations was evaluated using the Wilcoxon matched-pairs signed-ranks test. CD4+ T cell losses were calculated on regression lines based on least squares fit. A Cox proportional hazards regression model was used for the analysis of continuous variables such as days following onset of symptoms, CD4+ T cell count, HIV viral load, and time to rebound of viremia, as well as for the estimation of hazard ratios for the categorical variables of ELISA, Western Blot, coreceptor polymorphism, and GBV-C status. Statistical analyses of CD8+ and CD4+ T cell responses were based on a Student's t test, a multiparametric ANOVA test, a Wilcoxon matched-pairs signed-ranks test, or a Mann-Whitney U test, as indicated. p -values lower than 0.05 were considered to indicate statistical significance, and all reported p -values are two-sided. Statistical analysis and graphical presentation were performed using SPSS, SAS, and Prism software packages. Results Longitudinal Assessment of Control of Viremia following Treated Acute or Early Infection Fourteen patients identified at the time of acute or early infection ( Table 1 ; Figure 1 ) were entered into this protocol, and they were followed for a median of 5.3 y from the time of infection (range, 494–2,475 d). Patients underwent successive treatment interruptions after an initial treatment period of at least 8 mo (median, 508 d; range, 245–1,096 d) and were required to restart therapy when viral load exceeded 50,000 RNA copies/ml plasma on a single occasion, or 5,000 copies/ml for three consecutive weeks. For purposes of analysis, patients who dropped out of the study or who reinitiated therapy without meeting criteria were considered to have lost the ability to contain viremia. Figure 1 HIV-1 Viral Loads and CD4+ T Cell Counts in the 14 Study Participants Time zero corresponds to first institution of highly active antiretroviral therapy (HAART). Closed squares, HIV-1 plasma viral loads; open circles, CD4+ T cell counts; shaded areas, treatment with HAART according to protocol; diagonally shaded areas, patient restarted therapy without meeting criteria of virological failure; vertical dotted lines, virological failure without reinstitution of HAART. Patients are ordered by increasing number of supervised treatment interruptions. Using these criteria for reinitiation of therapy and to define failure, 11 of 14 patients (79%) were able to achieve virologic control to less than 5,000 RNA copies/ml plasma for at least 90 d after one, two, or three treatment interruptions ( Table 2 ). The period of longest containment was after one interruption for five patients, after two interruptions for eight patients, and after three interruptions for one patient ( Table 3 ). Table 2 Period of Viral Control Achieved Off Therapy Table 3 Time to Failure during the STIs a Numbers correspond to time until virological failure, unless otherwise specified Red indicates the longest time off therapy until failure or last follow-up b Last follow-up visit; patients still meeting criteria of virological control c Failure because patient restarted antiviral therapy without meeting criteria of virological failure d Virological failure due to HIV-1 superinfection Once control was achieved, the majority of the patients experienced a subsequent rise in viremia. The median time between cessation of therapy and rebound of viremia (having a viral load greater than 50 copies/ml) was 17 d (range, 7–169 d). Six of 14 patients (43%) achieved a period of control after stopping therapy for 1 y, but only three of 14 (21%) were able to control viremia off therapy at less than 5,000 RNA copies/ml plasma for more than 2 y. Duration of viremia control during successive treatment interruptions was highly variable, and there was no increase in the sustainability of viral containment during successive STI cycles. The three patients achieving control of viremia for more than 2 y did so during the first (AC-10), the second (AC-02), and the third (AC-14) treatment interruption, respectively ( Figure 2 A). A paired comparison (Wilcoxon matched-pairs signed-ranks test) showed no significant difference in the length of viremia control with subsequent treatment interruptions. Although patients experienced rebound viremia with discontinuation of therapy, none of the patients experienced recurrence of symptoms associated with acute HIV-1 infection. Figure 2 Evolution of Viral Load and CD4+ T Cell Counts during STI (A) Survival curves of time to virologic failure during the first three supervised treatment interruptions. Virologic failure was defined as having a viral load of greater than 5,000 copies RNA/ml plasma for 3 wk or greater than 50,000 copies once. Patients still achieving viral control at the last visit and individuals restarting therapy without meeting criteria or lost in follow-up are censored at the last evaluable time point. The horizontal axis represents the time off therapy since the beginning of the treatment interruption, the vertical axis corresponds to the number of patients maintaining control of viremia. The curves for first, second, and third STIs do not differ significantly from each other (log-rank test, p > 0.05). (B) Evolution of CD4+ T cell counts during the longest treatment interruption. Slopes of CD4+ T cell counts during the first year of the longest treatment interruption are shown for patients who experienced a cessation of therapy of at least 12 mo (all except AC13, AC25, and AC45), compared to the natural decline of CD4+ T cell counts in untreated patients of the MACS cohort with early chronic HIV-1 infection (CD4+ counts of >350 cells/mm 3 ). CD4+ T cell losses were calculated on a regression line based on least squares fit. The two groups differed significantly from each other (Mann-Whitney U test, p = 0.02). (C) CD4+ T cell count at intercept and CD4+ T cell slopes during the longest treatment interruption. The CD4+ T cell slopes of the same 11 patients shown in (B) and of untreated patients of the MACS cohort are represented according to the CD4+ T cell count at the intercept of the regression line based on least squares fit with the vertical axis (day 0 of treatment interruption). These data show that at least transient control of viremia to less than 5,000 RNA copies/ml plasma was achieved in the majority of study participants during at least one of the treatment interruptions, but that durable viral control in participants following treated acute infection occurred infrequently. Moreover, the data do not show a consistent pattern of augmentation of viral control with sequential treatment interruptions. Effect of Treatment Interruptions on CD4+ T Cell Counts Although viral load is a strong predictor of disease progression, CD4+ T cell loss is an additional, independent predictor [ 21 ]. Early treatment of acute HIV-1 infection led to normalization of CD4+ T cell counts in most patients (median, 753 cells/mm 3 ; range, 492–986), but the effect of treatment interruption was variable, even in those doing well, as defined by sustained low viral loads. Overall, 11 of 14 patients interrupted therapy for at least 12 mo, and these individuals were evaluated regarding the effect of treatment interruption on CD4+ T cell loss ( Figure 2 B and 2 C). The rate of change in CD4+ T cell counts during the first year of the longest period off treatment ranged from +157 to −438 cells/mm 3 /y (median, −192). Of the three patients who did not meet viral load criteria for restarting therapy for more than 2 y, one (AC-02) had an increasing CD4+ T cell count of 157 cells/mm 3 /y, one (AC-10) had a stable CD4+ T cell count (−9 cells/mm 3 /y) , and one (AC-14) experienced a decline of 344 cells/mm 3 /y. Comparison with data from the Multicenter AIDS Cohort Study (MACS) showed that the kinetics of CD4+ T cell loss was faster (Mann-Whitney U test , p = 0.02) than in untreated patients with early chronic HIV-1 infection (average loss of −67 cells/mm 3 /y in patients with a CD4+ T cell count of more than 350 cells/mm 3 at baseline). However, CD4+ T cell loss rate was in the same range as what has been described after treatment interruption in chronic HIV-1 infection [ 22 , 23 ]. Analysis of CD4+ T cell decline during the second year for the three individuals who controlled viremia for more than 2 y revealed similar trends in CD4+ T cell slopes, although they were less steep: AC-02, +88 cells/mm 3 /y; AC-10, +44 cells/mm 3 /y; and AC-14, −110 cells/mm 3 /y. When the first 3 mo off therapy were excluded in order to minimize the potential effects of recent treatment on CD4+ T cell number, the rate of change in CD4+ T cell counts during the first year off therapy no longer differed statistically from the MACS data (median, −207 cell/mm 3 /y; range, +119 to −699; Mann-Whitney U test, p = 0.07). A possible reason for steep CD4+ T cell slopes may be high CD4+ T cell counts at time of treatment interruption. Comparison with MACS data ( Figure 2 C) showed that several of the study participants still behaved as outliers when this factor was considered. These results indicate that periods of relative control of viremia were associated with declining CD4+ T cell counts in most patients. Correlation of Clinical and Genetic Markers with Duration of Viremia Control Although the study was small, we evaluated clinical and laboratory parameters to see if any was predictive of duration of viral control. Analyses included clinical and laboratory parameters at time of presentation with acute HIV-1 infection, genetic markers associated with different rates of disease progression, and the presence or absence of GBV-C coinfection. All patients presented with symptomatic acute infection. Time between onset of symptoms and institution of therapy did not affect duration of control following STI (Cox proportional hazards regression model, p > 0.05). The individuals who controlled viremia for a longer time either during the first STI or during any of the treatment interruptions were not different from those who experienced earlier breakthrough as measured by ELISA and Western blot status at initiation of HAART, coreceptor polymorphisms (CCR5delta32, CCR2 V64I), or the presence or absence of GBV-C coinfection (Cox proportional hazards model, p > 0.05 in all comparisons; data not shown). The only parameter that was predictive of prolonged viral control during the first treatment interruption was a low viremia at time of institution of therapy ( p = 0.01): there was a 2.8-fold increase in hazard per order of magnitude increase in viral load. This factor was no longer predictive when the period of longest control of viremia was considered. The time to rebound of viremia (>50 copies/ml or >400 copies/ml) did not correlate with the duration of viral control. Although 11 out of 14 individuals achieved at least transient control of viremia, and three experienced prolonged control, none of these patients possessed the HLA alleles B27 or B57 associated with better disease outcome [ 24 , 25 ]. Relationship of Magnitude and Breadth of HIV-1-Specific CD8+ T Cells to Duration of Viremia Control To assess the relationship between the clinical outcome and evolution of HIV-1-specific CD8+ T cells, we longitudinally analyzed the breadth and magnitude of CD8+ T cell responses using an interferon-γ ELISPOT and a panel of 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence. At the beginning of the first STI, HIV-1-specific CD8+ T cells were weak (median of 590 SFCs/10 6 PBMCs) ( Figure 3 A) and narrowly directed at a median of two epitopes ( Figure 3 B). CD8+ T cell responses increased significantly ( p < 0.05) during the first off-treatment period, reaching a median total magnitude of 2,725 SFCs/10 6 PBMCs and targeting a median of eight epitopes, and then were sustained when therapy was reintroduced. A further increase in the magnitude and breadth of HIV-1-specific CD8+ T cells was observed in the subsequent off-treatment periods, although these augmentations failed to reach statistical significance. The CD8+ T cell–mediated immune responses emerging during these consecutive cycles of treatment interruption were broadly directed, targeting all structural and most accessory and regulatory HIV-1 gene products (data not shown). However, the magnitude of HIV-1-specific CD8+ T cell responses at the beginning of the first ( r = 0.01, p = 0.76), second ( r = 0.16, p = 0.54), or third ( r = 0.1, p = 0.55) treatment interruptions was not predictive of the time the study participants were subsequently able to stay off therapy according to study criteria. Figure 3 Evolution of HIV-1-Specific CD4+ and CD8+ T Cell Responses during STI (A–D) Magnitude and breadth of increase of HIV-specific CD8+ T cells during supervised treatment interruptions. (A and B) Magnitude (A) and breadth (B) of HIV-specific CD8+ responses at the first day of treatment interruption (black bars) and at the last day off therapy (white bars). Data represent the mean and standard deviation. (C and D) Correlation between the increase of the magnitude (C) or breadth (D) of CD8+ T cell responses and the time off therapy during the first treatment interruption. (E and F) Evolution of CD4+ T helper cell responses during supervised treatment interruptions. (E) Magnitude of CD4 T helper cell responses at baseline and at the first day of treatment interruption (closed circles) and last day off therapy (open circles). Horizontal bars correspond to median values. An stimulation index greater than five was considered significant. (F) Correlation between the magnitude of p24-specific lymphocyte proliferative responses at the beginning of the first treatment interruption and the time patients were able to remain off therapy during the subsequent STI. The periods off treatment allowed for assessment of the relationship between exposure to virus and evolution of immune responses. There was a highly significant positive association between time until virologic failure during the first treatment interruption and change in the magnitude of HIV-1-specific CD8+ T cell responses ( r = 0.92, p < 0.001) ( Figure 3 C). Similarly, the longer a patient remained off therapy during the second and third interruptions, the greater the augmentation of the total magnitude of HIV-1-specific CD8+ T cell responses ( r = 0.83, p < 0.016; r = 0.74, p = 0.05, respectively). The increase in CD8+ T cell epitopes targeted during the first treatment interruption was also linearly correlated to the duration until virological failure ( r = 0.81, p < 0.001) ( Figure 3 D). However, no significant relationship was observed between the augmentation of epitopes targeted during the second and third treatment pauses and the time the study participants were able to remain off therapy in the respective treatment interruption. These data suggest that the duration of a treatment interruption, and therefore the duration of exposure to plasma virus, correlates positively with the magnitude and breadth of HIV-1-specific CD8+ T cell responses that emerge during off-therapy time periods. Yet, CD8+ T cell responses prior to treatment interruptions were not significantly predictive of the duration of time that patients are able to spontaneously control HIV-1 replication, as defined by the study criteria. Relationship of Magnitude of Lymphocyte Proliferative Responses to p24 Antigen to Duration of Viremia Control We next analyzed evolution of lymphoproliferative responses to recombinant HIV-1 p24 Gag protein in order to assess HIV-1-specific CD4+ T cell function. Most individuals had no detectable response at baseline prior to treatment, consistent with prior reports of patients with acute HIV-1 infection [ 20 ]. After initiation of therapy, all individuals generated HIV-1-specific lymphoproliferative responses ( Figure 3 E), which was a criterion for inclusion in the study. During treatment interruptions, there was a variable decline in magnitude, and comparisons between responses on the first day of treatment interruption and last day off therapy did not reach statistical significance (first STI, p = 0.72; second, p = 0.12; and third, p = 0.60, respectively). These HIV-1-specific CD4+ T cell responses also tended to rise with reinitiation of therapy, and some of them were very robust, with stimulation indices over 50 detected in several individuals ( Figure 3 E). Similar to CD8+ T cell responses, the magnitude of HIV-1-specific CD4+ T helper cell responses at the beginning of the first ( r = 0.05, p = 0.43) ( Figure 3 F), second ( r = 0.16, p = 0.54), or third ( r = 0.1, p = 0.55) treatment interruption was not statistically predictive of the time the study participants were subsequently able to stay off therapy according to study criteria. Discussion Although early treatment of acute HIV-1 infection followed by treatment interruptions may enhance control of viremia [ 6 , 8 ], the durability of this control remains unclear. Here we analyzed the long-term impact of initiation of antiviral therapy during acute HIV-1 infection followed by STIs in a cohort of 14 patients. Although initial control of viremia to less than 5,000 RNA copies/ml plasma was achieved in the majority of the individuals studied, a gradual increase in viremia and decline in CD4+ T cell counts was observed in most patients, even after a year or more of viral containment. Durable virologic control occurred infrequently, despite the presence of robust HIV-1-specific CD4+ and CD8+ T cell responses detected by standard assays. Moreover, even during periods of successful control of viremia, progressive loss of CD4+ T cells was frequently observed. These data indicate that although early treatment of acute and early infection is frequently associated with transient control of viremia after STI, ongoing low-level viral replication is associated with ultimate virologic breakthrough in most patients. The standard immunologic assays and virologic assessments in this cohort revealed considerable heterogeneity among the study participants, and did not show a consistent pattern in duration of viremia control during successive treatment interruptions. Eleven of 14 patients (79%) were able to maintain a viral load of less than 5,000 copies/ml for at least 90 d, but progressive loss of control ensued in the majority of patients and only three patients (21%) were able to maintain control for more than 2 y. These three patients did so during the first (AC-10), the second (AC-02), and the third (AC-14) STI. Clinical, genetic, and immunological parameters did not distinguish these three individuals from the other 11 patients, nor did they predict the duration of control following treatment interruption. Indeed, the longer a patient was off therapy, the stronger and more broadly directed the CD8+ T cell responses became, but these were still not sufficient to maintain prolonged control in most patients. Although three patients did not complete the study as initially intended (patient AC-45 withdrew from the study after viral breakthrough on the first STI, AC-13 restarted therapy despite a viral load of less than 5,000 copies/ml during both the first and second STIs and then withdrew, and AC-05 restarted therapy prematurely during the second STI but then failed to control during the third STI), the results are not substantially different if these three are censored rather than considered to have failed to control. Loss of viral control in this cohort occurred not only in the presence of strong CD8+ T cell responses, but in most cases also in the presence of virus-specific CD4+ T cell responses, although the CD4+ T cell responses often declined during periods of viremia. In addition, total CD4+ T cell numbers were also monitored and declined in most patients over time, including one of the three patients who were able to maintain low viral loads for at least 2 y. Mechanisms leading to rapid CD4+ T cell loss need to be further studied in future STI trials. Other parameters including chemokine receptor polymorphisms [ 26 ] and GBV-C coinfection [ 27 , 28 ] similarly failed to explain the different courses following treatment interruption. The only parameter found to be associated with longer control of viremia during the first treatment interruption was a lower viral load at time of institution of antiviral therapy. Given the multiplicity of comparisons made, the true significance of this finding is uncertain. The reasons for progressive loss of control despite augmentation of virus-specific CD4+ and CD8+ T cell responses remain to be defined. In one individual (AC-06), HIV-1 superinfection in the setting of strong and broadly directed HIV-specific cellular immune responses was associated with the loss of viral control, as previously reported [ 29 ]. No other cases of superinfection have been identified in these patients (data not shown). The immunologic studies performed failed to show an association between increases in viral load and loss of immune responses, but this may be due to the use of the current standard IFN-γ assays to quantify immune function. Numerous studies now indicate that IFN-γ production alone is not associated with viral load [ 19 , 30 , 31 ] but rather that functional characteristics of CD4+ and CD8+ T cells may be better associated with viral control [ 32 , 33 , 34 , 35 ]. Such studies will be important to pursue. In particular, even a low level of viremia correlates with a low or undetectable frequency of interleukin-2-producing HIV-1-specific memory CD4+ T cells endowed with proliferative capacity in vitro [ 36 , 37 , 38 , 39 ], thus abrogating CD4+ T cell help crucial to maintain efficacy of CD8+ T cell functions. In an interim study of a subset of six of the 14 patients presented here (patients AC-02, AC-05, AC-14, AC-15, AC-25, and AC-46), a fully differentiated effector phenotype of HIV-1-specific CD8+ T cells for selected epitopes was found to be associated with better control of viremia [ 10 ]. Other factors that may contribute include functional defects in antigen-specific cell-mediated immunity [ 35 , 37 , 40 , 41 , 42 ], and progressive immune escape [ 43 , 44 , 45 ]. HIV-1-specific humoral immunity can also affect viral control after treatment interruption [ 46 ], and viral factors including viral fitness [ 47 , 48 ] and infection with multiple viral variants [ 49 ] can influence viral set point and the rate of disease progression. Virus sequencing studies currently in progress in this cohort indicate that viral breakthrough is associated with sequence changes within and outside known CTL epitopes (data not shown). Full evaluation of the relationship between immune escape and viral breakthrough will require extensive additional analyses, including detailed analysis of responses to autologous virus [ 50 , 51 ]. Assessing the changes in CD4+ and CD8+ T cell functions over time as well as viral evolution under immune selection pressure will be important to evaluate immune correlates in this cohort. These data are important in light of other recent data on treatment interruption in both acute and chronic infection. In chronic HIV-1 infection, STI studies showed only marginal, if any, improvements of HIV-1 viremia control following a number of treatment interruptions cycles, despite at least transient increases in HIV-1-specific CD8+ and CD4+ T cell responses [ 4 , 5 , 52 , 53 , 54 , 55 ]. In the setting of infection with a multidrug-resistant virus, this strategy may even be deleterious [ 56 ]. Other studies of STI after treated acute HIV-1 infection have shown limited benefits [ 9 ], including recent trials such as the PrimSTOP trial [ 57 ] and the QUEST study [ 58 ]. However, little is known about the relationship between scheduling of HAART and treatment interruptions and the characteristics of viral rebound after therapy has been discontinued. Although durable control of viremia was not achieved, it is noteworthy that the majority of patients were able to achieve transient relative containment of viremia, providing rationale for future studies aimed at further enhancing immune control. Early treatment alone should still be considered an important therapeutic option. Therapeutic vaccinations administered after treated acute HIV-1 infection and before cessation of therapy have given disappointing results thus far [ 9 ], but the availability of new and more potent immunogens requires reassessment of this approach. Indeed, the ability to enhance CD4+ T helper cell responses in the chronic phase of infection has been demonstrated [ 59 ], but whether this will enhance CD8+ T cell function requires additional studies. Some promising results have been obtained using immunomodulatory drugs, including cyclosporine [ 60 ] and hydroxyurea [ 61 ], in combination with antiviral therapy, presumably because of the limitation of T cell activation. Administration of granulocyte-macrophage colony-stimulating factor blunted the viral rebound following interruption of HAART, and largely prevented a decrease of CD4+ T cell counts in an STI trial in chronic HIV-1 infection [ 62 ]. These additional therapeutic interventions deserve further investigation in future STI studies. Although the present study shows progressive viral breakthrough, it was not designed to address whether there might be a change in set point viremia achieved or overall clinical benefit through transient early treatment of acute HIV infection. The definition of failure chosen for this study was a viral load of greater than 5,000 RNA copies/ml plasma, which at the time the study was initiated corresponded to the level of viremia at which treatment was recommended. Larger randomized trials will be needed to determine the potential clinical and virologic benefit of approaches based on STIs. In studies of untreated infection, there is only a 5-fold difference in viremia separating the quartile with the slowest disease progression from the quartile with the most rapid progression [ 63 ], suggesting that small differences in steady-state viremia may influence clinical outcome. In the meantime, STI probably should be avoided outside the setting of controlled clinical trials. The data in this study may also be relevant to current efforts to develop a therapeutic AIDS vaccine designed to retard disease progression rather than prevent infection, since they suggest that durable maintenance of low-level viremia may be difficult to achieve. Supporting Information Protocol S1 Study Protocol (68 KB DOC). Click here for additional data file. Protocol S2 Protocol Amendment (47 KB PDF). Click here for additional data file. Protocol S3 Patient Consent Form (187 KB PDF). Click here for additional data file. Protocol S4 Institutional Review Board Approval (36 KB PDF). Click here for additional data file. Patient Summary Background Highly active antiretroviral therapy (HAART), which is used to treat patients with HIV, can have nasty side effects and is expensive. As a result, for the last five years scientists have been trying to determine if it is possible to give patients breaks from taking antiretroviral drugs, without patients' health suffering. It is likely that such a treatment strategy (called “supervised treatment interruption,” or STI) will not work in patients who have been infected with HIV for a long time. However, until now, the jury was out about whether STI could be of benefit to patients who had only recently been infected with HIV. Some research suggested that if newly infected patients had short “holidays” from taking HAART, it might help to boost their immune system—which in turn might help to keep HIV at bay. What Did the Researchers Do and Find? The researchers studied 14 patients who had recently been infected with HIV. Patients were treated until their viral load was below the limit of detection by using a very sensitive method. As they were then watched after treatment interruption, most of them were able to maintain a low, though detectable, viral load for some time. The researchers then stopped the treatment and carefully watched the patients over a period of up to five years. If the number of viruses in the patients' blood rose too high, the researchers gave the patients HAART again until the number of viruses fell again. In about half the patients, treatment needed to be restarted within a year because the viruses had started becoming more numerous in the blood. The doctors had hoped that it would take much longer for the number of viruses to reach this level. In other words, giving patients “drug holidays” did not help to keep the virus at bay for long periods of time. What Are the Limitations of the Study? It is relatively rare for doctors to be able to diagnose HIV shortly after an individual becomes infected with the virus. Many people have HIV for a long time before they see a doctor. Therefore, even if the STI strategy had worked, it would only have been relevant to a few patients. In addition, the study was small and preliminary, so we have to be careful about reading too much into the results. A limitation in the study is that it did not address whether there might be a long-term clinical benefit despite a gradual increase in viral load. What Does This Study Mean for Patients? It is important to remember that this was an experimental trial—patients with HIV should not stop taking antiretroviral drugs unless their doctor specifically tells them to do so. Although it looks as though STI may not work in the way everyone had hoped, these results may help scientists develop an HIV vaccine that is designed to keep the disease at bay. Whether early treatment of acute infection has an overall benefit in terms of time-to-development of AIDS or need for long-term treatment after drug discontinuation will need to be answered in larger clinical trials designed to answer these important questions. Resources on the Web. AIDSinfo: http://www.aidsinfo.nih.gov/ AIDSmap: http://www.aidsmap.com/ Medline Plus AIDS Information: http://www.nlm.nih.gov/medlineplus/aids.html

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HIV, malaria and beyond: reducing the disease burden of female adolescents

In sub-Saharan Africa the highest overlap between malaria and HIV infections occurs in female adolescents. Yet control activities for these infections are directed to different target groups, using disparate channels. This reflects the lack of priority given to adolescents and the absence of an accepted framework for delivering health and health-related interventions to this high-risk group. In this paper it is argued that female adolescents require a continuum of care for malaria and HIV – prior to conception, during and after pregnancy and that this should be provided through adolescent services. The evidence for this conclusion is presented. A number of African countries are commencing to formulate and implement adolescent-friendly policies and services and disease control programs for malaria and HIV will need to locate their interventions within such programs to ensure widespread coverage of this important target group. Failure to prioritize adolescent health in this way will seriously limit the success of disease control programs for malaria and HIV prevention.

Background The current generation of adolescents – over one billion – is the largest in history [ 1 ] and, far from representing a picture of health [ 2 ], many will suffer untimely disease and death. HIV and malaria are responsible for much of the disease burden affecting female adolescents, who suffer disproportionately from these combined infections relative to other age groups. This is due to a high HIV incidence during the period when many adolescents become pregnant for the first time – an event which greatly increases their susceptibility to Plasmodium falciparum malaria [ 3 ]. Biological interactions between malaria and HIV in pregnancy complicate therapy, which can also be compromised by inappropriate adolescent health-seeking behaviour. The public health importance of HIV and malaria synergism is only now emerging. It has led to a recommendation by the World Health Organization (WHO) that the two disease programs should collaborate to ensure integrated service delivery, especially within the framework of reproductive health services [ 4 ]. A case has also been made for linking disease control programs as a more efficient and effective way for lowering the malaria and HIV disease burden [ 5 ]. Nevertheless, it is difficult to seen how these goals will be attained unless adolescents are accorded a much higher priority by disease control programs. In this paper we consider the justification, as well as the challenges, of using common approaches to improve delivery of HIV and malaria interventions to female adolescents. HIV and malaria burden in pregnant and non-pregnant adolescents Prevalence estimates for HIV infection [ 1 ] for 35 sub-Saharan African countries, for young males and females (15–24 years), are shown in Figure 1 . Estimations are often derived from sentinel surveillance of women attending antenatal care (ANC) and tend to overestimate prevalence in adolescents due to the selection of sexually active [ 6 ] and less-educated groups [ 7 ]. Accepting this bias, in every country, listed HIV prevalence is two to three times higher among females than males. Having an older male partner is associated with a modest increased HIV risk [ 8 ] but probably does not explain a sex difference that occurs uniformly across all countries and cultures and at all levels of HIV endemicity. In Rakai, Uganda, only 12.4% of HIV cases in 15–19 year olds was attributed to relationships with men 10 years older [ 9 ]. Other sexually transmitted infections (STIs) increase susceptibility to HIV but do not account for the sex difference [ 10 ]. Host biological factors may be critical [ 11 ]. Sexual maturation takes several years to complete, but menarche is delayed amongst socio-disadvantaged girls, and many will be sexually active before biological maturation is complete. Interventions that delay the onset of sexual activity could reduce exposure at a time of peak biological susceptibility to HIV and highest risk of malaria in pregnancy. Figure 1 Female and male HIV prevalence among young people 15–24 years of age in sub-Saharan Africa. Compiled from UNFPA 1 Malaria is an important cause of adolescent hospital admissions in many sub-Saharan African countries with stable malaria transmission [ 12 ]. Malaria mortality in African adolescents (both sexes) aged 10–14 years has been estimated at over 45,000 deaths per annum [ 13 ]. The highest prevalence of P. falciparum infection after childhood is also in young, mostly adolescent, women. Data from Malawi (Table 1 ) shows that non-pregnant and pregnant adolescents had significantly higher parasite rates than women above 19 years. Primigravidae also have a markedly increased susceptibility to malaria [ 3 ]. Young age and nulliparity in the same individual lead to greatly increased malaria risk. This partly relates to reduced acquisition of acquired malaria immunity in young individuals [ 14 ] as well as the lack of parity-specific malaria immunity acquired during the first pregnancy [ 15 ]. Risk factors for symptomatic malaria in adolescents have not been studied, but both symptomatic and asymptomatic malaria infection will contribute to the development of adolescent and newborn anaemia [ 16 ]. HIV infection increases P. falciparum prevalence during pregnancy and this is marked during the first pregnancy [ 17 , 18 ], which in Africa, is nearly always in adolescence. Table 1 Malaria prevalence in non-pregnant and pregnant adolescents and adults in the Shire Valley, Malawi Adolescent Adult Non-Pregnant Pregnant * Non-Pregnant Pregnant* % (n) % (n) % (n) % (n) 41.4(29)† 45.9 (122)‡ 20.3 (74) 35.4 (345) * At delivery. Includes women who had received anti-malarials during pregnancy † Difference with non-pregnant adults χ 2 = 4.8; p = 0.028 ‡ Difference with pregnant adults χ 2 = 4.3; p = 0.04 HIV and malaria interventions during adolescence For both HIV and malaria there are effective interventions for disease control. HIV and malaria frequently occur in the same African populations and the same individuals, yet control activities are directed to different target groups, using different channels. Adolescent HIV prevention, with its focus on behavioural change, has taken place largely outside conventional health settings. Few HIV strategies have been directed at pregnant adolescents and/or their partners – an omission noted by a WHO Technical Consultation on married adolescents [ 19 ]. In some African countries, by the age of 19 years, half of all female adolescents were in marital or permanent consensual unions [ 9 ] that will almost certainly lead to pregnancy. In contrast, malaria control efforts for women have focused on pregnancy, but not on adolescents and certainly not on adolescents before they become pregnant. As a result, in many sub-Saharan African settings, adolescents are often parasitaemic and anaemic when they first become pregnant. The problem is that currently no overall strategy for adolescent health has been approved for most African countries. The burden of infection and disease among adolescents is of sufficient magnitude that a way must be found to provide services to this population. The existence of an appropriate strategy would allow both HIV and malaria interventions to be delivered within a common framework. In traditional cultures, menarche often marks the transition from childhood to adulthood because it signifies reproductive potential. Nevertheless, adolescence is a physical process that takes a number of years and is usually completed by the late teens. This developmental process should be encompassed within health care provision. Pregnancy may punctuate that process, but during it, the adolescent continues to grow physically and mentally [ 20 ]. Strategies to reduce adolescent malaria or HIV should, therefore, provide a continuum of care – before conception, during and after pregnancy – encompassing appropriate information, preventive and curative services. For prevention, adolescents need access to condoms and bednets, information on how to use them and, when and where to go to deal with infection. Services must be available for infection monitoring and treatment, including HIV testing and drug therapy for malaria, anaemia and HIV-related opportunistic infections. System improvements may not be strictly about adolescent health services, but about support for ANC. Adolescents need good access to pregnancy care and this includes attendance for early pregnancy assessment and screening for HIV and other sexually transmitted infections (STIs). ANC presents an opportunity for HIV prevention, support for discordant couples and care and referral for HIV-infected adolescents. Early assessment is important as the HIV positive individual needs to plan for anti-retroviral treatment (ART) at delivery to prevent mother-to-child transmission. It is also important for malaria control in pregnancy. Intermittent preventive antimalarial treatment (IPT) is the key malaria prevention policy that improves birthweight outcomes and reduces pregnancy anaemia [ 21 ]. Its effectiveness depends on the prevalence of HIV infection [ 22 ]. It also depends on adherence to the treatment regimen. Coverage with the standard two dose intermittent sulphadoxine-pyrimethamine preventive malaria treatment was lower in younger adolescents in the Shire Valley, Malawi ≤ 17 years, 35.1% versus 18–19 years, 53.7%; p = 0.028, Verhoeff, personal communication), and more than half of all pregnant adolescents received inadequate antimalarial treatment. This demonstrates the importance of pre-conceptual malaria health education for girls. Adolescent mothers need instruction on how to recognize and respond to infant malaria infection and encouragement for using bednets for themselves and their babies. A major challenge for malaria control is to improve adolescent health prior to the first pregnancy. For non-pregnant adolescents there are several conundrums. On account of drug resistance, malaria combination therapy with artemisinin derivatives is being introduced into an increasing number of sub-Saharan African countries. Artemisinin may be foeto-toxic and should be avoided, except for symptomatic infections with multi-drug resistant malaria, in the first trimester of pregnancy [ 23 ]. Inadvertent use or self-treatment by adolescent girls could frequently occur in early pregnancy, before they recognise their pregnancy. The issue of pregnancy testing adolescent girls before treatment with potentially foeto-toxic drugs will need to be considered. A further issue is that the benefits of IPT extend to pregnant but not nulliparous adolescents who may be anaemic or parasitaemic at the start of their pregnancy. The use of permethrin-treated bednets would protect against anaemia and parasitaemia during the first trimester, but adolescent pregnant girls and their newborns are the least likely to be net users [ 24 ]. Reaching the target population The growing literature on adolescent-friendly health services shows that adolescents have substantial concerns about the delivery of interventions through health services [ 25 ]. Almost universally, adolescents complain of lack of information, confidentiality issues and judgmental attitudes of service providers. There may be barriers of physical inaccessibility, service fees and non-supportive community attitudes. A WHO consultation in Africa concluded that adolescents had "a right to access health services that can protect them from HIV/AIDS and from other threats to their health and well-being, and that these services should be made adolescent friendly" [ 26 ]. A WHO Global Consultation on Adolescent Friendly Health Services stated that government ministries should take appropriate action to ensure service provision for adolescents, taking into account cost, epidemiological factors and national health priorities [ 27 ]. The role of non-governmental organizations and the private sector in spearheading adolescent projects was acknowledged, but alone, these organizations cannot obtain sufficient coverage of the adolescent population to substantially reduce the HIV and malaria burden. Governments and disease control programs must also consider training health care providers and modifying service delivery, so as to reduce barriers to adolescent uptake. The only country in Africa that has scaled up adolescent health services is South Africa [ 28 ]. The South African National Adolescent-Friendly Clinic Initiative has strengthened the public health sector's ability to respond to adolescent health needs by building capacity and establishing good clinical services which, eventually, will be maintained by district and provincial health authorities. It has demonstrated that an adolescent strategy is achievable if there is political will. The South African approach may not be suitable or affordable for lower income countries. Well-focused services, training of key staff at selected sites, outreach preventive services and good quality, adolescent-oriented ANC are potential service reconfigurations that could be used to extend coverage and increase uptake of both HIV and malaria interventions without creating a separate adolescent service. What about young men? Available evidence suggests that young men are not attracted to clinic services which are perceived as "female "spaces [ 29 ]. In Ghana, for example, 76–89% of all adolescent clinic users were female [ 30 ]. Malaria is a reproductive health issue for females, but not for males, and the disease burden of both malaria and HIV infections is much lower in young males. Adolescent females require more health-service based care than males, and this should be reflected in service configuration. Creating an enabling environment to promote adolescent access to HIV and malaria interventions Behavioural change, including appropriate health-seeking practices, is essential for disease reduction. A decline in HIV prevalence amongst adolescents has been reported from Zambia [ 7 ], Uganda [ 31 , 32 ] and Tanzania [ 33 ] and is attributed to changes in sexual behaviour. In Zambia, between 1996 and 1999, urban adolescent males reported less frequent sexual activity, fewer partners and were more likely to have used a condom at the last sexual encounter. Changes in these indices were less marked, or even deteriorated, among rural female adolescents [ 7 ]. Whether behaviour change is the result of HIV programme interventions or other factors is unclear. Recent surveys in sub-Saharan Africa reported that higher female education was associated with lower HIV prevalence and lack of schooling with higher HIV incidence [ 6 ] and lower condom use [ 34 ]. In western Uganda the proportion of illiterate women fell from 41.6% to 24.6% over six years and may have facilitated behavioural changes, but this link has been little investigated. Illiterate females are likely to be young and married, and to be in a situation where they cannot reduce frequency of intercourse, negotiate condom use or delay having children [ 19 ]. In a large pregnancy study in southern Malawi, 73% of 469 nulliparous adolescents were illiterate [ 35 ]. They made significantly fewer ANC visits, were less likely to have a supervised delivery and had a higher risk of low birthweight. Reducing illiteracy and increasing educational and vocational opportunities for female adolescents may be critical enabling actions to promote risk reduction, to enhance their social status and to decrease female vulnerability. [ 36 ] Developing policies to support HIV and malaria control for female adolescents Few developing countries have well-defined policies for delivering care to the non-pregnant or pregnant adolescent, especially for sexual health. Existing malaria guidelines also do not mention adolescents specifically [ 12 ]. An important aspect is the issue of the adolescent's right to sexual and reproductive information and services vis à vis the legitimate rights of parents to act in the best interests of a child, and the health care provider's right to work within the law. These are issues of consent and confidentiality for adolescents considered "minors" and under parental control, or wives under their husband's or husband's family's jurisdiction. When these issues are ignored, adolescents cannot be assured of confidentiality, and health workers are neither bound, nor protected by, the legal system. Rights established in law, or by signed Conventions (eg Convention of the Rights of the Child), may not be implemented. Health policy falls down because it is not explicit about the vulnerability of female adolescents and does not challenge cultural and social norms that can perpetuate the disease burden. Similarly, encouragement for improving female literacy and education may be lacking, reducing the supportive environment for promoting uptake of adolescent health services and safer sex. Advocacy is essential to co-ordinate law, custom and health practice. At another level, it is important to acknowledge that interventions for female adolescents raise ethical issues that are avoided when they are excluded from care. The policy of avoiding treatment of adolescent girls (and adult women) because they might be in the first trimester of pregnancy, raises issues of gender discrimination and has been a serious issue for parasitic disease control programs [ 37 ]. Reducing the necessity for first trimester treatments for malaria can be achieved by increasing efforts to redress the malaria burden prior to pregnancy, but this requires reconfiguring service delivery. HIV-positive adolescents who are at the start of their sexual and reproductive lives might be prioritized to receive anti-retroviral therapy, but their relative poverty and lack of social status may lead to their exclusion from access to this intervention. Conclusions For both HIV and malaria, although interventions are available to reduce the disease burden, adolescents are not prioritized. Effective policies and services are needed to reduce the substantial disease burden they experience from these infections. To secure this goal, concerted action across HIV and malaria disease control programs is required. This will reinforce support for adolescent programs rather than weakening them by multiple separate activities, which can confuse implementation. A schedule of activities is required that is reinforced by other systems (e.g. education) to facilitate embedding it into a way of life. These issues need to be addressed head-on by the adolescent health community to achieve the focus needed, and to establish the realistic steps forward. In the short term, developing services to deliver HIV and malaria interventions can act as a catalyst for addressing broader adolescent health service needs. In the medium-term adolescent-friendly health services provide the required platform for the evaluation and delivery of future vaccines and drug therapies for HIV, malaria [ 38 ] and other diseases. In the longer term an inter-sectoral adolescent strategy provides a basis for breaking the cycle of maternal-child ill health. Authors' Contributions Both authors made substantial contributions to the analysis, interpretation, drafting and revision of this article. Both have agreed to authorship.

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Transcriptome analysis of haploid male gametophyte development in Arabidopsis

A transcriptome analysis of male gametophyte development in Arabidopsis uncovers distinct temporal classes of gene expression and opens the door to detailed studies of the regulatory pathways involved.

Background Development of eukaryotic cells towards particular cell fates is regulated by complex and dynamic changes in gene expression. These changes, when monitored on a genome-wide scale, provide a detailed framework for the analysis and modeling of cellular development. To monitor patterns of gene expression it is important to be able to isolate cells at precise stages along a developmental pathway. Well-developed procedures for cell culture and single-cell PCR techniques have allowed genome-wide changes in gene expression to be monitored during animal cell differentiation [ 1 - 4 ]. Transcriptomic studies of single cell types in plants have focused on diploid sporophytic cell types, including undifferentiated cell suspensions [ 5 , 6 ], leaf epidermal and mesophyll cells [ 7 ], stomatal guard cells [ 8 ] and cultured mesophyll cells [ 9 , 10 ]. These studies have provided valuable information about gene expression in single cell types; however, their coverage of the transcriptome has been limited and/or hampered by low RNA yields from individual cells, requiring mRNA preamplification steps that can bias the complementary RNA (cRNA) [ 11 , 12 ]. Moreover, such studies have not involved the use of the most comprehensive tools for monitoring gene expression that are now available for Arabidopsis - which include the Affymetrix ATH1 gene arrays. Recently, a significant advance in transcriptome analysis of plant cell types has been achieved through fluorescence-activated cell sorting of cell-type marked and protoplasted root cells using Affymetrix ATH1 micorarrays [ 13 ]. This has provided a near-comprehensive transcriptomic view of cell-fate determination at three developmental stages in five different domains of the root apex. In contrast to such enabling technologies and procedures developed for sporophytic cell types there have been no studies that provide a genome-wide perspective of cell fate determination and differentiation during haploid gametophyte development. The haploid male gametophyte generation of flowering plants has a simple and well-defined pathway of development and consists of two- or three-celled pollen grains that deliver two sperm cells via the pollen tube to the embryo sac at fertilization. The highly reduced cell lineage and functional specialization of the male gametophyte are thought to be key factors in the reproductive fitness and evolutionary success of flowering plants. Moreover, pollen ontogeny provides an attractive model of cellular development in which to dissect the regulation of cell growth and division, cellular differentiation and intercellular communication (for reviews see [ 14 - 17 ]). Recent progress in understanding of molecular and cellular aspects of pollen development has emerged from genetic studies that have identified mutants in Arabidopsis that affect all phases of pollen development [ 18 - 29 ]. In parallel, cDNA libraries and databanks have been obtained for sperm cells in maize, lily, tobacco and Plumbago zelanica [ 30 - 33 ]. Despite such advances there is limited information about developmental changes in gene expression associated with particular phases of male gametophyte development. Our objective was to develop procedures to enable the isolation of populations of microspores and developing pollen grains at precise developmental stages in Arabidopsis and to analyze changes in gene expression from unicellular microspores to mature differentiated pollen grains. A particular advantage of the male gametophyte generation is that developing microspores and pollen grains are symplastically isolated. This facilitates access to viable cell populations at different stages of haploid development without contaminating sporophytic cells. Some initial progress has been made towards the definition of the male gametophytic transcriptome of Arabidopsis using serial analysis of gene expression (SAGE) [ 34 ] and Affymetrix AG microarrays that harbor probes for approximately 8,000 different genes [ 35 , 36 ]. These studies have provided valuable insight into the complexity of gene expression in mature pollen and the extent of overlap between male gametophytic and sporophytic gene expression (reviewed in [ 37 ]). However, these studies monitored the expression of only 30% of the annotated genes in Arabidopsis and analyzed mRNA populations only in mature differentiated pollen grains. These studies do not, therefore, provide a developmental perspective of gene expression during development and differentiation of the male gametophyte. Here we describe spore isolation procedures for Arabidopsis and the use of Affymetrix ATH1 Genome Arrays to analyze transcript expression profiles throughout four successive stages of male gametophyte development in Arabidopsis . Isolated spore populations were large enough to enable RNA extraction for direct microarray hybridization without any preceding amplification step that could lead to bias in expression signals between stages or between genes within individual stages. Progression from proliferating microspores to terminally differentiated pollen was characterized by large-scale repression and the activation of a unique collection of late-program genes during pollen maturation. Putative male gametophyte-specific genes and distinct clusters of coexpressed genes are identified, including key groups of regulatory factors including cell cycle, transcription and translation factors. Bioinformatic and experimental data are used to address the importance of transcription and translation during pollen germination and tube growth Results Isolation and characterization of developing spores Transcriptome profiling throughout microgametogenesis in Arabidopsis required the introduction of a procedure for the isolation of homogeneous populations of viable spores at precisely defined stages of development. The method was based on centrifugation of isolated mixed spores in a Percoll step-gradient [ 38 , 39 ]. Large homogeneous spore populations at three developmental stages were collected: uninucleate microspores (UNM), bicellular pollen (BCP) and immature tricellular pollen (TCP). In addition, a homogeneous mature pollen grain (MPG) population was isolated from open flowers according to Honys and Twell [ 35 ]. Microscopic examination of isolated spore populations revealed no contaminating sporophytic cells and little or no other cellular debris (Figure 1a ). Vital staining revealed more than 90% viable spores at each stage (data not shown). The purity of spore populations was evaluated by DAPI staining (Figure 1b-e ). The UNM population was the most homogeneous, containing 95% uninucleate microspores, 2.5% tetrads and 2.5% bicellular pollen. The BCP population was 77% pure, but also contained some tetrads (3.5%), microspores (12%) and tricellular grains (7.5%). The TCP population comprised 88% tricellular pollen and 12% bicellular pollen. The MPG population was 100% pure with approximately 2% aborted pollen. Developmental changes in the male gametophytic transcriptome Arabidopsis ATH1 Genome Arrays were used to explore the dynamics of gene expression throughout male gametophyte development in comparison with sporophytic tissues. Microarrays were hybridized with cRNA probes made from total RNA purified from isolated spores. Hybridization data from two biological replicates derived from independently grown populations of plants were compared. Only genes with a positive hybridization signal and a detection call value of 1 in both experiments were scored as expressed. Microarray data from each pair of replicates were highly correlated, with correlation coefficients of 0.986 (UNM), 0.972 (BCP), 0.991 (TCP) and 0.971 (MPG). Complete microarray data are publicly available at the European Arabidopsis Stock Centre (NASC) microarray database [ 40 ]. Sporophytic ATH1 Genome Array datasets were downloaded from the NASC website [ 41 ]. This provided transcriptome data for seedlings at open cotyledon stage (COT, stage 0.7 [ 42 ]), leaves (LEF, stage 6.0), petiole (PET, stage 3.9), stems (STM, stage 6.1), roots (ROT), root hair zone (RHR, stage 1.02), and suspension cell cultures (SUS). Genes that were consistently expressed in replicate sporophytic datasets were identified using the same algorithm used for gametophytic data. We have previously confirmed and validated the expression pattern of 15 putative pollen-specific genes identified using Affymetrix AG arrays by reverse transcription-PCR analysis [ 35 ]. Similarly we validated the current ATH1 datasets by RT-PCR analysis in two separate experiments that included analysis of 41 genes encoding predicted glycosylphosphotidylinositol-anchored proteins (GAPs) [ 21 ] and 16 cation/proton exchanger proteins [ 43 ]. In both experiments the expression patterns of all genes tested that were identified as pollen-expressed, or pollen-specific by ATH1 analysis were confirmed by RT-PCR. The ATH1 Genome Array harbors oligonucleotide probes representing 22,591 genes based on the Arabidopsis Genome Initiative annotation. This represents 80.7% of the most recent estimate of 28,000 protein-coding genes in Arabidopsis [ 44 ]. Of these, 13,977 genes gave a consistently positive expression signal in at least one stage of male gametophyte development, representing 61.9% of the unigene targets on the microarray. The majority of these were expressed in the two earliest developmental stages; 11,565 in microspores and 11,909 in bicellular pollen (Figure 1f ). After pollen mitosis II, there was a sharp decline in the number of diverse transcripts to 8,788 in tricellular pollen and 7,235 in mature pollen. To identify genes expressed preferentially or specifically in developing male gametophytes, hybridization data was compared with sporophytic ATH1 datasets (COT, LEF, PET, STM, ROT and RHR; see Additional data file 1). Transcripts with a consistent positive expression signal in at least one stage of male gametophyte development and a zero signal in any sporophytic dataset were considered male gametophyte-specific. In total, 1,355 specific transcripts were identified, representing 9.7% of the male gametophytic transcriptome. The number of male gametophyte-specific transcripts ranged from 857 (BCP) to 625 (MPG). Thus, in contrast to the decline in the total number of diverse transcripts expressed, the representation of male gametophyte-specific transcripts increased, from 6.9% and 7.2% at UNM and BCP-stages to 8.0% and 8.6% at TCP and MPG-stages respectively. Analysis of the distribution of transcripts among three abundance classes: high (up to 10-fold less than the maximum signal), medium (10- to 100- fold less) and low (more than 100-fold less) (Figure 1f ), revealed a decrease in the proportion of transcripts forming the high-abundance class during development from 20% to 12%. On the contrary, there was sharp increase in the proportion of mRNAs forming the low-abundance class after pollen mitosis II from 4% (UNM) to 14% (MPG). Moreover, 55% of low-abundance transcripts at MPG stage represented repressed mRNAs expressed more abundantly at earlier stages. Thus, the dramatic decrease in the number of transcripts expressed between bicellular and tricellular stages is paralleled by redistribution of mRNA from the high to the low abundance classes. These changes may be associated with reduced cellular activities and cell differentiation processes together with preferential expression of certain classes of genes during pollen maturation. This finding is in accord with the over-representation of cytoskeleton, cell-wall and signaling-related genes that comprise 26% of the high-abundance transcripts at MPG stage. In particular, the average expression signals of cytoskeleton, cell-wall and signaling-related transcripts were increased by 3.1, 3.7 and 2.3-fold, respectively, compared with the UNM stage. Scatter-plot analysis was used to examine in more detail the complexity of the mRNA decline after PMII. The expression levels of individual genes were normalized using a scale of 0 to 100. Genes coexpressed in pairs of datasets were plotted using a logarithmic scale and a correlation coefficient ( R value) calculated (Figure 2 ). There was an extremely high correlation ( R = 0.96) between the transcriptomes of UNM and BCP, the two earliest developmental stages (Figure 2a ). These stages are closely related, with a moderate increase in the expression of a number of genes at BCP stage. The profiles of the two latest developmental stages, TCP and MPG, were also very similar (Figure 2c , R = 0.858), but with greater deviation than the early stages. The scatter plot of TCP and MPG revealed the shift between extreme mRNA abundance classes as described above. This was more evident when bicellular and tricellular stages were compared (Figure 2b ). The scatter of gene expression values and the low correlation ( R = 0.541), provide evidence that the major quantitative shift in transcriptome size between BCP and TCP stages is not simply the result of repression, but also involves the activation of new groups of genes associated with pollen maturation. The lack of correlation ( R = 0.194) between gene expression profiles in uninucleate microspores and mature pollen (Figure 2d ), also reflects the pronounced change in cell status from proliferating microspore to terminally differentiated pollen. The relationship between cell proliferation activities and transcriptome profiles was examined by comparison of early UNM and late MPG stages with a publicly available suspension cell culture dataset. These comparisons demonstrated that the microspore transcriptome was significantly more similar to that of cell suspensions ( R = 0.474) than to mature pollen ( R = 0.194). This is also in accord with the lack of correlation between transcriptome profiles of mature pollen and cell suspensions ( R = 0.13). Co-regulated clusters of gametophytic genes To identify gametophytic genes that may form co-regulated clusters, all 13,977 male gametophyte-expressed genes were hierarchically clustered using EPCLUST clustering and analysis software. Application of a threshold value of 0.05 resulted in the definition of 39 gene clusters covering all phases of male gametophyte development (Figure 3 ; see also Additional data files 1 and 2). Cluster 37 contained 735 early genes (5.3% of all gametophytic genes) with positive expression signals only at UNM stage. Transcriptome data reflect steady-state mRNA profiles that result from the combination of transcription and mRNA turnover rates. In this regard, some transcripts grouped in early cluster 37 may be inherited through meiosis and/or from the tetrad stage. The majority of male gametophyte-expressed genes (52%) were grouped into four clusters (25, 27, 29 and 35) comprising early expressed genes repressed after PMII. Several large gene clusters collectively containing 1,899 genes (13.6%) were associated with pollen maturation. These were activated or upregulated between BCP and TCP stages, forming clusters 5, 7, 11, 13, 18-24, 26, 28, 38 and 39. In contrast, a discrete set of 298 genes forming cluster 17 was upregulated only after TCP stage. In total, 3,342 late genes (24%), forming clusters 1-3, 6, 8 and in particular, cluster 17, encode proteins that are likely to function during post-pollination development. Expression of regulatory genes throughout male gametophyte development We focused our further analysis on three key categories of genes with likely regulatory significance in male gametophyte development; core cell-cycle genes, transcription factors and core translation factors (Figure 4 ). Core cell-cycle genes [ 45 ] were defined according to TAIR [ 46 ]. Genes comprising Arabidopsis transcription factor families were derived by compilation of data available at The Ohio State University Arabidopsis Gene Regulatory Information Server [ 47 ], data published in [ 48 ] and databases homology searches. Recent annotations of the MADS-box and bHLH transcription factor gene families were defined according to [ 49 , 50 ], respectively. Core translation factors [ 51 ] were defined according to TAIR [ 46 ]. Core cell-cycle genes Among 61 core cell-cycle genes, 55 genes were present on the ATH1 GeneChip and 45 (82%) of these were expressed in the male gametophyte (see Additional data file 1). Representative(s) of all families and subfamilies were expressed. The majority of gametophytic core cell-cycle genes showed similar expression profiles (Figure 4a ), with a decline in mRNA abundance after UNM stage to zero (or low levels) at TCP and MPG stages. This pattern is consistent with the termination of proliferation of the microspore and generative cell before pollen maturation. Putative transcription factors We identified 1,594 genes encoding putative transcription factors that were divided into 34 gene families (see Additional data file 1). Their representation on the ATH1 GeneChip was 1,350 (85%). Of these, 608 (45%) were expressed in the male gametophyte, including 54 (15.7%) that were male gametophyte-specific. There were distinct differences in the representation of large transcription factor families (with over 25 members) in the gametophyte. Among those over-represented were the p-coumarate 3-hydroxylase (C3H) family (67% of family members present on the ATH1 GeneChip), the CCAAT family (64%), C2H2 zinc finger proteins (57%), the WRKY family (53%), the bZIP family (51%), the TCP family (50%) and the GRAS family (50%). In contrast, the AUX/IAA (20%), HSF (33%), bHLH (34%), NAC (34%), AP2-EREBP (35%), HB (36%), R2R3-MYB (37%), MADS (37%) and C2C2 zinc finger (37%) gene families were all under-represented. The dominant expression pattern of transcription factor genes reflected the general repression of mRNA diversity between BCP and TCP stages (Figure 4b ). Besides a limited number of constitutively expressed genes, two major transcription factor gene groups could be distinguished. One contained a major group of early-expressed genes and the second a smaller group of genes that were more abundantly expressed late during pollen maturation. The same general tendency was apparent when the profiles of individual transcription factor families were analyzed (exemplified by the C3H family, Figure 4d ). Several gene families comprised predominantly early-expressed genes. These were the NAC, WRKY, TCP, ARF, Aux/IAA, HMG-box and Alfin-like gene families (Figure 4c-e , Additional data file 3). Complete lists of transcription factor gene families and their expression profiles are presented in Additional data files 1 and 3. Core translation factors Among 100 annotated core translation factor genes, 82 were present on the ATH1 GeneChip and 75 (91%) of these were expressed in the male gametophyte (see Additional data file 1). The vast majority of translation factor genes belonged to the early group and these were strongly expressed (Figure 4g ). Reflecting the constitutive requirement for protein synthesis, only six genes showed male gametophyte-specific expression. These were: AtPAB3 (At1g22760), AtPAB6 (At3g16380), AtPAB7 (At2g36660), AteIF2 - B3 (At3g07920), AteIF4G -like (At4g30680) and AteIF6 - 2 (At2g39820). There was a striking over-representation of poly(A)-binding (PAB) proteins among the male gametophyte-specific genes; seven out of eight PAB genes were male gametophyte-expressed, three of which were specific. Moreover, two of these gametophyte-specific PAB genes were among the few late pollen genes encoding translation initiation factors (Figure 4h ). Integrating transcriptomic and experimental data The rapid decline of mRNAs encoding translation initiation factors after bicellular stage and the parallel de novo synthesis of a new set of late pollen transcription factors, suggested storage of translation factors and ongoing transcription after pollen germination. Therefore we investigated the dependence of Arabidopsis pollen germination and tube growth on transcription and translation. Pollen was cultured with increasing concentrations of actinomycin D and cyclohexmide to examine the importance of transcription and translation, respectively. Actinomycin D had only moderate effects on both pollen germination and tube growth even at high concentrations (Figure 5a ). Similar results were observed when another transcription inhibitor, cordycepin, was used (data not shown). In contrast, cycloheximide had a dramatic effect on pollen tube growth (Figure 5b ). The presence of 0.1 μg/ml cycloheximide only inhibited pollen germination by 40%, but pollen tube growth was inhibited by 90%. At higher concentrations, 40% of pollen was still able to germinate, but further pollen tube growth was blocked. We conclude that active pollen tube growth is strictly dependent upon protein synthesis, and that pollen germination and tube growth are relatively independent of transcription. Discussion To identify patterns of gene expression involved in Arabidopsis male gametophyte development, we compared the transcriptomes of isolated spores at four discrete developmental stages using ATH1 microarrays. ATH1 microarrays harbor probe sets for 22,591 annotated genes [ 52 ]. Of these, 61.9% (13,977 genes) gave positive hybridization signals in at least one stage of male gametophyte development. A comparable proportion of active genes was reported for isolated root cells which expressed 10,492 genes (46%) on ATH1 microarrays [ 8 ]. Moreover, in similar studies of animal cell development, 53% of 13,179 arrayed genes were found to be expressed during early murine adipocyte differentiation [ 1 ]. As the proportion of known genes embedded on the ATH1 array is 80.7%, we estimate the total number of genes expressed throughout Arabidopsis male gametophyte development to be more than 17,000. Similarly, the total number of genes expressed at individual developmental stages is estimated to be 14,300 at UNM stage, 14,800 at BCP stage, 10,900 at TCP stage and 9,000 at MPG stage. Previous gene-by-gene approaches identified only 21 different genes expressed during Arabidopsis male gametophyte development (for a review see [ 16 ]). Moreover, only three of these genes were shown to be expressed at microspore stage [ 53 - 55 ]. The data sets reported here include more than 11,000 microspore-expressed genes, representing a 3,600-fold increase in knowledge of gene expression in haploid microspores. Two recent studies of the Arabidopsis mature pollen transcriptome using Affymetrix 8K AG arrays led to the identification of 992 and 1,584 pollen-expressed mRNAs, respectively [ 35 , 36 ]. Results obtained with ATH1 and AG arrays are considered comparable and largely independent of the different probe sets used [ 56 ]. However, there was a significant discrepancy in the number of incorrectly annotated genes between both arrays, with 6.3% of probe sets on the AG array being incorrectly annotated in comparison with only 0.4% on the ATH1 array [ 56 ]. Therefore, results from ATH1 arrays are more accurate as well as more comprehensive. Accordingly, the use of the more complete ATH1 array and more accurate microarray normalization protocols led to an increase in the estimated total number of genes expressed in mature pollen from around 3,500 [ 35 ] to around 9,000 (this study). The proportion of these genes that are considered male-gametophyte specific is strongly dependent on the choice of the set of reference sporophytic datasets. In the work reported here, the availability of more comprehensive sporophytic datasets and the application of more stringent criteria therefore led to a decrease in the estimated number of putative pollen-specific genes from around 1,400 [ 35 ] to around 800 (this study). This number could be reduced further if cell-type-specific expression within an organ limits detection of overlap with pollen expression. Our data highlight the extensive overlap between sporophytic and gametophyte gene expression and reveal the subset of the transcriptome that is strongly enhanced or specifically expressed during male gametophyte development. Considering all stages of microsporogenesis the total number of putative male-gametophyte-specific genes was 1,355 with the proportion of specific genes increasing from 6.9% at UNM-stage to 8.6% at MPG-stage. Among the male-gametophyte-specific genes identified there was an increase in the collective proportion of cell-wall, cytoskeleton, signaling and transport-related genes from 22% at UNM stage to 34% in MPG stage. This reflects the increasing functional specialization of mature pollen in preparation for a dramatic change in the pattern of cell growth during pollen germination and pollen tube growth. Developmental analysis of transcriptome data revealed two striking features, a sharp reduction in transcript diversity after BCP stage and a major shift in mRNA populations between BCP and TCP stages. The decline in mRNA diversity after BCP stage is associated with terminal differentiation as well as the documented phenomenon of protein storage in pollen (see [ 57 ], and this study). Moreover, this large-scale repression associated with termination of cell proliferation after PMII is accompanied by the selective activation of new groups of genes that are likely to function during pollen maturation and post-pollination development. It is interesting that the expression profiles of UNM stage and BCP stages are similar despite the presence of two different cell types in pollen grains at BCP stage - the larger vegetative cell and the smaller generative cell. Given the limited volume of cytoplasm associated with the generative cell, developmental changes in gene expression in the gametic or male germline cells are likely to be masked by the predominant contribution of the vegetative cell cytoplasm. Therefore, our male gametophytic gene expression profiles largely reflect the passage of the microspore through cell division and changes in gene expression associated with the differentiation of the vegetative cell. Large-scale changes in gene expression occur between BCP and TCP stages, and therefore do not coincide with asymmetric division of the microspore. UNM expression patterns persist into the bicellular stage, which is consistent with experiments that demonstrate that vegetative cell fate is specified independently of cell division at pollen mitosis I [ 58 ]. In contrast, generative cell fate appears to be dependent on asymmetric division at pollen mitosis I [ 25 , 58 ]. Sperm-cell cDNAs and databanks recently established in maize, lily, tobacco and Plumbago zelanica [ 30 - 33 ] provide valuable gametic gene-expression data in other species. Although our data do not provide direct information about gametic gene expression in Arabidopsis , further development of cell gamete isolation sorting [ 36 ] would allow genome-wide identification of generative- and sperm-cell-specific genes in comparison with the datasets generated here. Hierarchical cluster analysis provided detailed evidence for the dramatic switch between early and late developmental programs. We identified 39 gene clusters that could correspond to co-regulated genes. These included early clusters, several clusters of late genes, those with constitutive expression profiles and clusters showing transient expression with peaks at BCP or TCP stages. The large size of cluster 29 (4,464 genes) documents the homogeneity in expression profiles of most early genes. In contrast, late gene clusters included a significant number of genes with similar profiles between BCP and TCP stages, followed by expression profiles that deviated between TCP and MPG stages. Cluster 1, and in particular cluster 17, contained genes strongly upregulated in TCP and MPG, with likely functions in post-pollination events. The differential fate of certain late gene clusters is likely to be a feature of their requirement during pollen maturation or post-pollination events. Our analysis revealed completely different expression profiles of transcription factors when compared to core translation factors. The majority of core translation factors belonged to the early-group genes with few that were male gametophyte-specific. This may be expected, given that many genes are involved in general cellular activities. However, genes encoding PAB proteins did not follow the general trend. Seven out of eight Arabidopsis PAB mRNAs were gametophytically expressed. Three PAB genes ( PAB3 , PAB6 and PAB7 ) appeared to be male gametophyte-specific and PAB5 was preferentially expressed in pollen. Moreover, PAB3 and PAB5 are the most abundant early and constitutive PAB mRNAs and PAB6 and PAB7 belong among the few late core translation-factor genes. Although these data suggest-specific expression, our data do not rule out expression in other sporophytic tissues, particularly in flowers. Indeed, previously published expression data confirmed the expression of these PABs in other reproductive tissues together with pollen [ 59 ]. Conversely, transcription factors showed more diverse spectra of expression profiles including early, constitutive and late. There was a considerable variation in the expression profiles of individual transcription factor families. The most over-represented was the C3H family, members of which are known to have roles in lignin and other phenylpropanoid pathways in plants [ 60 ]. Although sporopollenin synthesis is believed to be under strict sporophytic control (see [ 16 ]), the diversity of gametophytic C3H transcription factors might suggest a function for these genes in regulating chemical interactions between phenylpropanoid precursors secreted by the tapetum. One candidate is the At1g74990 gene encoding a putative RING finger protein, which is abundantly and preferentially expressed at UNM and BCP stages. The majority of core translation factors belonged to the early gene clusters. In contrast, a significant number of transcription-factor genes were strongly expressed during pollen maturation. These data alone did not obviously support the fact that pollen germination and early tube growth in many species are largely independent of transcription, but vitally dependent on translation [ 61 ]. Similarly, we found that Arabidopsis pollen germination and tube growth were relatively independent of transcription, and that active pollen-tube growth, and to a lesser extent pollen germination, were dependent upon protein synthesis. It is known for some plant species that mRNAs and rRNAs accumulate during pollen maturation and are stored for use during pollen germination [ 62 , 63 ]. Our results show that Arabidopsis pollen is charged with a diverse complement of stored mRNAs that could be used to support pollen germination and pollen tube growth. Moreover, the early synthesis of mRNAs encoding translation factors strongly suggests that these are preformed and stored in mature pollen grains to support rapid activation upon hydration and germination. We also suggest that some abundant late transcription factors could regulate maturation-associated genes or act as repressors of inappropriate transcription in growing pollen tubes. Conclusions The key impact of this work is that it provides a genome-wide view of the complexity of gene expression during single cell development in plants. Analysis of the male gametophytic transcriptome provides comprehensive and unequivocal evidence for the unique state of differentiation that distinguishes the developing male gametophyte from the sporophyte. Male gametogenesis is accompanied by large-scale repression of gene expression that is associated with the termination of cell proliferation and the selective activation of new groups of genes involved in maturation and post-pollination events. Development is associated with major early and late transcriptional programs and the expression of about 600 putative transcription factors that are potential regulators of these developmental programs. This wealth of information lays the foundation for new genomic-led studies of cellular functions and the identification of regulatory networks that operate to specify male gametophyte development and functions. Materials and methods Plant material and spore isolation Arabidopsis thaliana ecotype Landsberg erecta plants were grown in controlled-environment cabinets at 21°C under illumination of 150 μmol/m 2 /sec with a 16-h photoperiod. Isolated spores from three stages of immature male gametophyte were obtained by modification of the protocol of Kyo and Harada [ 38 , 39 ]. After removal of open flowers, inflorescences from 400 plants were collected and gently ground using a mortar and pestle in 0.3 M mannitol. The slurry was filtered through 100 μM and 53 μM nylon mesh. Mixed spores were concentrated by centrifugation (50 ml Falcon tubes, 450 g , 3 min, 4°C). Concentrated spores were loaded onto the top of 25%/45%/80% Percoll step gradient in a 10-ml centrifuge tube and centrifuged (450 g , 5 min, 4°C). Three fractions were obtained containing: (1) microspores mixed with tetrads; (2) microspores mixed with bicellular pollen; and (3) tricellular pollen (Figure 1 ). Fraction 2 was diluted with one volume of 0.3 M mannitol loaded onto the top of a 25%/30%/45% Percoll step gradient and centrifuged again under the same conditions. Three subfractions of immature pollen were obtained: (2.1) microspores; (2.2) microspores and bicellular pollen mixture; and (2.3) bicellular pollen. Spores in each fraction were concentrated by centrifugation (eppendorf tubes, 2,000 g , 1 min, 4°C) and stored at -80°C. The purity of isolated fractions was determined by light microscopy and 4',6-diaminophenylindole (DAPI) staining according to [ 25 ]. Viability was assessed by fluorescein 3',6'-diacetate (FDA) treatment [ 58 ]. Mature pollen was isolated as described previously [ 35 ]. Pollen tubes were cultivated in vitro for 4 h according to [ 21 ]. Pollen was scored as germinated when pollen tubes were longer than half a pollen grain diameter. Pollen-tube growth was scored by counting those with tubes longer than two pollen grain diameters. RNA extraction, probe preparation and DNA chip hybridization Total RNA was extracted from 50 mg of isolated spores at each developmental stage using the RNeasy Plant Kit (Qiagen) according to the manufacturer's instructions. The yield and RNA purity was determined spectrophotometrically and using an Agilent 2100 Bioanalyzer at the NASC. Biotinylated target RNA was prepared from 20 μg of total RNA as described in the Affymetrix GeneChip expression analysis technical manual. Double-stranded cDNA was synthesized using SuperScript Choice System (Life Technologies) with oligo(dT) 24 primer fused to T7 RNA polymerase promoter. Biotin-labeled target cRNA was prepared by cDNA in vitro transcription using the BioArray High-Yield RNA Transcript Labeling Kit (Enzo Biochem) in the presence of biotinylated UTP and CTP. Arabidopsis ATH1 Genome Arrays were hybridized with 15 μg labeled target cRNA for 16 h at 45°C. Microarrays were stained with streptavidin-phycoerythrin solution and scanned with an Agilent 2500A GeneArray Scanner. Data analysis Sporophytic data from public baseline GeneChip experiments used for comparison with the pollen transcriptome were downloaded from the NASC website [ 41 , 64 ]. The list of dataset codes was as follows: COT (three replicates), Cornah_A4-cornah-wsx_SLD_REP1-3; LEF (three replicates), A4-LLOYD-CON_REP1-3; PET (three replicates), Millenaar_A1-MILL-AIR-REP1-3; STM (two replicates), Turner_A-7-Turne-WT-Base1-2_SLD; ROT (two replicates), Sophie_A1-Fille-WT-nodex_SLD, Sophie_A5-Fille-WT-nodex_SLD; RHR (two replicates), Jones_A1-jones-WT1, SLD, Jones_A1-jones-WT2_SLD; SUS (three replicates), A1-WILLA-CON-REP1-3. All gametophytic and sporophytic datasets were normalized using freely available dChip 1.3 software [ 65 ]. The reliability and reproducibility of analyses was ensured by the use of duplicates or triplicates in each experiment, the normalization of all 26 arrays to the median probe intensity level and the use of normalized CEL intensities of all arrays for the calculation of model-based gene-expression values based on the Perfect Match-only model [ 66 , 67 ]. A given gene was scored as 'expressed' when it gave a reliable expression signal in all replicates. Expression signal value '0' means that the detection call value was not 'present' in all replicates provided. All raw and dChip-normalized gametophytic datasets are available at the Institute of Experimental Botany AS CR website [ 68 ]. Although a RT-PCR validation of microarray data was not performed specifically for the purpose of this publication, our confidence in the quality of the data presented is based on our previously published RT-PCR validation of the expression of 70 genes [ 21 , 35 , 41 ]. Microsoft Excel was used to manage and filter the microarray data. For annotation of genes present on the ATH1 Array, the Arabidopsis Genome Annotation Release 3.0 published by The Institute for Genomic Research [ 52 ] was used. Genes were sorted into functional categories created according to data mined from the Munich Information Center for Protein Sequences Arabidopsis thaliana Database [ 69 ], Kyoto Encyclopedia of Genes and Genomes [ 70 ] and TAIR [ 46 ]. Hierarchical clustering of expressed genes was performed using expression-profile data clustering and analysis software EPCLUST [ 71 ], with correlation measure based distance and average linkage clustering methods. Additional data files The following additional data is available with the online version of this article: Additional data file 1 is an Excel file containing the following items. The table Data contains the complete transcriptomic datasets used. Data were normalized using dChip 1.3 as described in Materials and methods. Expression signal value '0' means that the detection call value for particular gene was not 'present' in all replicates provided. In the column 'Cluster', the appropriate cluster for each male gametophyte-expressed gene is shown. The table Clusters gives the number of genes comprising all 37 clusters of genes coexpressed during male gametophyte development. The table Cell-cycle data lists core cell-cycle genes showing their expression values in male gametophytic datasets. Genes were defined according to [ 21 ]. The chart shows expression profiles of male gametophyte-expressed core cell-cycle genes. The table Transcription data lists transcription-factor genes, showing their expression values in male gametophytic datasets. Genes comprising Arabidopsis transcription factor families were derived by compilation of data available at the Ohio State University Arabidopsis Gene Regulatory Information Server [ 47 ], data published in [ 22 ] and database homology searches. MADS-box and bHLH gene families were defined according to [ 23 ] and [ 24 ], respectively. The table Translation data lists core translation-factor genes showing their expression values in male gametophytic datasets. Genes were defined according to the FIAT database [ 51 ]. The chart shows expression profiles of male gametophyte-expressed core translation-factor genes. The Transcription table summarizes transcription factor gene families showing the number of genes expressed during male gametophyte development. Additional data file 2 lists a complete set of 39 clusters of genes coexpressed during male gametophyte development. Clusters were determined using EPCLUST software with a threshold value of 0.05. The list of genes comprising each cluster is given in Additional data file 1 . Additional data file 3 gives the expression profiles of male gametophyte-expressed transcription factors sorted into individual gene families. Expression data are given in Additional data file 1 . Supplementary Material Additional data file 1 The complete transcriptomic datasets used Click here for additional data file Additional data file 2 The complete set of 39 clusters of genes coexpressed during male gametophyte development Click here for additional data file Additional data file 3 The expression profiles of male gametophyte-expressed transcription factors sorted into individual gene families Click here for additional data file

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539341

Multiple Campylobacter Genomes Sequenced

In 1995, the first complete bacterial genome sequence was published. Now, nearly 200 bacterial genomes have been completed, and a new one hits the scientific press most weeks. This burgeoning industry is not just scientific “stamp collecting,” however. Having all these genome sequences may provide useful clues about why some bacteria cause human disease, how to control their spread, and how to treat the infections caused by them. By comparing genome sequences, scientists can learn much more about what makes a bacteria tick than they can learn from a single sequence. Derrick Fouts and his colleagues have taken this comparative approach with Campylobacter . Infection with a Campylobacter species is one of the most common causes of human bacterial gastroenteritis. In the US, 15 out of every 100,000 people are diagnosed with campylobacteriosis every year, and with many cases going unreported, up to 0.5% of the general population may unknowingly harbor Campylobacter in their gut annually. Diarrhea, cramps, abdominal pain, and fever develop within 2–5 days of picking up a pathogenic Campylobacter species, and in most people, the illness lasts for 7–10 days. But the infection can sometimes be fatal, and some individuals develop Guillain-Barré syndrome, in which the nerves that join the spinal cord and brain to the rest of the body are damaged, sometimes permanently. Campylobacteriosis is usually caused by C. jejuni , a spiral-shaped bacterium normally found in cattle, swine, and birds, where it causes no problems. But the illness can also be caused by C. coli (also found in cattle, swine, and birds), C. upsaliensis (found in cats and dogs), and C. lari (present in seabirds in particular). Disease-causing bacteria generally get into people via contaminated food, often undercooked or poorly handled poultry, although contact with contaminated water, livestock, or household pets can also cause disease. Genome sequencing and comparison of four species of Campylobacter In 2000, C. jejuni was the first food-borne pathogen to be completely sequenced, but we still know little about how Campylobacter species cause disease. In their search for clues, Derrick Fouts and coworkers have completely sequenced the genome of C. jejuni strain RM1221 (isolated from a chicken carcass) and compared it with the previously sequenced C. jejuni strain NCTC 11168 and with the unfinished sequences of C. coli strain RM2228 (a multi-drug-resistant chicken isolate), C. lari strain RM2100 (a clinical isolate), and C. upsaliensis strain RM3195 (taken from a patient with Guillain-Barré syndrome). The researchers describe numerous differences and similarities between these different Campylobacter strains and species. For example, there are major structural differences between the genomes caused by the insertion of new stretches of DNA. Some of these pieces of DNA may carry genes that improve bacterial virulence or fitness, so their presence could help to explain the different biological behaviors of these strains. There are also major variations in the genes responsible for synthesis of molecules that are important for the interaction of Campylobacter with the environment. Such differences could underlie the host specificity of the different species. Differences between the Campylobacter species in genes that are likely to be involved in aspects of bacterial virulence, such as adherence, motility, and toxin formation, are all detailed by Fouts et al., who also describe a new putative Campylobacter virulence locus. Further work is needed to relate these genomic differences to functional differences, but this detailed comparative genomic analysis provides the core blueprint for this important family of human pathogens. And in doing so, it lays the foundation for the development of new ways to monitor and control Campylobacter in the food chain and in human infection.

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539355

The DiAMOND trial protocol: a randomised controlled trial of two decision aids for mode of delivery among women with a previous caesarean section [ISRCTN84367722]

Background Caesarean section (CS) has become an increasingly common method of delivery worldwide, rising in the UK from 9% of deliveries in 1980 to over 21% 2001. This increase, and the question of whether CS should be available to women on request, has been the subject of considerable debate, and national reports and guidelines have specifically highlighted the importance of patient choice in the decision making process. For women who have already experienced CS, the UK National Institute of Clinical Excellence recommends that the decision should consider maternal preferences and priorities in addition to general discussion of the overall risks and benefits of CS. Decision aids for many different medical treatment and screening decisions have been developed and evaluated, but there is relatively little evidence for the use of decision aids for choice of mode of delivery among women with a previous CS. The aim of the study is to evaluate two interventions to assist decision making about mode of delivery among pregnant women with one previous CS. Methods/design Women with one previous CS are recruited to the trial during their booking visit at approximately 12–20 weeks' gestation in participating maternity units in Bristol, Weston and Dundee. Using central randomisation, women are allocated to one of three arms: information programme and website; decision analysis; usual care. Both interventions are computer-based, and are designed to provide women with detailed information about the potential outcomes for both mother and baby of planned vaginal delivery, planned CS and emergency CS. The decision analysis intervention additionally provides a recommended 'preferred option' based on maximised expected utility. There are two primary outcomes (decisional conflict and actual mode of delivery), and five secondary outcomes (anxiety, knowledge, perceptions of shared decision making; satisfaction with decision making process, proportion of women attempting vaginal delivery). Primary follow up for the questionnaire measures is at 36–37 weeks' gestation, and a total of 660 women will be recruited to the study. The primary intention-to-treat analyses will comprise three pair-wise comparisons between decision analysis, information and usual care groups, for each of the two primary outcomes. A qualitative study will investigate women's experiences of the decision making in more depth, and an economic evaluation from the perspective of the NHS will be conducted. Discussion Provision of information to women facing this decision appears variable. The DiAMOND study aims to inform best practice in this area by evaluating the effectiveness of two interventions designed to aid decision making.

Background Over the last 20 years caesarean section (CS) has become an increasingly common method of delivery. The CS rate in the United Kingdom rose from 9% of deliveries in 1980 to 21% in 2001 [ 1 ]. This increase, and the question of whether CS should be available to women on request, has been the subject of considerable debate [ 2 ]. The optimal mode of delivery for women who have experienced a previous CS is complicated by the difficulty in balancing the risks of repeat CS with those of vaginal birth after caesarean section (VBAC). An evaluation of caesarean delivery by the American College of Obstetricians and Gynecologists reported that first time mothers with term singleton cephalic pregnancies and women with a previous CS account for two thirds of all caesarean deliveries in the US [ 3 ]. The Changing Childbirth report has emphasised the importance of patient choice when decisions need to be made in relation to the management of pregnancy and childbirth [ 4 ]. However the views of women who have experienced CS and their preferences for future deliveries have received little attention to date. Obstetricians tend to focus on the risks of uterine rupture and emergency caesarean section in labour [ 5 ], which may influence the advice they give to women uncertain about future mode of delivery. Others have focused on the increased morbidity following CS [ 6 ] and on the negative impact of operative delivery on first postnatal contact between the mother and her baby [ 7 ]. In Chile, where there is a very high rate of CS in the private sector, only a minority of women reported that they had wanted this method of delivery [ 8 ]. In a Scottish study, more women who delivered by elective CS reported they were satisfied with their involvement in the decision making process compared with women who underwent emergency CS [ 9 ]. The advantages and disadvantages of planned CS versus planned vaginal delivery has also been debated in north America in response to a growing number of requests for elective CS [ 10 ]. In an attempt to ensure appropriateness of CS in the UK, a set of evidence-based guidelines on indications for CS have recently been published [ 11 ]. The guidelines, commissioned by the UK National Institute for Clinical Excellence, make a specific recommendation that for women with a previous CS, the decision should consider maternal preferences and priorities in addition to general discussion of the overall risks and benefits of CS. It is essential that the process of decision making about future mode of delivery is evaluated and enhanced to achieve the safest and most satisfactory outcome for both mother and baby. It has been proposed that the way in which clinical decisions are made lies on a continuum, from paternalistic (clinician decides) through partnership (clinician and patient share the decision) to informed (patient decides) [ 12 ]. Although proposed as the preferred approach of determining patients' treatments [ 13 ], some problems with the concepts, terminology and practice of shared medical decision making have recently been highlighted [ 14 ]. The appropriateness of the shared model may also depend on the clinical context as well as patients' and clinicians' preferences for involvement in decision making [ 15 ]. In addition, there is some evidence that patients and health professionals often have different treatment preferences, potentially making agreement on a treatment strategy more difficult [ 16 ]. Decision aids are designed to help people select between various treatment strategies by providing information on the options and outcomes relevant to a person's health. Decision aids for many clinical conditions have been developed [ 17 ], and evaluations of these decision aids have been the subject of systematic reviews [ 18 , 19 ]. A north American trial found no difference in terms of VBAC rate between written versus personal counselling interventions that actively promoted vaginal delivery [ 20 ]. An Australian trial of a paper-based decision aid for women who have previously experienced CS is currently underway [ 21 ]. The specific content of decision aids may vary, but in general they aim to present more than one strategy for clinical management, help people understand the probable outcomes of treatment choices and allow people to consider the personal value they place on benefits versus harms. Decision aids can take several formats, such as leaflets, interactive videodisks, individualised decision analysis, personal counselling sessions and audio workbooks. Interventions to assist patient decision making can improve knowledge about treatment options, make patients more realistic in their expectations, reduce decisional conflict and increase active involvement in decision making [ 18 ]. As an intervention to aid patient decision making, individualised decision analysis has so far received limited attention. By explicitly combining patients' values regarding treatment outcomes and individual probability information, decision analysis attempts to provide a rational framework to guide patient decision making. The use of individualised decision analysis has been debated [ 22 ], but empirical evidence demonstrates that it is feasible and acceptable and has value as an aid to patient decision making [ 23 ]. Aim The aim of this paper is to describe the protocol for a randomised controlled trial of two interventions to aid decision making about mode of delivery among pregnant women with one previous CS. The interventions being assessed are (1) Decision analysis, and (2) Information programme and website. Both decision aids will be compared with usual care given by the obstetric team. The interventions will be assessed in terms of decisional conflict, planned and actual mode of delivery, anxiety, knowledge, perception of shared decision making and satisfaction with the decision making process. Development and piloting of the interventions took place in 2003–2004, and the main phase of the study started in May 2004 and will continue until December 2006. Ethical approval for the study was obtained from the UK South West Multi-Centre Research Ethics Committee. Methods/design Recruitment and allocation of participants The sample will comprise pregnant women with one previous lower segment CS (all parities will be included, but the most recent delivery must have been CS), no current obstetric problems and delivery expected at ≥ 37 weeks. Women are being recruited to the study by research midwives during their initial booking visit at approximately 12–20 weeks' gestation. Recruitment takes place from maternity units in St Michael's and Southmead Hospitals in Bristol, Weston General Hospital, and Ninewells Hospital in Dundee. The current CS rates for these units range between 18 and 24% and are representative of rates for other units throughout England and Scotland. The women are informed that although both vaginal delivery and repeat CS carry their own benefits and risks, the best method of presenting this information in order to assist women in reaching a decision is not known. Women expressing an interest in participating in the trial at the booking visit are given an information sheet, a written consent form and a baseline questionnaire to take home. Following receipt of the baseline questionnaire and written informed consent to enter the trial, women are randomised to one of three arms as detailed below. Allocation is stratified by maternity unit and preferred mode of delivery and blocked (using random permuted blocks of sizes 6, 9, 12 and 15) to ensure reasonable balance between the trial groups through time. The randomisation sequence was generated by a member of the study team (AAM), and allocation of participants is performed by a staff member with no other involvement in the study. Interventions Both interventions are computer based. Women allocated to receive an intervention have an appointment with a researcher to allow the decision aid to be delivered using a laptop computer, usually in the woman's own home or workplace. (i) Information programme and website This intervention provides information about the outcomes associated with planned vaginal delivery, planned CS, and emergency CS. This includes descriptions of outcomes for both mother and baby, and the probabilities of these outcomes based on the best available evidence. Both the probabilities of having and not having the event are given, and all probabilities are presented in both numerical and pictorial format [ 24 ]. The programme easily allows women to choose the information that they view, and the information each women accesses is logged. At the end of the initial appointment with a researcher, women are given a password that allows them to access the information programme via the internet as often as they wish. Womens' use of the programme via the internet is also logged. (ii) Decision analysis There are generally four main steps involved in a decision analysis. The first is to draw up a decision tree that maps out the likely outcomes of the strategies in question [ 25 ]. These outcomes are then assigned utilities that represent how an individual values a particular outcome. A utility is a number between 0 and 1, often representing the outcomes 'death' and 'perfect health' respectively [ 26 ]. Probability information is then included in the tree to represent the chance of each outcome occurring [ 26 ]. Finally, strategies are compared by calculating the weighted sum of the utilities of all possible outcomes [ 27 ]. The recommended strategy is that with the highest expected utility value, or in other words, the one that gives an individual the best chance of achieving an outcome that is valued. The decision analysis intervention in the trial proceeds according to the principles described above. First, women are given information about the outcomes associated with planned vaginal delivery, planned CS, and emergency CS. This includes descriptions of outcomes for both mother and baby, but not the probabilities of these events. These are embedded in the decision tree which is not visible to users. Second, women are required to rate (assign a utility value between 0 and 1) each possible outcome using a visual analogue scale. Finally, the programme combines the elicited utilities and the probabilities of each outcome in a decision tree to produce a recommended 'preferred option' based on maximised expected utility. Each woman is given a computer printout of the outcome of the decision analysis. (iii) Usual care This comprises care normally given by the obstetric and midwifery team. Women allocated to decision analysis or information programme receive these interventions in addition to usual care. Women in both intervention arms are contacted by letter at 35 weeks' gestation. The purpose is to encourage discussion of the intervention with their obstetrician and/or midwife when they attend the clinic at 36–37 weeks to finalise their planned method of delivery. Participation in the study is recorded in the medical records of all women in the trial. Outcome measures There are two primary outcomes: (1) Decisional Conflict Scale (DCS) [ 28 , 29 ]. This is a 16 item questionnaire that measures degree of uncertainty about which course of action to take and the main modifiable factors contributing to uncertainty. Previous research indicates that effect sizes of about 0.35 to 0.5 standard deviations can discriminate between individuals who make a decision and those who delay or are unsure [ 28 ]. Assuming a standard deviation of 15 points [ 18 ], this is equivalent to differences of 5.25 to 7.5 points on the total DCS 100 point scale. (2) Actual mode of delivery (vaginal, elective CS, or emergency CS). Unlike a previous trial [ 20 ], we are not seeking to promote one method of delivery over another. However differences between the arms in the proportions of different modes of delivery may have important healthcare resource implications. There are five secondary outcomes: anxiety; [ 30 ] knowledge; perception of shared decision making; satisfaction with decision making process; proportion of women attempting vaginal delivery. Follow up The primary and secondary outcomes are assessed in all three groups at baseline, and approximately two weeks after randomisation. The questionnaire at two weeks will constitute a secondary follow up, and will enable sufficient time for delivery of the appropriate interventions. As part of usual care, women in the trial normally attend the clinic at around 36–37 weeks' gestation to finalise plans for their preferred method of delivery. Questionnaire outcomes are measured again after this visit, and this constitutes the primary follow up for this trial. Actual and attempted mode of delivery (cross-checked with hospital records) and satisfaction with choice are assessed by a further postal questionnaire at approximately six to eight weeks after giving birth. Justification of sample size As noted above, differences in excess of 0.35 standard deviations have been considered as important for the total DCS score, and differences of this magnitude are feasible for interventions of this kind [ 23 ]. With regard to mode of delivery, UK data indicate that about 33% of women with one previous CS are delivered vaginally [ 1 ]. The sample size calculation for a previous trial of written versus verbal counselling in north America presumed a vaginal delivery rate of 30% for a minimal intervention, and in the event observed that 51% of women achieved vaginal delivery for the trial groups overall [ 20 ]. A change from 30–33% to 51% corresponds to an odds ratio of about 2.1–2.4, and this would certainly be considered as clinically important. With two-sided 1% alpha, a total sample size of 600 provides 82–99% power to detect a standardised difference of 0.35–0.5 in total decisional conflict score between the groups, and 84–95% power to detect odds ratios of 2.1–2.4 in women achieving vaginal delivery. A pair-wise alpha of 1%, corrected for multiple comparisons using Tukey's procedure, maintains an overall study-wise alpha of 3.4%. In order to allow for pre-term deliveries, malpresentations, and losses to follow-up, we will therefore recruit 660 women to the trial. Statistical analysis Data analysis will proceed according to CONSORT guidelines for randomised controlled trials. The first stage of the analysis will be to use descriptive statistics to describe the group of individuals recruited to the trial in relation to those eligible, and to investigate comparability of the trial arms at baseline. The primary analyses will comprise three pair-wise intention-to-treat comparisons between decision analysis, information and usual care groups, for each of the two primary outcomes. These comparisons will use appropriate (that is, standard or logistic) multivariable regression models, adjusting for maternity unit, initial preference regarding mode of delivery, and value of the outcome variable at baseline. Full attention will be paid to the estimates and confidence intervals for these comparisons as well as the p-values, with the latter adjusted for multiple comparisons using Tukey's procedure. Secondary outcomes will then be analysed in the same way, using appropriate multivariable regression models depending on the nature of the outcomes. Other secondary analyses will involve investigation of the short-term effects of the interventions using data from the two week follow up, and the effects at 36–37' weeks gestation adjusted for both baseline and two week follow up. Pre-planned subgroup analyses employing appropriate interaction terms in the regression models will be used to ascertain any differential effects of the interventions on the two primary outcomes across the following categories of women: previous caesarean section occurring before or after labour; previous successful vaginal delivery; stated preferred mode of delivery. Since the trial is powered to detect overall differences between the groups rather than interactions of this kind, the results of these essentially exploratory analyses will be presented using confidence intervals as well as p-values, and interpreted with due caution. Finally, we will investigate the effect of differential use of the information intervention via the internet using descriptive statistics and appropriate comparisons with the other groups. Qualitative study Qualitative research methods will be used in order to explore aspects of the interventions and women's experiences of the decision making in more depth. Specifically, semi-structured interviews will be conducted with a sample of women from each of the intervention arms (Decision Analysis and Information), across the research sites in Bristol and Dundee. These interviews will explore: (1) Women's views and experiences of the intervention and its delivery – for example, the quality and relevance of the decision aid/information, what they felt about the risk information presented to them, and which particular aspects of each intervention were helpful or unhelpful. (2) Which factors women felt had most influenced their preferences regarding method of delivery. (3) Whether the women had prior preferences about method of delivery, whether/how these changed during their pregnancy and in the case of the decision analysis, what they felt about the method of delivery proposed by the intervention compared to any prior preferences. (4) Any other information sources women sought and used to help them make a decision about method of delivery (for example, information from health professionals, partner/family/friends, internet, media, books). (5) Women's views and feelings about their actual method of delivery compared with any prior preferences and the method of delivery suggested by the intervention (for decision analysis). A small number of interviews may also be conducted with women in the usual care arm of the trial to explore what support and advice was normally provided during pregnancy to those who did not receive an intervention. A sample of approximately 30 women across the two intervention arms will be interviewed in depth to ensure a thorough exploration of emergent themes and concepts. Within each arm, women will be purposefully chosen to include those with different parities/ages/socio-economic backgrounds, and where possible different methods of delivery/outcomes, following a maximum variation sampling strategy [ 31 ]. The interviews will take place a short time after the primary follow up, to avoid any influence of the interview on these measures. A subset of the women will be interviewed a second time six to eight weeks after birth in order that they can reflect upon their decision regarding preferred mode of delivery with the actual mode of delivery. In addition, a small number of interviews may take place closer in time to receipt of the intervention if this is deemed (a) feasible given recruitment and (b) worthwhile in terms of providing new information to that gleaned from qualitative interviews undertaken in the development phase of the trial. Interviews will be conducted in the womens' homes or other suitable setting chosen by them. A check-list of topics will be used to ensure that the primary issues are covered, whilst allowing flexibility for new issues to emerge from each interview. Interviews will be recorded on mini-disc, fully transcribed and anonymised to protect confidentiality. Transcripts will be studied in detail and a list of common themes and concepts drawn up. Data collection and analysis will run in parallel and the coding index added to or refined and coded material regrouped as new themes and categories emerge from subsequent interviews [ 32 ]. Further analysis will employ the constant comparison method of grounded theory in which the textual data is scrutinised for differences and similarities within themes keeping in mind the context in which mention of these themes arose in each interview [ 32 ]. In addition to the interviews with women, a small number of focus groups with health professionals (e.g. obstetricians, midwives) in each research site may be conducted near the end of the trial, resources and time permitting. Focus groups are often used in evaluations of new services/interventions and are useful for exploring group views, concerns and preferences (e.g. consensus or disagreement about an issue) [ 33 ]. They may be valuable for exploring professionals' views about the intervention and issues around implementation into routine practice. Economic evaluation The aim of the economic evaluation is to compare the costs and benefits of the two interventions with usual care. The analysis will be from an NHS perspective and will be based on the costs incurred during pregnancy, delivery and 6–8 weeks following delivery. Incremental cost effectiveness ratios will be formed comparing (i) the cost per point improvement on the Decisional Conflict Scale (DCS) and (ii) the cost per extra patient able to make a decision (represented by a 0.5 standard deviation change on the DCS). We will also compare the average cost of care per patient in each arm of the trial with patient satisfaction. Differences in the cost of care of women receiving the two interventions will be compared with usual care from the point at which patients are randomised. The analysis will include all resources under the control of the NHS that may differ as a result of the interventions and will include resources used by both mother and baby. The costs identified as being of relevance are: antenatal appointments in addition to routine appointments, including both primary and secondary care; mode of delivery and related hospital stay for mother and baby; follow up care for mother and baby, including primary and secondary care appointments, outpatient appointments, inpatient stays, A&E visits, and prescribed medication. Data on resource use will be collected principally by two questionnaires completed by the women. The first questionnaire, completed at 36–37 weeks' gestation, will provide information on all antenatal appointments, and the proportion of these that involved discussion about mode of delivery. Women will be asked how often mode of delivery was addressed at (i) routine appointments, and (ii) appointments initiated by them to discuss mode of delivery. A sample of 10 hand-held records from each arm at each main centre (St Michaels, Southmead and Ninewells, total 90) will be scrutinised to validate the information given in response to the questionnaire. The second questionnaire, completed six to eight weeks after delivery, will provide information on mode of delivery and all non-routine postnatal health service contacts and prescriptions for both mother and baby. The principle of opportunity cost will underlie the valuation of resource use. In many cases, market rates will act as a proxy for opportunity cost. National data sets such as the Unit Costs of Health and Social Care [ 34 ] and the British National Formulary [ 35 ] will be used to value primary care consultations and prescribed medication. Secondary care contacts will be coded according to Healthcare Resource Group (HRG) and valued using the Department of Health National Reference Costs [ 36 ]. Costs and outcomes will not be discounted, as the economic evaluation will be limited to a period of 12 months. Sensitivity analysis will be conducted in areas where there is uncertainty around assumptions about resource use measurement and/or valuation. Variation in resource use and the effectiveness of the intervention is not captured by a cost-effectiveness/utility ratio. We will use bootstrapping to address this, and construct a cost effectiveness acceptability curve. Discussion Women with an uncomplicated pregnancy and expected term delivery who have previously delivered by CS face a choice between repeat elective CS or attempted trial of labour. Guidelines in the UK emphasise the importance of involving women in the decision making process and taking account of maternal preferences and priorities, but the type and amount of information available to women facing this choice appears variable. The DiAMOND study is a randomised trial that aims to inform best practice in this area, by evaluating the effectiveness of two interventions to assist decision making in terms of decision quality and actual mode of delivery. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Alan Montgomery, Deirdre Murphy, Ali Shaw and Sandra Hollinghurst drafted the manuscript, with input from the other members of the DiAMOND Study Group. The Decision Aids for Mode Of Next Delivery (DiAMOND) Study Group comprises the following members: Clare Emmett, Tom Fahey, Peter Gregor, Sandra Hollinghurst, Claire Jones, Beverley Lovering, Alan Montgomery, Irene Munro, Deirdre Murphy, Roshni Patel, Tim Peters, Ian Ricketts, Anne Schlegelmilch, Ali Shaw, Kav Vedhara, Kate Warren. Pre-publication history The pre-publication history for this paper can be accessed here:

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526275

i-Genome: A database to summarize oligonucleotide data in genomes

Background Information on the occurrence of sequence features in genomes is crucial to comparative genomics, evolutionary analysis, the analyses of regulatory sequences and the quantitative evaluation of sequences. Computing the frequencies and the occurrences of a pattern in complete genomes is time-consuming. Results The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase. The database supports various complete genomes including human, yeast, worm, and 128 microbial genomes. Conclusions This investigation presents and implements an efficiently computational approach to accumulate the occurrences of the oligonucleotides or patterns in complete genomes. A database is established to maintain the information of the sequence features, including the distributions of oligonucleotide, the gene distribution, the distribution of repetitive elements in genomes and the occurrences of the oligonucleotides. The database can provide more effective and efficient way to access the repetitive features in genomes.

Background During the last decade, many genomes have been successfully and completely sequenced. Summarized information about the oligonucleotides in genomes provides biologists, who interests in the evolution and growth of genomes, to work in comparative genomics, oligonucleotide probe design, primer design and the analyses of genomic repetitive features. The computation of the occurrences and the frequency of all oligonucleotides in a complete genome is very elaborate and time-consuming, especially when the genome size is very large, such as the human and mouse genomes. A database that summarizes the occurrences and the frequencies of oligonucleotides in complete genomes can facilitate the biological and the statistical analyses of genomes. The contents of the database can be used in many biological applications, such as comparative genomics and evolution analyses [ 1 , 2 ], the prediction of regulatory sequences by detecting the over-represented oligonucleotides [ 3 - 8 ] and primer/probe design based on the uniqueness of oligonucleotides [ 9 ]. Table 1 shows the biological applications of the database entries. The entries in the database are divided into two types, namely, the occurrence positions of oligonucleotides and the frequencies of oligonucleotides. The oligonucleotide occurrences and the oligonucleotide frequencies in both the coding regions and the non-coding regions are summarized. For instance, these information can be used in statistical analyses to study the over-representation of the regulatory sequences in upstream promoter regions in genes. van Helden et al. systematically searched the promoter regions of potentially co-regulated genes for over-represented oligonucleotides which may be transcription factor binding sites [ 3 ]. They presented a simple and fast method for isolating DNA binding sites for transcription factors from families of co-regulated genes, illustrating their results using Saccharomyces cerevisiae. Although conceptually simple, the algorithm efficiently extracted the upstream regulatory sequences that had been previously been determined experimentally for most of the yeast regulatory families already analyzed. Other studies [ 4 - 8 , 10 - 12 ] on the prediction of gene regulatory sequences have been based on oligonucleotide analysis. Table 1 Applications and the relevant data in the database. Database entries Entry types Biological applications Oligonucleotide occurrences Positions 1. Oligonucleotide analysis for regulatory sequences 2. Oligonucleotide probe design 3. Primer design Oligonucleotide frequencies Counts 1. Oligonucleotide analysis for regulatory sequences 2. Evolutionary analysis 3. Oligonucleotide probe design 4. Primer design Gene coding regions Positions 1. Oligonucleotide analysis for regulatory sequences 2. Evolutionary analysis 3. Oligonucleotide probe design 4. Primer design Repetitive element frequencies (LINE, SINE, Alu, and so on) Counts Evolutionary analysis Repetitive element occurrences Positions Evolutionary analysis Tandem repeats Positions Prediction for genetic disease marker Hsieh et al. [ 1 ] investigated the oligonucleotide distributions of typical microbial genomes and found that the microbial genomes have the statistical characteristics of a much shorter DNA sequence. This peculiar property supports an evolutionary model in which a genome evolves by random mutation but grows primarily by random segmental duplication. Repetitive elements, including LINEs, SINEs, LTR and Alu, can be investigated in evolution analysis [ 2 ]. It has been estimated that at least 43% of the human genome is occupied by four major classes of interspersed repetitive elements – LINEs, SINEs, LTR elements and DNA transposons [ 2 ]. Their analysis has yielded some insights into the evolution of the human genome. The tandem repeats provided in our database can be used for forensic analysis and the study of genetic diseases [ 13 - 16 ]. Another application of the established database is to facilitate the design of primers to amplify specific regions of the genomic sequence. The basic concept is that the sequences of primers from 15 to 40 bps should be unique, unlike the repetitive oligonucleotides in our database. Additionally, our database maintains the repetitive oligonucleotides that facilitate the design of oligonucleotide probes to allow the selection of signature oligonucleotides when identifying different organisms using DNA arrays [ 9 ]. The user can query oligonucleotides whose lengths exceed a threshold, such as 15 bps, to determine whether the oligonucleotides are repetitive. The non-repetitive regions of the target sequences without repetitive oligonucleotides can be used as the signatures for the target genomes. Construction and content Data sources and contents The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The data sources including the complete genomes and the gene annotation information are obtained from GenBank [ 17 ]. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase [ 18 ]. The database supports a range of complete genomes including human, yeast, worm, and 128 microbial genomes. The Appendix lists the organisms supported in the database [see additional file 1 ]. The occurrences and the frequencies of oligonucleotides from one to 50 base pairs are generated and accumulated from each of the complete genome sequence. Inputting the sequence of the oligonucleotide returns the positions of the oligonucleotides. Additionally, both the occurrences and the frequencies of the repetitive elements such as LINEs, SINEs, Alu and LTR are provided by computationally scanning whole genome sequences. The tandem repeats are computationally detected by the tandem repeat finder [ 19 ]. The database also provides the gene annotation information. For instance, Table 2 presents the number of occurrences of repetitive oligonucleotides in yeast. The oligonucleotide "ACCCTA" occurs 2,724 times in the yeast genome, 822 times upstream of a gene (-600 ~-1 bp, +1 bp denotes the gene translational start postion) and 793 times in the coding regions. The counts of the occurrence of ecah oligonucleotide between one and 50 bps are present in the database. Table 2 Number of occurrences of the repetitive oligonucleotides in yeast genome Repetitive oligonucleotide Amount of occurrences Repetitive oligonucleotide Amount of occurrences Genome Up-streams Coding region Genome Up-streams Coding region ACCCTA 2,724 822 793 CAATCCA 1,895 655 343 ACCCTC 2,917 881 795 CGTCTCC 592 199 148 AGTACT 3,073 933 879 CGTCTGA 652 196 165 AGTAGA 6,673 1,970 1,798 ACAAACTA 594 179 183 AGTAGC 4,912 1,545 1,299 ACAAACTC 514 175 112 GATACC 4,829 1,638 1,005 CACAGAAAC 146 38 46 GATAGA 7,030 2,163 1,807 CACAGAAGA 164 57 39 TGGTAA 10,513 3,493 2,214 ACATATAAAAA 54 9 29 TGTAAA 11,364 3,439 3,418 ACATATAAAAC 139 34 56 AAGGGGA 1,172 299 421 ACATATAAAAG 36 7 22 AAGGGGC 626 142 256 ACTTATGTCATC 57 17 23 AGAGTGG 983 310 271 ACTTCTAGTATA 159 44 67 AGAGTTA 1,859 610 441 ACTTTTTTTTCT 32 5 21 CAATCAG 1,358 445 320 ACTTTTTTTTTC 50 6 33 Data Generation A software is implemented to index systematically a complete genome sequence into a suffix-array using a perfect match approach [ 20 ]. This index is only able to find the perfect match for any oligonucleotide. The user can thus use it to find the positions of a designated oligonucleotide in a genome sequence. For each genome, the occurrences of all oligonucleotides shorter than 50 bps can be efficiently searched for. The occurrence is the position of the oligonucleotide in genome. The frequency is the count of oligonucleotide occurrences in a region. The regions are the complete genome, the coding regions and the non-coding regions. Frequencies of oligonucleotides with different lengths are stored in different flat-files. For example, the two chromosomes of the Vibrio cholerae genome are processed separately to allow the computation of the occurrences of oligonucleotides in each chromosomal sequence. RepeatMasker [ 21 ] and the repetitive element database, Repbase [ 18 ], are used to search the instances of the repetitive elements in eukaryotic genomes. The tandem repeat finder (TRF) is used to find the tandem repeats in genomes [ 19 ]. The TRF and RepeatMasker can find the instances of repetitive elements with imperfect matches. The settings used here for each software is described in below. The Tandem Repeat Finder uses seven parameters. These are match score, mismatch score, indel score, probability of match and insertion, minimum score of alignment and the maximum of tandem repeat pattern size. The corresponding values used here are 2, 7, 7, 80, 10, 20 and 500. The values are the default suggestions found in Tandem Repeat Finder documentation. The transposable elements (TEs) are detected by RepeatMasker. TEs in each genome are identified using the complete dataset available from REPbase Updates [Please add the citation]. The sensitivity and the speed of RepeatMasker are set as the default values. Utility Table 3 presents the two output formats – flat-files and the web query interface with a filtering function. In the flat-file format, the fields of each oligonucleotide (in a chromosome) include the sequences, the number of occurrences in the chromosome, the number of occurrences in the coding regions and the number of occurrences in the non-coding regions. The user that requires a large amount of such data can download them in this format [ 1 ]. Table 3 Output styles of the database. Database entries Entry types Output formats Oligonucleotide occurrences Positions Web interface Oligonucleotide frequencies Counts Web interface and flat-file Gene coding regions Positions Web interface Repetitive element frequencies (LINE, SINE, Alu, and so on) Counts Web interface and flat-file Repetitive element occurrences Positions Web interface Tandem repeats Positions Web interface The web interface enables users to query the occurrences of an oligonucleotide in a genome and the number of occurrences in each chromosome. Figure 1 shows this approach. The occurrences of the repetitive elements and the tandem repeats in the established database can also be queried via the web interface, as in the example given in Fig 2 . Figure 3 depicts the flat-file format of oligonucleotide frequencies. The first row in the flat-file presents the basic information for the oligonucleotide frequencies and the fields are the chromosome sequence/NCBI accession number, the length of the chromosome sequence, the size of coding regions, the size of non-coding regions, the length of the oligonucleotides and the minimum number of copies of the oligonucleotide. The directories labeled C10 are the files that contain the counts of oligonucleotides with at least ten occurrences in genome. Each file name includes the sequence/NCBI accession number, the length of oligonucleotides and the minimum occurrences of oligonucleotides. For example, "NC 000913 L30 C10" is the oligonucleotides, which are 30 nucleotides in length and have at least 10 occurrences in the genome. Figure 1 Web query interface (1/2). Figure 2 Web query interface (2/2). Figure 3 Database entries in flat-file format. Figure 4 shows the web interface for the occurrences of a specific oligonucleotide. The user submits the query oligonucleotide and selects particular species; the positions of the oligonucleotide are then shown. The first line is the user submitted data. Following this information are the positions of oligonucleotides in forward strand. This is followed by the same information for the reverse strand. Figure 4 The occurrence positions of the oligonucleotide are found by Oligos Locator. Conclusions We have constructed the databases of both the oligonucleotide occurrence locations and their frequencies in the coding and the non-coding regions in complete genomes. The data in flat-file format can be downloaded directly for further analyses in several biological applications. The user may also use the web interface to query and access the database contents. The database also provides a filtering function for retrieving the information about oligonucleotides under search conditions specified by the users. Furthermore, the database provides the occurrences and the frequencies of other repetitive elements, such as LINE, SINE, Alu and tandem repeats in genomes. Availability and requirements The database is now available at Authors' contributions FML implements the software and refinements the system. HDH conceives of the study and drafted the manuscripts. YCC and JTH participates the design and coordination. All authors read and approved the final manuscripts. Supplementary Material Additional File 1 Appendix listing the organisms supported in the database. Click here for file

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535534

Recombinant activated factor VII (rFVIIa) as salvage treatment for intractable hemorrhage

Background Recently, there has been an increased use of recombinant activated factor VII (rFVIIa) to promote hemostasis in various hemorrhagic conditions. The objective of this study was to determine the outcome of patients treated with rFVIIa who had intractable bleeding associated with cardiac surgery (CSP) or as a result of other causes (OBP). Methods The medical records of 40 consecutive patients treated with rFVIIa were retrospectively reviewed for blood product use before and after treatment. In all patients, rFVIIa was given only after all other measures to stop bleeding had failed. The number of transfused units of red cells (R), platelets (P), fresh frozen plasma (F), and cryoprecipitate (C) were determined both before and after administration of rFVIIa, and the results compared. Mortality at 4 hours and 30 days was assessed. Patients dying within 4 hours of rFVIIa administration were not evaluable for response. Patient characteristics were also assessed as risk factors for mortality. Results Twelve of 24 CSP survived for more than 4 hours. These 12 patients required an average of 17 units (U) of R, 18 U of P, 18 U of F and 15 U of C pre-treatment compared to an average of 6 U, 10 U, 9 U and 4 U of R, P, F and C respectively, post-treatment. These differences were statistically significant. For the OBP, 11 of 16 survived more than four hours. These 11 patients required an average of 10 U of R, 11 U of P, 14 U of F and 10 U of C pretreatment compared to an average of 1 U, 2 U, 2 U and 0 U of R, P, F, and C respectively, post-treatment. With the exception of C, there was a statistically significant decrease in blood product use following treatment with rFVIIa. Of the survivors in each group, 6 of 12 CSP and 2 of 11 OBP died between 3 and 30 days post-treatment from causes other than bleeding. Mortality at 30 days for CSP and OBP survivors was 50% and 18% respectively, whereas overall 30 day mortality was 75% for CSP and 44% for OBP. Conclusions rFVIIa is effective in decreasing blood product use and promoting hemostasis in patients with intractable bleeding associated with cardiac surgery and a variety of other causes.

Introduction Recombinant factor VIIa (rFVIIa), originally developed for the treatment of acquired inhibitors associated with hemophilia [ 1 , 2 ], has been successfully used for bleeding due to acquired or congenital thrombocytopathy [ 3 , 4 ], extensive trauma and a variety of surgical procedures, including anecdotal reports of use in cardiac surgery patients [ 5 - 9 ]. It reverses the effect of warfarin in healthy volunteers [ 10 ], and corrects the prothrombin time in patients with hepatic failure [ 11 ]. Most critically ill patients with trauma suffer profound coagulopathy. Coagulation abnormalities in these patients have been attributed to multiple factors including disseminated intravascular coagulation (DIC), excessive fibrinolysis due to release of tissue plasminogen activator (tPA), dilutional coagulopathy from fluid replacement and massive blood product transfusion, dysfunctional platelets, and metabolic abnormalities including acidosis and hypothermia [ 12 - 18 ]. In addition, the use of cardiopulmonary bypass in patients undergoing cardiac surgical procedure may exacerbate the coagulopathy [ 19 ]. Although the mechanism of action of rFVIIa remains unclear, many investigators have suggested that it binds to the surface of activated platelets and directly activates Factor X, thus bypassing the early steps of the coagulation cascade. Activated Factor X (Xa) then combines with activated Factor V (Va) on the platelet surface, leading to rapid conversion of prothrombin to thrombin [ 20 , 21 ]. Hemostasis is promoted through high concentrations of thrombin generated near activated platelets at the site of vascular injury. Based on its mechanism of action, rFVIIa may be effective in controlling hemorrhage due to trauma, surgery and other causes. The present report is a retrospective review of 40 consecutive patients treated with rFVIIa (NovoSeven, Novo Nordisk, Denmark) for intractable hemorrhage. Twenty-four of these patients had bleeding associated with cardiac surgery, and the remaining 16 suffered with bleeding from other causes. The need for transfusion of blood products both before and after treatment and the mortality at 4 hour and 30 day were assessed. Patient characteristics were also assessed as risk factors for mortality. Methods Patients The study was approved by the Institutional Board Review of the Washington Hospital Center, Washington DC, USA. The medical records of 40 consecutive patients treated with rFVIIa between July 2001 and June 2003 at the Washington Hospital Center, a tertiary care teaching hospital and level 1 trauma center, were reviewed. The patients were divided into two groups: 24 patients who had undergone cardiac surgical procedures (CSP), and 16 patients who had bleeding from other causes (OBP). There were more male patients in both groups (19 males and 5 females in CSP, 10 males and 6 females in OBP). The median age in the CSP was higher at 65 years (range 26–85 years) compared to that of OBP (median age 42; range 23–82 years). The type of surgery in the CSP group included 15 coronary artery bypass grafts (CABG), 3 aortic arch repairs either elective or for dissection, 2 aorta rupture repair (one also underwent CABG), 3 valve replacements and 1 ventricular rupture. In the OBP group, 5 patients had suffered either stab or gunshot wounds, 2 had traumatic liver lacerations, 2 had undergone joint replacement, 3 had clotting factor abnormalities (acquired Factor VIII inhibitor, congenital Factor VII deficiency, acquired VWF deficiency), and one each had post partum hemorrhage, warfarin overdose, bowel resection, and trauma from a motor vehicle accident. Administration of rFVIIa Commercially available rFVIIa, (Novo-Seven, Novo Nordisk, Denmark) was used in all patients. There were no predetermined criteria for administration. In most patients, treatment was given after extensive blood product support had failed to control hemorrhage, and the decision to treat was made jointly by the surgeon, hematologist and blood bank pathologist. rFVIIa was administered as a bolus dose of 90 mcg/kg. The first two patients received a second dose of 90 mcg/kg 6 hours after the first. Endpoints and Assessment of Risk The total number of products given before and up to twenty-four hours after rFVIIa administration, or until death, whichever came first, was quantified. Patients who died within 4 hours of treatment were not evaluable because blood product support was suspended shortly after rFVIIa administration in these hemodynamically unstable patients deemed non-salvageable. Also, given the short half-life of rFVIIa, this time frame did not allow for an accurate determination of efficacy. The number of patients surviving at 4 hours and 30 days after the administration of rFVIIa was compared. Thirty days was chosen as an end point since this is the operative mortality cut off point, as defined by the Society of Thoracic Surgeons, for inpatient and out of hospital death. For patients surviving more than 4 hours, charts were reviewed for thromboembolic complications during hospitalization. In an attempt to determine risk factors for poor outcome, patients were assessed for age, gender, comorbid conditions, left ventricular ejection fraction (LVEF), use of cardiopulmonary bypass (CPB), hemoglobin, platelet count, arterial blood gas results and coagulation parameters including prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and D-Dimers. Laboratory parameters were not available for some patients. Statistical Considerations The Paired Student's t-Test was used to compare the number of blood product transfusions before and after treatment with rFVIIa, and, since the number of pair groups was less than 50, this result was confirmed with the Wilcoxon Matched-Pairs Signed-Ranks Test. Results Cardiac Surgery Patients (CSP) Outcome Twelve of the 24 patients survived for more than 4 hours after the administration of rFVIIa, and the results of their pre- and post-treatment blood product use are listed in Table 1 . From the start of surgery until the administration of rFVIIa, patients received on average 17 U of R (range 5–39), 18 U of P (range 6–37), 18 U of F (range 6–33), and 15 U of C (range 0–50). Post rFVIIa treatment, an average of 6 U of R (range 0–28), 10 U of P (range 0–19), 9 U of F (range 0–28) and 4 U of C (0–20) were transfused. The difference between the pre- and post-transfusion requirement was significantly lower for all blood products: R (p = 0.007), P (p = 0.038), F (p = 0.009) and C (p = 0.03). Eight of the 12 patients showed a rapid hemostatic response to rFVIIa, as measured by a decrease in R transfusion to an average of 2 U (range 0–6). The other 4 patients (Patient # 24, 27, 32 and 35) continued to require blood product support beyond 24 hours after rFVIIa administration. Table 1 Summary of rFVIIa treated cardiac surgery patients (CSP) who survived >4 hours P# Age sex Procedure Co-morbidities Response to rFVIIa Outcome CPB EF% Pre rFVIIa Post rFVIIa R P F C R P F C 1 75M CABG - >45 18 18 8 10 0 4 0 0 Survived 13 36M Aortic arch repair + >45 28 18 16 10 6 19 20 10 Died 14 64M Aortic dissection - >45 39 37 32 50 2 6 4 0 Survived 17 63M AVR/MVR repair - <25 7 18 16 15 1 12 6 0 Survived 18 70M CABG + >25 6 20 20 20 0 0 0 0 Survived 20 72M CABG - >25 21 12 10 0 0 0 0 0 Survived 24* 79M CABG + >25 10 12 12 10 5 4 4 0 Died 27* 64M CABG, IVAD + <25 30 30 33 20 28 16 28 20 Died 30 85M CABG, ruptured aorta + >45 13 12 6 10 3 6 8 0 Died 32* 83M CABG + >25 5 18 18 10 6 18 20 0 Died 33 78F Ruptured aorta + <25 14 18 20 20 3 12 6 0 Died 35* 54M Aortic arch repair + >45 9 6 18 10 15 24 12 20 Survived Average 17 18 18 15 6 10 9 4 P value .009 .038 .009 .03 P: Patient R: Red Blood Cell; P: Platelet; F: Fresh Frozen Plasma; C: Cryoprecipitate M: Male; F: Female CPB: Cardiopulmonary Bypass EF: Left Ventricular Ejection Fraction CABG: Coronary Artery Bypass Graft AVR: Aortic Valve Replacement MVR: Mitral Valve Replacement IVAD: Intraventricular Assisted Device * Patient continued to require blood transfusion past 24 hours post rFVIIa treatment. Six of the 12 patients who survived more than four hours were eventually discharged to home (Table 1 ). The remaining 6 patients (Patient # 13, 24, 27, 30, 32, and 33) died between 3 and 30 days after rFVIIa treatment from a variety of causes including multi-organ failure and infections. Amongst survivors beyond 4 hours, mortality at 30 days was 50% and overall mortality for the entire group was 75%. Of the 12 patients who died from multiorgan failure and hemodynamic instability within 4 hours of receiving rFVIIa, 11 died within 2 hours and 1 died within 4 hours (8 during surgery and 4 in the recovery room). Due to the limited time frame, it was not possible to determine if bleeding in these patients diminished after rFVIIa treatment. In the urgent setting in which these patients were treated, determining the amount and timing of blood loss and blood transfusion was difficult. Moreover, for patients deemed non-salvageable, blood product transfusion was suspended before response to rFVIIa could be determined. Risk factors and outcome in CSP Overall, 17 of 24 CSP were over the age of 60. Twenty underwent CPB, and 15 had a LVEF below 25%. Fourteen underwent mediastinal re-exploration for bleeding. Twenty-three patients had a significant coagulopathy defined as thrombocytopenia below 100,000/mm 3 , prolonged PT and PTT or both (data not given due to wide variations in the results). Seven of the 24 patients did not have a documented arterial blood gas (ABG) around the time of rFVIIa administration; however, among the other 17 patients, pH's of 7.0 in 1, 7.2 in 5, and 7. 4 in 11 patients were noted. With regards to rFVIIa administration, there was no correlation between pH, PT, PTT, thrombocytopenia, anemia and outcome. Of the 12 patients who died within 4 hours, all underwent CPB for over 30 minutes (time on CPB for 8 patients was more than 2.5 hours). All 12 had an LVEF of less than 25% and 7 had mediastinal re-exploration for bleeding. In contrast, only 3 of the 12 surviving patients had EF <25%, 8 had surgery with CPB and 7 underwent mediastinal re-exploration. The median age and coagulation parameters were not different between the two groups. Other Bleeding Patients (OBP) Outcome Eleven of the 16 patients with bleeding due to other causes survived for more than 4 hours after rFVIIa administration, and the results of their blood product use before and after rFVIIa are summarized in Table 2 . From the beginning of surgery or time of trauma until the administration of rFVIIa, patients received an average of 10 U of R (range 2–28), 11 U of P (range 0–36), 14 U F (range 0–34) and 10 U of C (range 0–70). Post administration of rFVIIa, an average of 1 U of R (range 0–6), 2 U of P (range 0–6), 2 U of F (0–10) and 0 U of C were transfused over 24 hours. The difference between the pre- and post-rFVIIa treatment blood product requirement was significantly lower for R (p = 0.004), P (p = 0.017), and F (p = 0.017), but not for C (p = 0.122). Nine of the 11 showed a rapid response to rFVIIa, as measured by a decrease in R transfusion to an average of <0.5 U (range 0–3). The other 2 patients (Patient # 2 and 15) continued to require blood product support beyond 24 hours post rFVIIa treatment. Table 2 Summary of rFVIIa treated other bleeding patients (OBP) who survived >4 hours P# Age Sex Procedure Co-morbidities Response to rFVIIa Outcome Pre rFVIIa Post rFVIIa R P F C R P F C 2* 48F Colectomy Colon cancer 4 6 24 70 2 0 2 0 Died 15* 26F Cystectomy, nephrectomy, pelvic fracture, termination of Pregnancy MVA, 20 weeks gestation 18 12 8 0 6 6 10 0 Survived 16 53M Left femur fixation Multiple myeloma 12 12 6 0 0 0 0 0 Survived 19 82M Shoulder replacement HTN, CAD, Renal failure 7 10 20 10 2 6 4 0 Survived 21 54F Liver laceration Trauma 23 17 34 20 3 6 10 0 Survived 22 24M Neck soft tissue trauma FVII deficiency 2 0 4 0 0 0 0 0 Survived 23 64F Liver laceration Dialysis 28 36 33 0 0 0 0 0 Died 28 30F Postpartum hemorrhage None 12 18 6 10 0 0 0 0 Survived 29 64M Gastrointestinal bleed FVIII inhibitor 2 0 0 0 0 0 0 0 Survived 38 60M Thyroidectomy VWF deficiency 4 12 12 4 0 0 0 0 Survived 40 40F Splenic subcapsular bleed Amyloidosis, coumadin overdose 4 0 8 0 0 0 0 0 Survived Average 10 11 14 10 1 2 2 0 P value .004 .017 .017 .122 P: Patient R: Red Blood Cell; P: Platelet; F: Fresh Frozen Plasma; C: Cryoprecipitate M: Male; F: Female EF: Left Ventricular Ejection Fraction VWF: von Willebrand Factor * Patient continued to require blood transfusion past 24 hours post rFVIIa treatment. Nine of the 11 surviving patients were discharged to home and 2 (Patient # 2 and 23) died between 3 and 30 days from a combination of infection, cardiac arrest and multiorgan failure. Amongst survivors beyond 4 hours, mortality at 30 days was 18% and overall mortality for the entire group was 44%. All five of the 16 patients who died within four hours of rFVIIa treatment had massive injuries either from gunshot or stab wounds. Upon arrival to the emergency room, these patients were hemodynamically unstable, with hemoglobin values ranging between 3–6 g/dL. rFVIIa was given as a last resort, but these patients were not evaluable since transfusions were suspended shortly after infusion of rFVIIa. Risk factors and outcome in OBP The median age of this group was 42 years. None underwent CPB, and two of the 7 patients tested had an EF <25%. Only 1 patient had a second exploratory laparotomy for bleeding. The patient suffering with a warfarin overdose had a splenic subcapsular bleed complicating systemic amyloidosis. At the time of rFVIIa administration, a wide variation in laboratory parameters was observed in these patients (data not shown). There was no difference in coagulation parameters between patients who died and those who survived. Eight of the 16 patients did not have an ABG documented. In the 5 patients who died within four hours, the pH was lower (6.9–7.0) than for those who survived (7.3–7.4). Thromboembolism after rFVIIa Only one of the 23 patients in both the CSP and OBP groups who survived more than 4 hours had a documented thromboembolic complication after receiving rFVIIa. Deep vein thrombosis (DVT) of the left subclavian vein was confirmed by a Doppler ultrasound 2 days after rFVIIa administration. The thrombus was related to a central line placed before rFVIIa treatment. No other clinical or laboratory evidence of thromboembolism was documented in the medical records of the other patients throughout their hospital stays. None of the other 17 patients in both the groups who died within 4 hours were evaluable for thromboembolic complications. Discussion This retrospective case series suggests that rFVIIa is effective in promoting hemostasis even when given as a last life-saving measure in poor prognosis patients with massive, transfusion-refractory hemorrhage. A statistically significant decrease in blood product transfusion was evident in 12 CSP and 11 OBP who survived more than 4 hours after rFVIIa infusion. Six of 12 CSP and 2 of 11 OBP died later between 3 and 30 days after rFVIIa infusion, but these deaths were due to causes other than hemorrhage. Despite the obvious limitations of a retrospective analysis, this is the largest series of such patients reported in the literature to date. Al Douri et al [ 8 ], described 5 patients who underwent open-heart surgery for valvular disease and experienced uncontrollable intraoperative or postoperative bleeding unresponsive to a blood product support protocol. The study was prospective, had well-defined criteria for "excessive bleeding" and excluded patients with a history of ischemic heart disease, stroke or venous thromboembolism. Hemostasis was achieved in all patients following a single dose of 30 ug/kg of rFVIIa, and there was no mortality from bleeding, although 1 patient died 3 days later of an unrelated cause. Overall, the 4-hour and 30 day mortality in the present series was high compared to that of Al Douri et al [ 8 ], but it is difficult to compare these two very different patient populations. Factors predictive of inferior outcome after cardiac surgery include age over 60, low LVEF, significant co-morbidity, the use of CPB and re-exploration after initial surgery [ 22 - 24 ]. The use of CPB increases the rate of fibrinolysis and induces an inflammatory response [ 19 , 25 ]. Moreover, prolonged time on CPB, defined as 2.5 hours or more, is associated with increased bleeding and need for re-exploration. Two-6% of patients undergoing CABG require re-exploration for bleeding and is associated with a mortality rate of up to 22% [ 19 , 26 , 27 ]. In this series, the CSP group had multiple poor prognostic factors. The median age was 65 years and in addition, all 12 of the CSP who died within 4 hours and 3 of 6 CSP who died later had preoperative LVEF of less than 25%. All of the 12 CSP who died within 4 hours in this series, required CPB and 8 of these patients were on CPB for more than 2.5 hours. More than half of our CSP (14 of 24) required re-exploration, which undoubtedly influenced the mortality rate. Both, the CSP and OBP groups had a significant decrease in blood product use after rFVIIa treatment but the short and long-term survival appeared to be worse in CSP probably due to multiple poor prognostic factors as discussed above. Poor outcome has also been independently associated with peri-operative transfusion of more than 7 units of R [ 28 ]. The CSP and OBP who survived received an average of 18 and 11 units of R in the peri-operative period, respectively. The R requirement of patients who died within 4 hours in both groups was probably higher but could not be reliably estimated. All 5 of the patients in the OBP group who died within 4 hours suffered from major blood vessel injury as a result of stabbing or gun shot wounds. These patients were unstable upon arrival to the emergency room with hemoglobins in the range of 3–6 g/dL. Excessive hemorrhage requiring massive transfusion can lead to hypothermia, DIC, excessive fibrinolysis, dilutional coagulopathy, and metabolic acidosis, which may further exacerbate bleeding and morbidity. [ 14 - 17 ] A reduction in the pH to 7.0 nearly abolishes rFVIIa activity as reported by Meng et al [ 18 ]. In this present series, there was no obvious correlation between the coagulopathy and mortality in both the CSP and OBP groups. Five of the OBP who died within 4 hours had a pH of 7 or less. However, acidosis did not appear to play an important role in CSP. The optimal dose and timing of administration of rFVIIa for these patients is unknown. The standard dose (90 mcg/kg) was first used in hemophiliacs and was based on the in vitro dose-dependent reduction in aPTT in plasma from these patients [ 1 , 29 ]. The 90 mcg/kg dose of rFVIIa corresponds to concentration of 1.25 mcg/ml FVIIa in plasma. Martinowitz et al [ 7 ], used a median dose of 120 mcg/kg (range 120–210 mcg/kg) to achieve hemostasis in 7 cases of trauma. In 2 patients, 120 mcg/kg was insufficient, and 2 additional doses of 60 mcg/kg were required to achieve hemostasis. Kenet et al. [ 30 ], have shown better efficacy with a 300 mcg/kg bolus dose followed by continuous infusion. The standard therapeutic dose of 90 mcg/kg of rFVIIa was used in this series of patients although a lower dose of 30 mcg/kg dose has also been shown to be effective [ 8 ]. However, in considering the data from the aforementioned studies, it would appear that higher initial doses, or additional doses, might improve outcome. It is also tempting to speculate that earlier treatment with rFVIIa may have prevented rapid clinical deterioration and complications associated with massive blood product transfusion in this series of patients. This question would best be addressed in controlled studies using standard dosing protocols and well defined criteria for intractable hemorrhage. Although rFVIIa is expensive, it would appear to be cost effective when compared with the combined cost of large amount of blood products. Previous clinical experience with rFVIIa supports a good safety profile in patients with hemophilia and trauma. Less than 1% of patients receiving rFVIIa had thrombosis and thrombosis related complications [ 21 , 31 ]. In our series, only one patient had a deep vein thrombosis of the left arm associated with an indwelling line and not considered to be due to rFVIIa. There were no other documented cases of thrombosis or microemboli. Conclusions This retrospective study suggests that rFVIIa can play a beneficial role as an adjunctive hemostatic agent in patients after cardiac surgery or extensive trauma who experience bleeding that cannot be controlled by conventional therapies. Prospective studies are necessary to determine optimal patient selection, and dose and timing of rFVIIa administration.

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548291

Human cytomegalovirus plasmid-based amplicon vector system for gene therapy

We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis -acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.

Background HCMV is a member of the betaherpesvirus family [ 42 , 48 ]. Other members of this family include human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7), and all are widely distributed in human populations. During productive replication, the 230 kilobase pair (kbp) viral genome replicates by a rolling circle mechanism, which generates long head-to-tail concatemers that are cleaved to unit length and packaged in capsids. The state of the HCMV genome during latency remains unidentified and is likely to be circular and extrachromosomal [ 6 ]. The HCMV genome has been detected in cells within the hematopoietic lineage as early as CD34+ progenitors and up through differentiated macrophages [ 23 , 29 , 38 , 54 ]. Defective HSV viruses created by high multiplicity serial passage of virus stocks have been described on numerous occasions and have been characterized in detail at the molecular level [ 13 , 18 , 31 , 43 , 52 , 67 ]. Naturally occurring defective HSV viruses and laboratory derived HSV amplicon vectors are composed of head-to-tail tandem reiterations of subgenomic regions containing a functional origin of DNA replication (Ori S or Ori L ) and a DNA cleavage/packaging signal [ 3 , 4 , 30 , 57 , 60 - 62 ]. These two cis -acting functions can be relatively small ranging from ca . 90–150 base pairs (bp) for the ori and ca . 250–300 bp for the a sequence. The functional HCMV oriLyt is much more complex than either of the HSV oris; the HCMV oriLyt consists of multiple direct and inverted repeats and extends over at least 1500 bp [ 1 , 2 , 24 , 37 ]. HCMV is unique among the herpesviruses in not having an origin binding protein homolog that is required for DNA replication [ 45 ]. The HCMV a sequence varies in size from ca . 550 bp to 762 bp, however, the conserved pac-1 and pac-2 cis -elements which determine the sites for cleavage of replicated viral DNA are present [ 15 , 28 , 58 , 64 , 65 ]. In contrast to HSV, HCMV does not efficiently produce defective virus genomes, this difference may be related to the distinct biology of the two viruses [ 45 ]. However two reports described the identification of what may potentially be HCMV defective viruses created by serial high multiplicity passage [ 47 , 59 ]. These reports characterized HCMV defectives as very large subgenomic DNA molecules, in some cases extending over two thirds of the genome. In addition to these replication defective HCMV viruses, a recent report by Borst et al . 2003 [ 7 ], described the construction of an HCMV amplicon. In this report we further utilized the HCMV amplicon for gene delivery to human CD34+ cells. HCMV infects cells of the hematopoietic lineage [ 34 , 38 , 39 , 55 , 68 ]. Viral genomes can be found in CD34+ cells from seropositive individuals and granulocyte-macrophage progenitors and differentiated macrophages can be infected experimentally [ 56 ]. We were interested in determining whether the tropism of HCMV can be exploited to construct defective HCMV virus vectors (amplicons) targeted to hematopoietic stem cells. The general feasibility of such an approach for other cell types has been shown using other herpesviruses, e.g. HSV, EBV, and HHV-7 [ 20 , 25 , 26 , 30 , 35 , 49 , 70 , 71 ]. Methods Cells and virus HCMV Toledo (passage 8, from Dr. S. Plotkin, Aventis Pasteur, Doylestown, PA), and HCMV Towne var RIT (passage 134, from Dr. Plotkin via Dr. Ed Mocarski, Stanford University), were propagated in human fibroblasts (HF) cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, Kans.). Recombinant HCMV, RC2.7EGFP, expressing enhanced green fluorescent protein (EGFP) (Clonetech, Palo Alto, CA), under the control of the major early 2.7 promoter, was constructed by cotransfection of plasmid pEAG2.7EGFP with a set of overlapping cosmid clones derived from HCMV Toledo (G.M. Duke, unpublished data). Plasmid constructions Plasmid pON205 (Spaete and Mocarski, 1985), contains the Towne strain a sequence, was obtained from Ed Mocarski (Stanford University). pEAG2.7EGFP was derived by cloning the EGFP gene from plasmid pEGFP-N2 (Clonetech, Palo Alto, CA) between the Eag I and Sma I site of the β2.7 gene taken from Toledo (G.M. Duke, unpublished data). HCMV amplicon plasmid Tn9-8 was derived by inserting the 6 kpb Dra I fragment of Towne var RIT (corresponding to nucleotides 91,166 – 95,909 relative to AD169) (Figure 1B ), spanning the HCMV oriLyt into the Eco RI site of pON205. Tn9-8 was partially sequenced by single-cycle and multicycle dideoxy-nucleotide chain termination method of Sanger et al ., [ 51 ]. The plasmids designated Tn9-8GF5 and Tn9-8GF7 incorporating the EGFP gene with the HCMV Major Immediate Early (MIE) promoter and SV40 poly A sequence was isolated as a 2,334 bp Nsi I fragment from plasmid pEGFP-N2 (Clonetech, Palo Alto, CA), and cloned into the HCMV amplicon Tn9-8 at the Pst I site in both orientations. The gpt gene in Tn9-8- gpt was derived by cloning a PCR fragment from Escherichia coli DH5α using the primer pairs 5'CTGCAGCTAGTCTAGACTGGGACACTTCACATGAGC3'and 5'CTGCAGCTATGTATCTAGAGCCAGGCGTTGAAAAGATTA3'. Figure 1 Schematic representation of amplified region near ori Lyt of the HCMV genome. A . Restriction enzyme map of minimal ori Lyt and adjacent region of heterogeneity (block). B . Region of heterogeneity shown as a dimer. Arrow indicates junction of the repeat segment. The number 91,166 to the left of the restriction map corresponds to the nucleotide position of the AD169 genome (EMBL accession number X17403). C . Autoradiograph of Southern blot utilizing a minimal ori Lyt probe, pON2623 (Kemble et al., 1996). Monomers and dimers are depicted with one and two arrows, respectively. Trimers and tetramers can be seen in the Towne var RIT viral stock. Generation of viral stocks containing amplicons Plasmid DNA was transfected by CaPO 4 precipitation of approximately 4 μg of Tn9-8 amplicon DNA. The Tn9-8 DNA was transfected into approximately 1 × 10 6 passage 16 human fibroblast (HF) cells. At 24 hours post transfection, the cells were infected with CMV Towne at a multiplicity of infection (MOI) of 5 plaque forming units (PFU) per cell. Fresh medium was added to cells four days after infection and cells were harvested at 6 to 7 days post infection as described previously (Spaete and Frenkel, 1982). Virus stocks are prepared by three freeze-thaw cycles. Serial passages of amplicon-containing viral stocks on fresh HF cells were superinfected with CMV Towne as a helper virus at a MOI of 1. Southern blot analysis Viral DNAs were digested with restriction enzyme, electrophoresed in 0.8% agarose gels, transferred to Hybond-N+ nylon membranes (Amersham Corp.), (Maniatis et al., 1989), and immobilized with a UV Crosslinker 1000 (Hoefer Scientific Instruments, San Francisco, CA). DNA on the membrane was probed with fluorescein-labeled pUC9 DNA using conditions previously described (Spaete and Mocarski, 1985). Isolation of CD34 + cells and infection with CMV The isolation of cord blood CD34+ stem cells was carried out by All Cells Inc. (Berkeley, CA) using CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA). The positive selection of the CD34+ cells was carried out using hapten-conjugated antibody to CD34+ followed by anti-hapten antibody coupled to MACS Microbeads. The magnetically labeled cells are enriched on positive selection columns in the magnetic field. The purity of the CD34+ population was >95% as analyzed by flow cytometry. The purified CD34+ cells were suspended in Iscove's modified Dulbecco's Minimal Essential Medium containing 5% fetal bovine serum. 2 × 10 5 CD34+ cells were used for each infection with TN9-8GF5 amplicon containing stocks, RC2.7EGFP virus, CMV Towne virus, or uninfected cell control. The cells mixed with virus were centrifuged at 500 × g for 10 mins at room temperature and were then placed in 37°C water bath for one hour. Following this the cells were cultured in 6-well cell culture plates (Costar) for 18–72 hours. At the end of the incubation the cells were harvested for CD34 staining. EGFP expression and immunostaining for flow cytometric analysis Amplicon containing viral stocks prepared from passage 1 were used to infect HF or human CD34+ cells maintained in 12 well culture plates. At 24 hour intervals post infection, the wells were observed for EGFP expression with a Nikon TE2000 microscope under UV illumination. Immunostaining for CD34+ cells was done using Phycoerythrin (PE)-conjugated anti-CD34 antibody (Becton Dickinson, San Jose, CA). Infected or control cells were incubated with 20 μl of PE-labeled anti-CD34 antibody for 45 minutes at room temperature and subsequently were washed twice with PBS containing 0.1% BSA. The cells were directly analyzed for EGFP and CD34+ staining on a FACSCalibur instrument (Becton Dickinson, San Jose, CA), at 18 and 36 hours post infection. Results In order to exploit the natural tropism of HCMV for cells of the hematopoietic lineage, in a nonlytic manner, an HCMV amplicon i.e. a plasmid containing the HCMV oriLyt and a sequences was constructed. Theoretically, due to the large size of the HCMV genome, an amplicon derived from this virus should be able to carry the large DNA inserts and be capable of efficient introduction into hematopoietic cells by infection. Heterogeneity at oriLyt During analysis of cosmid clones of HCMV strain Towne, sequence heterogeneity was observed in the Eco RI E fragment of Towne that was not present in the Toledo strain [ 27 ]. The Eco RI E region spans in part the complex oriLyt region [ 1 , 2 , 24 , 37 ]. Sequences in a 1.2 kbp repeat fragment were shown to give rise to the heterogeneity observed at this locus in the Towne genome (Figure 1 ). The coordinates of a single repeat unit starts at nucleotide 94,561 relative to the AD169 sequence and end at nucleotide 95,807 [ 10 ]. This segment can repeat at least three times in Towne strains from different passage histories (Fig. 1 ). This heterogeneity is different from the 189 bp repeat region previously described for the Towne strain oriLyt which occurs near the Bam HI sites in Figure 1 (nt 93337–93525 relative to AD169), [ 11 , 12 ]. Since Towne replicates to relatively high titers in cell culture, it was deemed advantageous to incorporate this heterogeneity in the origin containing sequences to be used in the amplicon construct. Construction of the HCMV amplicon As a test of the feasibility of the system, an HCMV amplicon was constructed which incorporated the two cis -acting functions required for the propagation of the defective virus genomes in the presence of helper virus (Figure 2 ). HCMV amplicon plasmid Tn9-8 was derived by inserting the 6 kpb Dra I fragment of Towne (corresponding to nucleotides 91,166–95,909 relative to AD169) [ 10 ] (Figure 1B ), spanning the HCMV oriLyt into the Eco RI site of pON205. The resulting amplicon was designated Tn9-8 (Figure 2 ), and was partially sequenced to verify its structure. Figure 2 Schematic representation of the HCMV amplicon plasmids Tn9-8 gpt , Tn9-8GF7 and Tn9-8GF5. The EGFP expression cassette was cloned in two orientations in the unique Pst I site in Tn9-8. The gpt expression cassette was cloned between the unique Pst I and Hind III sites. Generation of viral stocks containing amplicons As a test of the ability of this construct to function as an amplicon, plasmid Tn9-8 was transfected in human fibroblast (HF) cells, and subsequently infected with HCMV Towne strain at an MOI of 5 to provide helper virus replication functions. Seven days later, infected cells were harvested, sonicated, and viral stocks were prepared for passage to fresh HF cells. Fresh HF cells were infected with the progeny of the transfection/infection and incubated for 7 days. The DNA from these infected cells was harvested (designated passage 1), restricted with Hind III and Dpn I, and Southern blotted [ 36 ]. Southern blot analyses of DNA demonstrated that Tn9-8 was susceptible to digestion with Dpn I, consistent with replication of the plasmid in bacteria (Figure 3 , lane 2). In contrast, Tn9-8 in infected cells was resistant to Dpn I demonstrating that it had replicated in eucaryotic cells (Figure 3 , lanes 3–5). This observation is consistent with replication and packaging of Tn9-8 into infectious virions. These results demonstrate that foreign DNA sequences, exemplified by the plasmid pUC9, can be introduced into defective genomes that are packaged and propagated in serially passaged virus stocks. To examine whether these results were the consequence of amplicon replication and packaging or integration of the amplicon plasmid into the helper virus, DNA prepared from passage 1 infected cells was digested with Cla I, Xba I, Afl II and Dpn I. These enzymes digested the Towne helper virus DNA to fragments no larger than 13.6 kbp but do not cut within Tn9-8. The amplicon DNA was significantly larger than 23 kbp consistent with the amplicon being replicated as a concatamer (Figure 4 ). This result indicated that the high molecular weight DNA containing plasmid sequences was packaged independently and was not integrated into helper virus. Figure 3 Southern blot analysis of passage 1 of HCMV amplicon DNA probed with plasmid pUC9. Lane 1. Plasmid Tn9-8 linearized with Hind III serves as a marker for correct migration of monomeric repeats. Lane 2. Plasmid Tn9-8 restricted with Hind III and Dpn I as a control for non-replicating DNA. Lanes 3–5. Infected cell DNAs restricted with Hind III and Dpn I. The signal in lanes 3–5 (arrow) demonstrates authentic replication and packaging of amplicon Tn9-8 in eucaryotic cells. Figure 4 Southern blot analysis of high molecular weight HCMV amplicon DNA at passage 1 probed with plasmid pUC9. DNA prepared from passage 1 infected cells was digested with Cla I, Xba I, Afl II and Dpn I, Southern blotted and probed with plasmid pUC9. The samples are from those shown in Fig. 3, lanes 3 and 5. The high molecular weight DNA containing plasmid sequences (arrow) demonstrates the major hybridizing species migrating slower than the 23 kbp lambda DNA Hind III digest indicated as the marker on the left of the autoradiograph. Packaged defective viral genomes derived from Tn9-8 or a derivative containing a selectable marker (Tn9-8- gpt ), were serially passaged in HF cells. Defective viruses could be detected at passage 3 when probed with plasmid pUC9; however, the copy number appeared to diminish upon serial passage (not shown). Selection with mycophenolic acid on Tn9-8- gpt amplicons did not enhance recovery. Rescue of monomeric repeat units in bacteria Concatemeric DNA was prepared from passage 2 and 3 virus stocks containing the defective virus genomes (Tn9-8- gpt ), digested with Pst I and Hind III, respectively, in order to analyze monomeric repeat units. The Hind III-digested DNA was circularized by ligation and used to transform E. coli bacteria to analyze structure and to demonstrate shuttle vector capability between eucaryotic and bacterial hosts. A number of plasmids prepared from the rescue attempt had a restriction enzyme pattern indistinguishable from the input (Fig. 5A , lanes 1 and 2). Other plasmids however exhibited the expected restriction pattern consistent with a head-to-tail amplification of the a sequence (lanes 3 and 4). Digestion with Nae I produced a fragment of the predicted size of a unit length a sequence (762 bp), and this product hybridized with an a sequence specific probe ( Pst I- Sgr AI fragment from Tn9-8), (Figs. 2 and 5B , lanes 3 and 4). This type of amplification has been readily seen in restriction enzyme digested DNA preparations of parental genomes of both HSV and HCMV [ 31 , 40 , 41 , 58 , 64 , 69 ] and has also been observed in HSV amplicons using Southern blot hybridizations [ 15 ]. Figure 5 Tn9-8- gpt was rescued from concatamers following serial passage in HF cells. (A) Tn9-8- gpt was passaged in HF cells and monomer units were recovered by linearizing concatemeric DNA from serial passage 3 with Hind III and cloning in bacteria (lanes 2 and 4) and also by linearizing passage 2 DNA with Pst I and cloning in bacteria (lane 3). Lane 1 represents the unpassaged clone for comparison. Following rescue in bacteria, DNA was prepared and cut with Nco I. Fragments were separated on an agarose gel and visualized with ethidium bromide staining (left panel). The gel was transferred to a nylon membrane and probed with an a sequence specific probe (right panel). (B) Fragments from an Nae I digest were also separated on an agarose gel and visualized as in panel A. The gel was transferred to a nylon membrane and probed with an a sequence specific probe. Lane 5 shows a 100 bp ladder. Lanes 3 and 4 show a ca . 800 bp fragment that hybridizes to a sequences. Expression of heterologous genes in an HCMV amplicon To demonstrate that the HCMV amplicon could be used as a vector system to support the expression of a foreign gene, EGFP under the transcriptional control of the HCMV major immediate early (MIE) promoter was used as test reporter gene. Two resulting amplicon plasmids designated Tn9-8GF5 and Tn9-8GF7 both expressed EGFP following transfection of HF cells in the absence of helper virus, as expected (not shown). Packaged amplicons were generated by introduction of Tn9-8GF5 into cells and infecting with HCMV 24 hours later at an MOI of 5. Transfection-derived viral stocks were passaged onto fresh HF cells supplemented with Towne helper virus at an MOI of 1. Viral stocks prepared from passage 1 were used to infect HF cells and grown on 12-well tissue culture plates. A limited number (ca. 0. 1%) of brightly fluorescing cells could be seen by microscopic examination at 24, 72 and 96 hours post-infection (Figure 6 ). This demonstrates that a foreign gene can be expressed in the context of a HCMV amplicon viral stock in infected HF cells. Figure 6 Fluorescent Microscopic Analysis of TN9-8GF5 amplicon infected cells. Human fibroblast cells (HF) or human cord blood CD34 + cells were infected with TN9-8GF5 amplicon-containing stocks, or mock infected. Cells were observed at different time-points 24, 72 and 96 hrs post infection with TN9-8GF5 amplicon under the fluorescent microscope (Nikon TE2000 microscope). EGFP expressing fluorescent cells were observed in the TN9-8GF5 amplicon infected human fibroblast cells or human CD34+ cells at different time-points. Control uninfected cells were negative (not shown). To test the utility of the HCMV amplicon in gene therapy or gene delivery, we used packaged amplicons in viral stocks to infect and deliver an expressed gene into human CD34+ progenitor cells. Viral stocks containing amplicons carrying EGFP under the transcriptional control of the HCMV major immediate early (MIE) promoter prepared from passage 0 and passage 1 were used to infect CD34+ cells derived from cord blood. Starting at 24 h after infection, CD34+ cells were examined for EGFP expression by fluorescent microscopy. EGFP expression was observed in TN9-8GF5 amplicon-infected CD34+ cells starting at 24 h post-infection. The cells remained positive for EGFP expression for more than 96 hrs, at which point the cells were terminated (Figure 6 ). In a separate experiment, at 36 hours post infection, the cells were stained with PE-labeled anti-CD34 and analyzed for the CD34 marker and EGFP expression. EGFP expression was observed in the CD34+ population in the TN9-8GF5 amplicon (0.3%, 0.1%) (Figure 7a , & 7b ) or the CMV-EGFP virus (0.6%) (Figure 7c ) at 36 hours post infection. The CMV Towne control-virus infected cells or uninfected CD34+ cell control were negative for EGFP expression (Figure 7d , & 7e ). A small population (7–12%) of the cells lost expression of the CD34+ marker upon in vitro culture. EGFP expression was also observed in a CD34(-) population infected with either TN9-8GF5 amplicon containing viral stocks (0.8%, 0.1%) or with the CMV-EGFP virus, RC2.7EGFP (3.6%) (Figure 7a, 7b & 7c ). The CMV Towne infected cells or uninfected control cell also had a significant CD34 negative population but were negative for EGFP expression (Figure 7d , and 7e ). These results clearly demonstrate that CD34+ cells can be infected with replication competent or incompetent CMV vectors expressing a foreign gene. Figure 7 Flow cytometry analysis of human cord blood CD34 + cells infected with CMV amplicon containing stocks, virus, or uninfected cell control. TN9-8GF5 amplicon ( a,b ), CMV-EGFP (RC2.7EGFP) virus ( c ), CMV (Towne) infected ( d ), or control uninfected human cord blood CD34 cells ( e,f ), were stained 36 hours post-infection with PE-antiCD34 antibody ( a-e ), or were left unstained ( f ), and were analyzed for two-color cytometry analysis using a FACS Calibur instrument. The dot-plots are generated using Cell Quest software and reveal the EGFP + cells populations. Numbers in the upper right and lower right quadrants indicate percentage of the EGFP + CD34 + and EGFP + CD34 - cells respectively. A frequency lower than 0.01% is considered negative. Conclusions We have shown that a replication-defective virus vector system that is derived from HCMV is capable of delivering and expressing foreign genes in infected primary cells including progenitor stem cells such as human CD34+ cells. Further improvement and optimization of the system offers the potential to deliver gene-based therapies to multipotent cells. Advantages for use of the HCMV amplicon Foremost among the advantages of the vector system we have described is the potential ability to efficiently infect and deliver genetic information to hematopoietic stem cells (CD34+) and other dividing and non-dividing cell types which may support HCMV infection [ 34 , 38 , 39 , 55 , 68 ]. Genetic hematological disorders such as thalassemias and sickle-cell anemia and other hemaglobinopathies could therefore be targeted for therapy with this strategy. Another potential advantage for the system is that vector DNA could possibly be maintained as an episome with minimal concern for the potential consequences of random integration of vector DNA (i.e. activation of oncogenes or inactivation of tumor suppressor genes). In order to insure efficient segregation as an episome, the EBV latent replication origin, oriP, and the transactivator, EBNA-1, could be added as was previously shown for another hybrid herpesvirus vector [ 71 ]. However such a modification may not be necessary because HCMV genomes appear to be carried continuously in cells of hematopoietic origin in infected individuals. Yet another potential advantage as with other herpesviral vectors, is that the HCMV vector system should have the capacity for very large inserts. Infection of CD34+ cells with HCMV The infectivity of CD34+ cells from seropositive and seronegative subjects with HCMV has been tested both in vivo and in vitro [ 53 ]. Furthermore, hematopoietic stem cells are also reported as a site for HCMV latency. Efficient transduction of human CD34+ cells with retroviral and non-viral vectors has been unsatisfactory due to the lack of maintenance of high levels of expression of the transgene following engraftment of the engineered cells [ 16 ]. The HCMV MIE promoter may not be the right promoter for optimal expression in a CD34+ cells, since it has been shown that in the context of a lentiviral-based gene transfer system this promoter appeared to function less efficiently due to a cell-type specific expression defect [ 16 ]. The approaches to improving the efficiency of gene transfer into human cells have focused on improving gene delivery vectors and optimizing ex vivo culture conditions, which preserve the developmental properties of the stem cells [ 14 , 22 ]. Umbilical cord blood is recognized as a rich source of hematopoietic CD34+ stem cells [ 33 ]. In our experiments we used cord blood derived CD34+ cells for infection with HCMV amplicon containing stocks or HCMV-EGFP virus. However, bone marrow derived CD34+ cells have also been shown to be infectable in vitro with HCMV [ 34 ]. Gentry & Smith [ 21 ], reported a progressive loss of primitive cell properties including a reduction of CD34 expression upon in vitro culture of cord blood derived CD34+ cells. In a separate study, cord blood derived CD34+ cells cultured with IL-3 in vitro showed a progressive decline of the CD34+ population and more differentiated cells originating in the CD34(-) population [ 9 ]. In our experiments with >95% pure cord blood derived CD34+ cell population, a loss of CD34 expression in a small percent population (9–12%) of stem cells upon in vitro culture has been observed. EGFP expression was also seen in the CD34(-) population (Fig 7a, 7b , & 7c ). It is possible that HCMV infection of CD34 cells could induce cell differentiation and loss of primitive properties including reduction of CD34 expression. Further studies of HCMV infection in CD34 cells will help in defining whether CD34+ infected cells undergo cell differentiation by increased expression of other markers such as CD33, CD38, HLA-DR or cytokines. It is also relevant to note here that HCMV virus carries homolog sequences for HLA-related and cytokine-related molecules and infection can induce cellular cytokines [ 5 , 8 , 19 , 32 , 44 , 46 , 50 , 66 ]. The HCMV amplicons contain only the cis -acting ori and packaging sequences, and have no structural gene sequences. However, amplicon containing viral stocks are a mixture with HCMV replication competent helper virus. HCMV induced cell-differentiating effect, if any, might be minimized using a helper virus-free amplicon system. In this regard, it should be possible to test a number of strategies to prepare helper virus-free stocks [ 17 , 63 ]. These preparations would be useful for therapeutic applications in immuno-compromised patients. Competing Interests The authors of this publication are supported financially by salary and shares of MedImmune Inc., during the completion of this work. A patent application has been filed with the United States Patent Office relating to the content of this manuscript. The authors have assigned all rights of ownership to MedImmune Inc. The authors declare that they have no other competing interest. Authors' Contributions KM carried out the expression analysis in human fibroblasts and CD34 cells. MNP & GMD generated amplicon stocks and MNP did the ' a ' sequence analysis. GWK provided critical intellectual input. RRS provided the original idea and experimental design as well as cloning of the amplicon, generation of amplicon stocks and Southern analysis.

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509282

Increased contractile responses to 5-hydroxytryptamine and Angiotensin II in high fat diet fed rat thoracic aorta

Background Feeding normal rats with high dietary levels of saturated fat leads to pathological conditions, which are quite similar to syndrome X in humans. These conditions such as hypertriglyceridemia, hypercholesterolemia, obesity, and hyperglycemia might induce hypertension through various mechanisms. Metabolic syndrome and the resulting NIDDM represent a major clinical challenge because implementation of treatment strategies is difficult. Vascular abnormalities probably contribute to the etiology of many diabetic complications including nephropathy, neuropathy, retinopathy, and cardiomyopathy. It has been shown that in Streptozotocin induced diabetic animals there is an increase in maximal responses to 5-Hydroxytryptamine and Angiotensin II. The purpose of this study was to evaluate High fat diet fed rats for the development of hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia and hyperglycemia and to assess their vascular responses to 5-Hydroxytryptamine and Angiotensin II. Methods Male Sprague Dawley rats were used for this study and were divided into two equal groups. One of the groups was fed with normal pellet diet and they served as the control group, whereas the other group was on a high fat diet for 4 weeks. Body weight, plasma triglycerides, plasma cholesterol, and plasma glucose were measured every week. Intraperitoneal glucose tolerance test was performed after 4 weeks of feeding. At the end of fourth week of high fat diet feeding, thoracic aortae were removed, and cut into helical strips for vascular reactivity studies. Dose-response curves of 5-Hydroxytryptamine and Angiotensin II were obtained. Results There was no significant difference in pD 2 , with 5-Hydroxytryptamine and Angiotensin II in both groups but E max was increased. Conclusions These results suggest that hypertension in high fat diet rats is associated with increased in vitro vascular reactivity to 5-HT and Ang II.

Background Syndrome X comprises a plethora of conditions such as obesity, dyslipidemia, impaired glucose tolerance, insulin resistance and hypertension [ 1 ]. It places stress on multiple organ systems and plays a significant role in the development of other related cardiovascular disorders. Western style diet, which contains high levels of fat, is also considered to be one of the main factors in the development of obesity and insulin resistance. The most common reason for the development of hyperinsulinemia (decreased hepatic insulin clearance and/or increased insulin secretion) from insulin resistance is obesity. Excess fat deposits in the white adipose tissue affects insulin mediated glucose metabolism in non adipose tissues, causes disordered insulin response and increases lipid deposition [ 2 ]. However, pathophysiologies of vascular complications in syndrome X have not been fully understood. 5-hydroxytryptamine (5-HT) is shown to be related to pathogenesis of vasculopathy in diabetes. Various studies in humans and rabbits have reported increased plasma 5-HT levels and enhanced contraction to 5-HT in diabetes mellitus [ 3 - 5 ]. Another vasopressor peptide, Angiotensin II (Ang II) is involved in cardiovascular complications of diabetes mellitus. Many have reported that the vascular Renin angiotensin system is one of the key systems in the etiology of vascular alterations in early stages of diabetes. It is already established that 5-HT and Ang II responses are altered in the aortic rings of streptozotocin (STZ) induced diabetic rats [ 6 , 7 ] and high fructose diet fed rats [ 8 ]. However, very little information is available in the literature regarding the vascular contractile responses to vasopressor agents such as 5-HT and Ang II in HFD fed rats. The aim of this study was to elucidate the contractile responses to 5-HT and Ang II in HFD fed rat thoracic aorta which will provide an avenue for further exploratory studies. We have selected HFD fed rat model for our study because it is a useful model of the putative effects of excess fat intake in humans and it represents the major sub type of diabetes mellitus, non insulin dependent diabetes mellitus (NIDDM). Results Biochemical measurements HFD fed rats showed significant increase in body weight as compared to NPD fed rats (Table 2 ). In addition, the rats fed HFD showed significant elevation in the basal plasma glucose, triglyceride, total cholesterol and insulin levels at the end of four weeks of dietary manipulation as compared to NPD fed control groups (Table 2 ). The HFD fed rats exhibited significant elevation in basal fasting glucose and showed significant impairment in glucose tolerance to exogenously administered glucose (Fig 1 ). Estimation of AUC values indicated 28.4 % increase in plasma glucose of HFD fed when compared to NPD fed rats. Table 2 Various parameters of NPD and HFD fed rats Parameter NPD fed HFD fed Body weight (gm) 259.2 ± 5.3 315.0 ± 6.5*** plasma glucose (mg/dl) 87.5 ± 2.5 111.0 ± 0.9*** Plasma triglycerides (mg/dl) 41.3 ± 1.7 89.7 ± 7.2*** Plasma cholesterol (mg/dl) 48.9 ± 3.7 87.5 ± 2.6*** Plasma Insulin (ng/ml) 1.90 ± 0.15 3.05 ± 0.21* pD 2 of 5-HT 5.74 ± 0.09 6.00 ± 0.06 E max of 5-HT 30.0 ± 1.8 49.0 ± 2.0** pD 2 of Ang II 7.69 ± 0.08 7.31 ± 0.18 E max of Ang II 25.0 ± 3.4 34.0 ± 3.0* Each point is represented as mean ± SEM *p < 0.05, **p < 0.01 and ***p < 0.001 Vs NPD fed group Figure 1 Effect of HFD on intra peritoneal glucose tolerance test (IPGTT) in rats, as compared to NPD fed group. All values are expressed as mean ± SEM (n = 8) *p < 0.05, **p < 0.01, ***p < 0.001 Vs NPD fed group Vascular Responses Cumulative concentration response curves of 5-HT and Ang II for both HFD fed rat thoracic aortae showed an increase in E max (Fig 2 and 3 ) with out any change in pD 2 values when compared to NPD fed rats (Table 2 ). There was no significant change in both E max and pD 2 values with KCl in both groups (data not shown). The order of potency of agonists in both groups was Ang II>5-HT>KCl. Figure 2 Cumulative concentration response curve to 5-HT in helically cut aortic strip preparations obtained from NPD fed and HFD fed rats. Each point is represented as mean ± SEM (n = 5) *p < 0.05, **p < 0.01 Vs NPD fed group Figure 3 Cumulative concentration response curve to Ang II in helically cut aortic strip preparations obtained from NPD fed and HFD fed rats. Each point is represented as mean ± SEM (n = 5) *p < 0.05, **p < 0.01 Vs NPD fed group Discussion Obesity is a major risk factor for several metabolic diseases, frequently clustering to form the metabolic syndrome or syndrome X [ 9 ]. Obese people have increased incidence of NIDDM with high percentage of mortality and morbidity [ 10 ]. Western style diet, which is abundant with calorically dense and saturated fatty foods, is considered to be the main factor in the development of obesity and insulin resistance. Our studies have shown that HFD causes increase in bodyweight when compared to NPD after four weeks of dietary manipulation in rats. Hyperglycemia is observed in insulin resistance where glucose utilization is reduced. We saw significant elevations in blood glucose levels. Intraperitoneal glucose tolerance tests confirm severe glucose intolerance. Oversupply of dietary lipids causes insulin resistance in rats [ 11 ]. Randle glucose fatty acid cycle suggested that the body prefers excess lipid stores to glucose for metabolic oxidation in insulin resistance [ 12 ]. Our HFD model also exhibited high plasma triglyceride levels. Hence studies on our experimental model in compliance with Randle et al findings suggested that HFD feeding causes insulin resistance [ 13 ]. Insulin resistance with compensatory hyperinsulinemia is a prominent feature of metabolic syndrome. The most common reason for the development of hyperinsulinemia in insulin resistance is obesity. It stands as one of the major cardiovascular risk factors in patients with obesity. The present study on HFD rats demonstrated higher plasma insulin levels than control values. This marked hyperinsulinemia could be due to a combination of increased β-cell mass and decreased insulin clearance, as well as failure of insulin to suppress hepatic gluconeogenesis [ 14 ]. Elevated cholesterol is also observed in insulin resistant individuals. For this reason we measured plasma cholesterol levels which were found to be more than normal values. Previous studies have reported a down regulation of LDL receptors and associated decrease in LDL clearance, increased total cholesterol levels [ 15 ]. According to National Cholesterol Education Program's Adult Treatment Panel III (Third report) easily measured clinical findings for syndrome X includes increased abdominal circumference, elevated triglycerides, low high-density lipoprotein-cholesterol, and elevated fasting blood glucose and/or elevated blood pressure. Three of these five are required for diagnosis. Our study demonstrated three of the clinical parameters indicating conditions of syndrome X in HFD fed rats [ 16 ]. Insulin resistance along with other conditions of syndrome X might induce hypertension by a host of mechanisms involving insulin itself, increased sodium reabsorption and/or enhanced intra cellular concentration of free calcium in vascular smooth muscle [ 17 ]. Although the etiology of vascular disorders in metabolic syndrome has not completely been revealed, it is suggested that alterations in the reactivity of blood vessels to neurotransmitters and circulating hormones are responsible for the functional abnormalities of blood vessels. In order to elaborate the pathways that connect syndrome X to hypertension we have studied the contractile responses to 5-HT and Ang II in both HFD and NPD fed rat thoracic aortae. Previously we have demonstrated increased contractile responses with synthetic alpha adrenoceptor agonist, phenylephrine, in HFD fed rat thoracic aorta [ 18 ]. This enabled us to explore the role of these endogenous mediators in the same animal model. The present vascular studies demonstrated that the magnitude of responses to 5-HT and Ang II was significantly enhanced in HFD fed animals without change in pD 2 value. Endothelial denudation obviates any related mechanisms such as impairment of NO release, increased destruction of EDRF and substrate availability for the production of EDRF. Hence, the probable reasons for these enhanced 5-HT and Ang II responses may be due to receptor mediated or non-receptor mediated pathways. The role of non-receptor mediated contraction can be ruled out for there was no change in contractile response to KCl. Vascular studies have also shown functional evidence that hypertension developed in HFD fed rats may be associated with enhanced vasoreactivity to various vasoconstrictor agents. Further the enhanced responses to 5-HT in HFD fed rats could be due to increased PKC as previously reported with STZ and alloxan (ALL) induced diabetic animals [ 19 ]. Increased contractions to 5-HT can also be related to 5-HT 2A upregulation as observed in spontaneously hypertensive rats or due to serotonin acting through alpha adrenoceptors [ 20 ]. Increased Ang II responses in HFD fed rats are may be due to upregulation of Ang II receptors as observed in hyperinsulinemia or via amplified secondary messenger systems [ 21 ]. A proposed scheme of events is given in Fig 4 . Figure 4 Proposed events underlying syndrome X in HFD rats. HFD leads to condition, in rats, similar to syndrome X. This includes obesity, insulin resistance, hyperglycemia and dyslipidemia which all are interrelated. These events are the initial steps in the cascade towards hypertension. This could be mediated via increased contractile responses to various endogenous mediators such as 5-HT and Ang II. In summary, the present study has shown that HFD feeding in rats produces conditions similar to syndrome X. Increased vasocontractile responses observed in the model are not only mediated via alpha adrenoceptors but also due to 5-HT and Ang II (see Figure 4 ). More robust studies on the secondary messenger systems of 5-HT and Ang II will provide valuable insights into the mechanisms underlying increased vascular contractility in insulin resistance. Materials and Methods Tissue Preparation Male Sprague-Dawley rats (central animal facility, National Institute of Pharmaceutical Education and Research (NIPER), India), 160–200 g, were kept in controlled environmental conditions with room temperature 22 ± 2°C, humidity 55 ± 5% and 12-h light/dark cycles. All the animals had free access to food and water. The rats were divided into two dietary groups and fed with standard rat normal pellet diet (NPD) (3.8 kcal/g, carbohydrate 67%, protein 21%, fat 12% kcal) and HFD (5.3 kcal/g, carbohydrate 17%, protein 25%, fat 58% kcal). Composition of HFD is described in Table 1 . Table 1 Composition of HFD (g) Powdered pellet diet 364 Lard 310 Casein 250 Cholesterol 10 DL-Methionine 3 Yee-sac powder 1 Vitamin and mineral mix powder 60 Sodium chloride 2 Biochemical measurements Blood samples from the retro orbital plexus of anaesthetized rats (Pentobarbitone 45 mg/kg, i.p.,) were collected into the heparinized tubes and immediately centrifuged at 5000 rpm for the separation of plasma. Plasma was stored at -20°C until assayed. The plasma was used for the estimation of glucose (Qualigens, Mumbai, India), triglycerides, and cholesterol (Chema diagnostica, Jesi, Italy) by commercial kits. Plasma insulin was determined by radioimmuno assay using rat insulin as standard (Linco research, St. Charles, MO, USA) Intra peritoneal glucose tolerance test (IPGTT) Glucose tolerance tests were carried out after four weeks of feeding of both NPD and HFD. After an overnight fast, blood samples were collected from the retro orbital plexus. Glucose levels were measured at time zero (0 min) and glucose was injected into the rats (2 g/Kg/4 ml, i.p.,). Additional blood samples were taken at 15, 30, 60 and 120 min. following the glucose load. Plasma glucose levels were measured by the glucose oxidase reaction (GOD/POD) using commercial kit (Qualigens, Mumbai, India). Area under the curve (AUC) was calculated for both NPD and HFD fed rats. Vascular Studies After 4 weeks of feeding, rats were sacrificed by cervical dislocation. The section of the aorta from between the aortic arch and the diaphragm was removed from the euthanized rats and placed in oxygenated, modified Krebs-Henseleit solution (KHS; in mM: 118 NaCl, 4.7 KCl, 25 NaHCO 3 , 2.6 CaCl 2 2H 2 O 1.2 NaH 2 PO 4 , 1.2 MgCl 2 6H 2 O, 5.5 glucose). With the help of a steel rod, aortic endothelium was deliberately denuded. The aorta was cut into helical strips 3 mm wide, 20 mm long and then placed in a well-oxygenated (95% O 2 -5% CO 2 ) bath of 10-ml KHS with one end connected to a tissue holder and the other to an isotonic transducer (Bio Devices, Ambala, India). The tissue was equilibrated for 60 min under a resting tension of 1.0 g. At the beginning of each experiment, aortic strips were primed with depolarizing concentration (90 mM) potassium chloride (KCl). After the equilibration period, contractile responses to various concentrations of 5-HT (10 nM-30 μM) and Ang II (1 nM-300 nM) were recorded. Data analysis Contraction responses are expressed as percentage. For each contractile agent, both the maximal contraction (E max ) and the concentration necessary to produce 50% of its maximal response (EC 50 ) were determined. The EC 50 values were converted to the negative logarithms and expressed as pD 2 . Results were shown as mean ± SEM; n refers to the number of animals from which vessels were taken. Agonist potencies and maximal effects were compared by student's t test by using statistical software (GraphPad Prism 3.01). Values were considered significantly different at p < 0.05. Drugs and solutions The following drugs were used in this study: 5-HT (RBI, USA), Ang II (Bachem, Switzerland), All drugs were dissolved in KHS. Drugs were added to the organ chambers in volumes not greater than 0.2 ml. List of abbreviations HFD – High fat diet NPD – Normal pellet diet NIDDM – Non insulin dependent diabetes mellitus STZ – Streptozotocin 5-HT – 5-Hydroxytryptamine Ang II – Angiotensin II KHS – Krebs-Henseleit solution GOD/POD – Glucose oxidase/peroxidase AUC – area under the curve E max – Maximal response EC 50 – Concentration required producing 50% of maximal response pD 2 – -log EC 50 Authors' contributions SG carried out the all experimentations. PR has conceived the study and participated in its design and coordination. Authors read and approved the final manuscript.

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526261

Personalized versus non-personalized computerized decision support system to increase therapeutic quality control of oral anticoagulant therapy: an alternating time series analysis

Background The quality control of oral anticoagulant therapy (OAT) during the initiation and maintenance treatment is generally poor. Physicians' ordering of OAT (especially fluindione and warfarin ) can be improved by dose adjustment algorithms, taking into account the results of International Normalized Ratio (INR). Reminders at the point of care, computerized or not, have been demonstrated to be effective in changing physicians prescription behavior. However, few studies have addressed the benefit of personalized reminders versus non personalized reminders, whereas the personalized reminders require more development to access patient record data and integrate with the computerized physician order entry system. The Hospital Information System of George Pompidou European Hospital integrates an electronic medical record, lab test and drugs order entry system. This system allows to evaluate such reminders and to consider their implementation for routine use as well as the continuous evaluation of their impact on medical practice quality indicators. The objective of this study is to evaluate the impact of two types of reminders on overtreatment by oral anticoagulant: a simple reminder of text formatted dose adjustment table and a personalized recommendation for oral anticoagulant dose and next date of INR control, adapted to patient data. Both types of reminders appear to the physician at the moment of drug ordering. Methods The study is an alternating time series experiment with three 6 months periods, each one including every 2 months according to a Latin square scheme: a control period without any reminder, a period with the simple non personalized reminder, a period with personalized reminder. All patients hospitalized in departments using the computerized physician order entry system and ordered fluindione or warfarin , will be included in the study between November 2004 and May 2006. Main outcome will be the proportion of overcoagulation, as expressed by the proportion of observation time with INR over 4.5, assuming INR change linearly. Secondary outcome is the incidence of major haemorrhagic events. Data will be collected thanks to Hospital Information Systems databases. Data will be analyzed taking into account patient and physician clustering effect.

Background Iatrogenic effects of oral anticoagulants therapy According to a study carried out by French pharmacovigilance centres, haemorrhage subsequent to oral anticoagulant treatment (OAT) is the most common drug-related side effect resulting in hospitalisation in public hospitals in France (13% of such admissions, and 0.41% of the 3,137 admissions analysed). On the basis of these findings, the AFSSAPS (French Agency for Health Product Safety) has made the prevention of iatrogenic effects related to OAT one of its priorities. Many of these events are consequences of interactions between different drugs, resulting in inappropriate doses [ 1 - 3 ]. Implementation of a system of support when prescriptions are made out is likely to improve prescription practices and to decrease the frequency of side effects. It should be possible to integrate a support tool into the drug prescription system, by using nomograms to adjust OAT doses. Decision-making tools and appropriate practice The efficiency of reminders issued at the time of prescription has been demonstrated by a various studies [ 4 - 8 ]. These reminders can come in a paper, telephone [ 9 ] or computer form. Hunt et al . [ 10 ] reviewed all studies (randomised and quasi-randomised) evaluating the effect of computer-based clinical decision support systems on medical practice and treatment outcome. They showed that these systems are effective for drug prescription purposes and for the implementation of various medical strategies [ 10 ]. We have demonstrated the efficiency of reminders in the French situation, both in the form of "paper" reminders [ 11 ] and in the form of computer-generated reminders [ 12 ]. Computer-generated reminders appear the most promising given the development of computer programs including prescription aids for hospital usage. Several types of reminders can be issued at the time of prescription [ 13 ]: • simple, general information concerning the recommendations that should be taken into account (non-specific or non-personalised reminders), • "check list": includes questions or a precise list of practices that the doctor must tick to show that it has been done, • reminders including clinical data concerning a specific patient that must be taken into account for a given procedure (personalised reminders). The advantage of personalised reminders over non-personalised reminders has not been demonstrated in the literature. However, the production of personalised reminders necessitates better integration of existing information and is thus more expensive to develop. It is important to determine whether this personalised tool results in a better quality of care than non-personalised tools. Several randomised clinical trials have tried to evaluate decision support systems for the prescription of OAT, but failed to draw any conclusions about their efficiency for several reasons: heterogeneity and complexity of the systems evaluated, experimental designs difficult to apply and not necessarily adapted, and too few patients included [ 14 - 17 ]. Reminders issued at the time of prescription have been shown to be effective by experimental studies, but the difficulties of maintaining the effectiveness of interventions designed to improve clinical practices remains a major problem. We evaluated the effect of an active decision support system for the prescription of low molecular weight heparin as prophylaxis for venal thrombosis in an orthopaedic surgery department. In this study, the system was and was not used during alternate periods. It showed that such programs affect practices without affecting learning [ 12 ]. Other authors have looked at the problem of the routine use of computerised systems [ 18 , 19 ]. The long-term use of a computer program to modify medical practices necessitates a hospital information system that ties in prescriptions and the results of medical tests, and the continuous collection of indicators making it possible to evaluate use and effect. Georges Pompidou European Hospital (GPEH) Organisation of the information system at GPEH The hospital information system currently collates prescriptions and results of biological tests and imaging procedures. Eight hundred computers, both laptops and fixed posts, are used to in care procedures (in care departments and medical offices). The Dx-Care ® program is at the centre of care delivery. It is used by doctors and nurses: • to prescribe laboratory examinations and imaging tests for a patient, • to visualise the results of laboratory tests, • to establish and to consult nursing schedules, • to archive a structured observation, • to prescribe drugs. Dx-Care ® is integrated with other applications to allow circulation of information between departments, laboratories and the pharmacy. Prescriptions for laboratory tests are transmitted to the Netlab ® program which manages such tests (this program is used by all biochemistry, haemostasis and immunology laboratories, etc.) The laboratories return the results using this same program, which retransmits them to Dx-Care ® . Furthermore, prescriptions of drugs are transmitted to the Phedra program, which is used by the pharmacy to manage prescriptions. The prescription is validated by the pharmacy and this validation is then transferred to Dx-Care ® . The lab test prescription facility has been available in the hospital information system since 2000 and is used by all departments of the hospital. The drug prescription facility has been implemented later on, since January 2003, and its use is still increasing (currently by half of the hospital departments). The hospital information system thus allows to install decision support systems that are activated whenever a prescription is issued and routinely to collect evaluation criteria of prescription practices. If possible and validated, the use of the hospital information system to evaluate care procedures will make it possible to collect data regularly, and routinely to assess methods for the improvement of care practices. Organisation of quality assurance and system for recording undesirable events With the aim of quality of care and preventing risks, the hospital is developing a system, based on the Intranet network, of declaration of undesirable events. This system must be able to record all undesirable events and incidents linked to the use of health products (drugs, medical equipment, blood products, etc.) and care (e.g. falls, lost files, bedsores, long waiting times) as well as those due to the patient environment (building, security, malevolence, etc.) and the job of health care professionals (accidental exposure to blood, chemicals, radiation, etc.). When a health care professional decides to report an undesirable event, he or she must complete a dedicated form available on the Intranet with all relevant information. This incident form includes an item entitled "complications associated with anti-coagulants". When the doctor clicks on this item, a form specific to haemorrhagic accidents following anti-coagulant treatment appears (see form in appendix). Prevention of thrombosis at GPEH Among departments which already started to use the computerised drug order entry system, several (cardiology and vascular medicine departments) are heavy "consumers" of anti-thrombosis drugs (see Table 1 ). In 2001, a working group representative of all the hospital departments described a group of procedures. This was done by critically reading articles published in the literature. The procedures concerned the preventive and curative indications of anticoagulants (OAT and heparins) in arterial thrombosis and venous thrombosis, and the way in which patients receiving anticoagulants are monitored and handled in cases of overdose. The procedure concerning curative OAT included a nomogram for adjusting doses of fluindione and warfarin based on previous doses and the INR (see Tables 2 and 3 ). These nomograms are currently available on the hospital's intranet, although they are not widely used. Their inclusion in a computerised prescription aid could increase their impact. Study aims Main aims 1. To evaluate the effect on the frequency of overanticoagulation of the implementation in the computerised physician order entry system of two types of tool to adjust OAT doses (one personalised and one non-personalised). 2. To assess any advantages of using the personalised tool rather than the non-personalised tool. Secondary aims 1. To evaluate the frequency of haemorrhagic accidents in the context of the study. 2. To evaluate the feasibility of long-term implementation of the intervention. Methods Experimental design The study is an alternate time series experiment which consists of three successive six-month periods (timed to change with the changing of medical residents even though they will not be the only prescribers). Each phase will consist of: • a two-month period without active support during which evaluation criteria will be collected (period A), • a two-month period with non-personalised active support (period B), • a two-month period with personalised active support (period C). To limit the impact of a learning effect on appropriate OAT management practice within the department over time (possible for medical residents), the order of these three periods was determined by using a Latin square plan (see Figure). This experimental design can be considered valid for an impact study in this context whereas a randomised controlled design is difficult to apply with just one hospital [ 20 ]. Participants Patients This study will include all the patients who are prescribed OAT for any indication and are hospitalised in clinical care departments where physicians are using the hospital information system to prescribe drugs. The following table shows the number of INR examinations prescribed by these departments in a three-month period. It provides an estimation of the approximate proportion of overdoses among the INR that exceeded 2, which is supposed to be found in patients treated with OAT in these units. Physicians All doctors authorised to prescribe drugs in the participating departments will be included in the study: residents and fellows, registered and non registered university hospital doctors. Each six-month period will coincide with an internship semester. Intervention To prescribe a drug using Dx-Care ® , the doctor selects the required drug from an exhaustive list. This opens up a dialogue box in which the doctor types the dose, the frequency of intake and the mode of administration. From this window, it is possible to add a text comment or to consult particular protocols that have been defined by the departments. It is planned to integrate two types of decision support systems into the computerised prescription program: 1) non-personalised active system: when the drug is selected a window automatically opens giving the prescribing the nomogram for the adjustment of OAT doses in the form of a table (see Tables 2 and 3 ). 2) personalised active system: when the drug is selected a window automatically opens suggesting a dose recommended according to the nomogram (taking into account the doses previously received by the patient and the patient's INR), together with a date for next INR control and an explication. Definition of endpoints Overanticoagulation Proportion of patient observation time with INR results > 4.5, assuming linear change of INRs. Major haemorrhagic accidents Intra-cranial haemorrhage or spontaneous haemorrhage necessitating surgery or a transfusion or decreasing haemoglobin concentration by more than 2 g/dl. Assessment of evaluation criteria OAT overdose The Netlab ® application allows biological laboratories to receive prescriptions and to return results. All of the INR results can be extracted from the Netlab ® database accompanied by information making it possible to identify the patient, the treatment and dose received, the prescribing doctor, the hospitalisation unit, the date the test was prescribed. Data about overdoses can therefore be collected systematically by regular database searches. Furthermore, the storage of the information in a computerised tool will make it possible to determine previous doses and INR results each time a drug is prescribed. Haemorrhagic accidents When a health care professional decides to declare an undesirable event, he or she fills in a specific, pre-formatted form available on the Intranet. This form includes a list of events that must be declared at the GPEH. The declaration form includes an item entitled "complication of haemorrhagic accidents". When the doctor clicks on this item, a specific form for the declaration of a haemorrhagic accident associated with anti-coagulant treatment appears (see form in appendix). Determination of sample size The determination of the number of participants necessary requires the definition of the statistical unit of interest, information about the incidence of the evaluation criteria in the study population and a hypothesis about the efficiency of the intervention. Statistical unit In this study, the main aims are to guide each prescription and to reduce the number of anti-coagulant overdoses: the simplest statistical unit to study is therefore the INR result. This unit will be used to calculate the sample size. This choice is not, however, perfect and the efficacy results will be presented using other indicators of the quality control of anti-coagulant treatments: Given the low incidence of major haemorrhagic accidents (not currently measured at the GPEH but probably below 1%), it is not possible in this study to estimate the number of subjects necessary to demonstrate an effect of intervention on the "haemorrhagic accident" endpoint. Recording haemorrhagic accidents will give the frequency of such accidents, which will then be used for realistic estimates of power and sample size if further studies are carried out. In previous studies evaluating the efficacy of tools to aid the prescription of OAT, the unit considered was not always the same, taking into account the number of INR per patient and the time between INR measurements to greater or lesser extents. The most recent studies considered the number of patient-days according to the method described by Rosendaal [ 15 , 16 , 21 ]. This method can also be used to calculate the rate of haemorrhagic events as a function of the number of patient-days for a given range of INR values. We may also carry out an analysis for each prescribing doctor given that the intervention targets doctors directly. This will involve adjusting the effect of the intervention to the fact that intra-physician variability is a priori lower than inter-physician variability. Number of INR measurements and predicted frequency of overdoses During a six-month period (January to June 2004), 4 920 INRs were requested by the six departments which already routinely use the computerized physician order entry system. The frequency of overtreatment can approximately be estimated from the percentage of INR > 4.5 among INR >2. Among the 2620 INR > 2, 330 (12%) were higher than 4.5 (see Table 4 ). This frequency has been stable during these six months but differed considerably between departments (10% to 23%). Hypothesis about the efficacy of the intervention The number of INR tests necessary for a six-month period, with an α risk of 5% and a power of 80%, for the comparison of two percentages by classical methods (untreated group half the size of the treated group), for a basal incidence of the judgement criterion of 12% are and for the following hypotheses on relative reduction of the risk (RRR) of overdose, are: • RRR 30%: 2500 • RRR 40%: 1300 • RRR 50%: 800 • RRR 60%: 500 Carrying out approximately 5000 tests over six months will make it possible to detect an intervention effect of less than 30% in this period. The experimental design includes three six-month periods and should thus ensure adequate power. Statistical analyses Statistical analyses will be performed with the STATA statistical software (release 8, STATA corp, College station, Tex, USA) Standard statistical tests will be used to compare the baseline characteristics of the departments and patients. The main analysis concerns the effect of the intervention on the number of dangerously high INRs. The analysis will be carried out using a mixed effect analysis of variance model, in which the effect linked to the period will be considered fixed and that linked to the prescription tool will be considered random [ 22 ]. Rosendaal's method will be used to analyse the number of patient-days with INR over the target [ 21 ]. Regulatory aspects According to French policy, this study was exempt from medical ethics committee approval. The anti-coagulants being evaluated are prescribed as recommended by clinical studies validated within the GPEH. These recommendations are available on the hospital's Intranet and are thus accessible to all doctors. They conform to standard practices. Neither the patients nor the doctors will be randomised. The interventions are simply different means of giving valid information to physicians. Using funding from the PHRC (Hospital Clinical Research Program), we carried out two research studies related to this project. In the first (PHRC 95), an intervention aimed at modifying the way in which emergency department doctors handle ankle injuries, the study design was a randomised controlled study and the randomisation unit was the hospital [ 11 ]. In the second (PHRC 98), a computer-based decision-support system for the prevention of venous thromboses in orthopaedics, the experimental design was identical to that of our present study [ 12 ]. In both cases, the Ile de France branch of the Clinical Research Delegation considered that the project was exempt from medical ethics committee approval. List of abbreviations OAT: Oral Anticoagulant Therapy INR: International Normalized Ratio GPEH: Georges Pompidou European Hospital PHRC: Hospital Clinical Research Program Authors' contributions IC and PD, conceived, wrote the protocol and prepared the manuscript. GC is the statistical expert and performed the power calculations GC and ABR revised the protocol and the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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545936

Cough quality in children: a comparison of subjective vs. bronchoscopic findings

Background Cough is the most common symptom presenting to doctors. The quality of cough (productive or wet vs dry) is used clinically as well as in epidemiology and clinical research. There is however no data on the validity of cough quality descriptors. The study aims were to compare (1) cough quality (wet/dry and brassy/non-brassy) to bronchoscopic findings of secretions and tracheomalacia respectively and, (2) parent's vs clinician's evaluation of the cough quality (wet/dry). Methods Cough quality of children (without a known underlying respiratory disease) undergoing elective bronchoscopy was independently evaluated by clinicians and parents. A 'blinded' clinician scored the secretions seen at bronchoscopy on pre-determined criteria and graded (1 to 6). Kappa (K) statistics was used for agreement, and inter-rater and intra-rater agreement examined on digitally recorded cough. A receiver operating characteristic (ROC) curve was used to determine if cough quality related to amount of airway secretions present at bronchoscopy. Results Median age of the 106 children (62 boys, 44 girls) enrolled was 2.6 years (IQR 5.7). Parent's assessment of cough quality (wet/dry) agreed with clinicians' (K = 0.75, 95%CI 0.58–0.93). When compared to bronchoscopy (bronchoscopic secretion grade 4), clinicians' cough assessment had the highest sensitivity (0.75) and specificity (0.79) and were marginally better than parent(s). The area under the ROC curve was 0.85 (95%CI 0.77–0.92). Intra-observer (K = 1.0) and inter-clinician agreement for wet/dry cough (K = 0.88, 95%CI 0.82–0.94) was very good. Weighted K for inter-rater agreement for bronchoscopic secretion grades was 0.95 (95%CI 0.87–1). Sensitivity and specificity for brassy cough (for tracheomalacia) were 0.57 and 0.81 respectively. K for both intra and inter-observer clinician agreement for brassy cough was 0.79 (95%CI 0.73–0.86). Conclusions Dry and wet cough in children, as determined by clinicians and parents has good clinical validity. Clinicians should however be cognisant that children with dry cough may have minimal to mild airway secretions. Brassy cough determined by respiratory physicians is highly specific for tracheomalacia.

Background Cough is the most common symptom presenting to medical practitioners in Australia, the UK and USA [ 1 - 3 ]. Cough quality, specifically dry versus we t[ 4 ] or productive cough, is often used in epidemiological [ 5 - 7 ] and clinical research [ 8 , 9 ]. Clinically, physicians also often differentiate between dry and wet cough [ 10 - 12 ] but there are no studies that have evaluated if these are reproducible descriptors. In adults, productive cough is usually obvious but children however often swallow their sputum and hence a 'wet cough' is used inter-changeably with 'productive cough' to describe cough quality in young children who are unable to expectorate [ 10 , 13 ]. It is known that nocturnal cough is unreliably reported in both children [ 14 ] and adults [ 15 ] but there is no data on cough quality. Wet and dry cough are determined subjectively as there are no 'gold standards'. To date there are no human studies that have identified the objective relationship of the cough descriptors to mucus secretory states. The sound of a cough is due to vibration of larger airways and laryngeal structures during turbulent flow in expiration [ 16 , 17 ]. It is not known which generation of the airways is involved when the human ear identifies a wet cough and currently there are no validated human models that allow measurement of increased airway mucus. Mucus hypersecretory states in human diseases can occur from a variety of mechanisms which include; hypersecretion of stored mucin, hypertrophy or hyperplasia of goblet cells and/or increased synthesis from over-expression of mucin genes [ 18 ]. In disease states, it is not known which mechanism or site of production is the most important but in smokers with chronic bronchitis, a common cause of productive cough in adults, the larger bronchi (bronchi of diameter >4 mm ie segmental bronchi and above) [ 19 ] are the site of greatest inflammation [ 18 ]. Flexible bronchoscopy allows an in-vivo visual assessment of larger airways usually to the 3 rd (lobar bronchi) or 4 th generation (segmental bronchi) in young children. The study aims were to compare (1) cough quality (wet vs dry and brassy vs non-brassy) with bronchoscopic findings of secretions and tracheomalacia respectively and, (2) parent(s) vs clinician's evaluation of the cough quality (wet and dry). We hypothesised that clinical assessment of cough is good compared to bronchoscopic findings and that a wet cough is related to presence of airway secretions. Methods Children electively admitted for bronchoscopy without a known underlying respiratory diagnosis were seen by a member of the research team 0.5–3 hours prior to bronchoscopy. The clinician's assessment of cough quality (wet or dry) was recorded on a standardised sheet (based on the cough present on the day of the bronchoscopy), before the parent(s) independently evaluated the current (the morning of, or last 12 hours) cough quality (wet or dry) of their child. For clinician's assessment of wet/dry cough, when no spontaneous cough was heard or if child was too young to elicit a cough, cough quality (wet or dry) was deemed 'non-assessable'. Clinicians also rated cough as 'brassy' or 'non-brassy' based on coughs heard anytime before bronchoscopy. For assessment of reliability of cough quality (wet/dry and brassy/non-brassy), 21 cooperative children had their coughs digitally recorded (Acer Pocket PC n11, Taiwan) using music compact disc quality format (44.1 kHz, 16 bit) on the morning of their bronchoscopy. These stored cough sounds were later replayed (using headphones 30–10,000 Hz, Lanier, Japan) from a computer and re-scored in a blinded manner (blinded to the child's name and cough quality assigned earlier) for wet/dry and brassy/non-brassy qualities. Written consent was obtained from a parent and the study approved by the hospital's ethics committee on human research. Bronchoscopy and quantification of secretions seen during bronchoscopy Flexible bronchoscopy was performed under general anaesthesia as previously described [ 20 - 22 ]. Briefly, anaesthesia was induced with sevoflurane in 100% oxygen administered through a Jackson Rees T piece circuit, the vocal cords and upper trachea then sprayed (4 mg/kg lignocaine via a Cass needle). Atropine was given intravenously to most children aged <12 months. In all children a video flexible bronchoscope (BF 3C160, Olympus, Tokyo, Japan) entered the circuit via the port of a swivel right angle connector attached to a facemask. Images were projected onto a monitor (Sony Trinitron, Tokyo, Japan). A respiratory consultant (ABC or IBM) blinded to the child's history and cough quality scored the bronchoscopy sheet quantifying the amount of secretions at the time of the bronchoscopy in real time. When no scorer was available, the session was videotaped and played back. A secretion quantification card (figure 1 ) was visible to the scorer at all times. Secretions were quantified according to amount of mucus in the airways in relation to lumen size (fig 1 ) and scored from the trachea to the level of lobar bronchi (total of 9; trachea, right main stem, right upper lobe, right middle lobe, right lower lobe, left main stem, left upper lobe, left lingula, left lower lobe). When segmental bronchi were seen, the worst segment (ie segment with most secretions) was scored. These scores were used to obtain a final grade of bronchoscopic secretions (BS) from grades 1 to 6; Figure 1 Bronchoscopic secretion quantification card. BS Grade 1 = Nil secretions BS Grade 2 = Near dry = Bubbles only in < half total number of bronchi involved BS Grade 3 = Minimal = Bubbles found in > half total number of bronchi involved or Secretion type-I in < half total number of bronchi involved BS Grade 4 = Mild = Secretion type-I, > half total number of bronchi involved or Secretion type-II, < half total number of bronchi involved BS Grade 5 = Mod = Secretion type-II, > half total number of bronchi involved or Secretion type-III, < half total number of bronchi involved BS Grade 6 = Large = Secretion type-III, > half total number of bronchi involved Inter-rater reliability of BS grading was assessed by replaying the videotapes of the recorded bronchoscopy of 20 children, with the 2 nd assessor blinded to the child's condition. BAL was obtained from the macroscopically most abnormal lobe; when changes were generalised, BAL was obtained from the right middle lobe. Cell count was performed on the cell suspension, cytocentrifuge slides were prepared and stained (modified Wright's stain) for cell differential profile. All cellular examinations were performed by cytologists blinded to the children's medical history. Statistics Data were not normally distributed and thus non parametric analyses were used; medians and inter-quartile range (IQR) were used for all descriptive data and Kruskal Wallis for comparisons between groups. Cohen's kappa (K) with 95%CI was utilised for inter and intra-observer reliability and graded from 'poor' (K<0.2) to 'very good' (K = 0.81–1.0)[ 23 ]. For calculation of sensitivity and specificity, negative and positive predictive values (NPV, PPV); cough quality was assigned to dry when a history of cough was absent and bronchoscopy findings at two cut offs (grades 3 and 4) of BS grades were taken as the 'gold standard' eg for cut-off at BS grade 3, BS grades 1–2 were defined as no secretions and BS grades = 3 defined as secretions present. To determine if cough quality (wet/dry) was predictive of amount of secretions found during bronchoscopy, a receiver operating characteristic (ROC) curve was generated [ 24 ] where cough quality wet/dry was considered the true positive/negative and the bronchoscopic secretion scoring (1 to 6) as the ordinal rating scale. Two tailed p value of < 0.05 was considered significant. SPSS ver 11.1 was utilised for most statistical calculation. Results Median age of the 106 children (62 boys, 44 girls) enrolled was 2.6 years (IQR 5.7). Indications for bronchoscopy were chronic cough (n = 44, 41.5%), wheeze (n = 21, 19.8%), stridor (n = 16, 15.4%), investigation of persistent radiological changes (n = 14, 13.5%), recurrent pneumonia (n = 6, 5.8%), suspicion of aspiration lung disease (n = 3, 2.9%), BAL and suspected foreign body (n = 1 each, 2%). In four children, BS grades were not obtained (session was inadvertently not recorded and 'blinded' clinician not present at bronchoscopy). Scores of BS were done in real time in all but 9 children. In 30 children, cough was non-assessable. Agreement between clinicians and parents assessment of cough quality (wet/dry) was good (K = 0.75, 95%CI 0.58, 0.93). For cough quality of 'wet/dry', cough assessed by clinicians had the highest specificity, sensitivity, NPV, PPV and positive likelihood ratio for both BS cut-offs (tables 1 and 2 ). Parent(s) assessment were less precise but only marginally so. The area under the fitted ROC curve (figure 2 ) was 0.85 95%CI 0.77, 0.92. The specificity, NPV and likelihood ratio for brassy cough assessed against gold standard bronchoscopic finding of tracheomalacia was good (table 1 ) but less than that for cough quality of wet/dry. Table 1 Assessment of cough quality vs bronchoscopic findings with BS cut off at grade 3* Assessment type (clinical vs bronchoscopic findings) Sensitivity Specificity NPV PPV Positive LR Clinician 1.00 0.55 1 0.64 2.21 Cough quality (wet/dry) assessed by clinician (n = 96) Parent(s) 0.95 0.54 0.93 0.61 2.06 Cough quality (wet/dry) assessed by parents (n = 92) Combined *(n = 100) 0.98 0.54 0.97 0.62 2.10 Tracheomalacia (n = 81)# 0.57 0.81 0.84 0.52 3.12 *Cough quality (wet/dry) assessed by clinicians combined with parents. When cough was non-assessable by clinician and child has current cough, parental assessment of the cough (wet or dry) was taken. If child has no history of current cough, cough was assigned 'dry'. LR = likelihood ratio. Specificity, sensitivity of dry and wet cough was assessed against bronchoscopic findings as the gold standard where *BS grades ≥ 3 were considered abnormal (secretions present) and ≤ 2 considered normal (no secretions). #That for tracheomalacia was assessed using clinicians record of presence/absence of brassy cough with bronchoscopic findings of tracheomalacia.[21] Table 2 Assessment of cough quality vs bronchoscopic findings with BS cut off at grade 4* Assessment type (clinical vs bronchoscopic findings) Sensitivity Specificity NPV PPV Positive LR Clinician 0.79 0.75 0.82 0.72 3.22 Cough quality (wet or dry) assessed by clinician (n = 96) Parent(s) 0.78 0.71 0.80 0.67 2.69 Cough quality (wet or dry) assessed by parents (n = 92) Combined * (n = 100) 0.77 0.73 0.80 0.69 2.88 *Cough quality (wet/dry) assessed by clinicians combined with parents. When cough was non-assessable by clinician and child has current cough, parental assessment of the cough (wet or dry) was taken. If child has no history of current cough, cough was assigned 'dry'. LR = likelihood ratio. Specificity, sensitivity of dry and wet cough was assessed against bronchoscopic findings as the gold standard where BS grades ≥ 4 were considered abnormal (secretions present) and ≤ 3 considered normal (no secretions). Figure 2 ROC curve with 95%CI relating cough quality (wet/dry) to bronchoscopic secretion (BS) grades from 1–6. There was little difference in sensitivity and specificity between children grouped by indication for bronchoscopy (cough or other indications). Values were marginally better in older children (tables 4 and 5 in supplementary data additional file 2 ). Area under the fitted ROC curve was similar for both age groups (aged ≤ 2 years = 0.811, 95%CI 0.79, 0.84; age >2 = 0.84, 95%CI 0.74, 0.95). Agreement for clinicians vs parents cough quality (dry/wet) was better in children aged ≤ 2 years (K = 0.85, 95%CI 0.57, 1.0; n = 42 but 18 non-assessable) than that for those age >2 years (K = 0.70, 95%CI 0.49, 0.92; n = 64, but 12 non-assessable) (see additional file 1 ). Using recorded coughs, kappa scores were 'very good' for both intra-observer and inter-clinician agreement for wet and dry cough (K = 1.0 and 0.88 [95%CI 0.82–0.94] respectively). There was only one disagreement in wet and dry cough between clinicians and in this child the cough was mildly wet (BS grade of 3). Kappa scores for intra-observer and inter-observer clinician agreement for brassy cough was good, K in both was 0.79, 95%CI 0.73, 0.86. Inter-rater agreement for BS grades was 'very good' (weighted K = 0.95, 95%CI 0.87–1). Cellularity for total cell count, percentages of neutrophils and macrophages were significantly different between children grouped by BS grade cut-offs of 3 and 4 as well as wet/dry cough (table 3 ). Table 3 Cellular differential profile in BALs Median TCC (IQR) % M IQR) % N (IQR) % Lym (IQR) % Eos (IQR) BS cutoff at grade 3 ≤2 (n = 31) 195 (290) 82.0 (15.8) 5.0 (7) 13.5 (15.8) 0 (0) ≥3 (n = 70) 334.0 (425) 66.0 (45) 12.0 (38) 11.0 (16.0) 0 (0) p value^ 0.038 0.001 0.006 0.605 0.758 BS cutoff at grade 4 ≤3 (n = 52) 176 (257) 81.0 (17.0) 6.0 (8.0) 13.0 (16.0) 0 (0) ≥4 (n = 49) 368 (574) 51.5 (59.8) 20.0 (47.0) 11.0 (15.0) 0 (5) p value^ 0.0001 0.0001 0.0001 0.445 0.613 Cough quality * Wet (n = 45) 365 (522) 51.5 (49.8) 25.0 (43) 13.0 (16) 0 0 Dry (n = 25) 176 (315) 80.5 (24.8)) 5.5 (13.0) 1.8 (16.0) 0 (0) No history (n = 28) 80 (310) 15 (16.5) 1 (7.5) 1 (11.5) 0 (0) 310 16.5 7.5 11.5 0 p value^ 0.017 0.0001 0.001 0.242 0.769 ^p value = examined using Kruskal Wallis test. *assessed by clinician TCC = total cell count; N = neutrophils, M = macrophages, L = lymphocytes, Eos = eosinophils, Discussion We have shown that clinical assessment of cough quality of wet/dry cough generally relates to bronchoscopic secretions determined using a standardised scoring system (BS grades). When cough is wet, secretions were always present; when cough was dry secretions if present, were usually minimal or mild. Clinicians were marginally better than parents at assessing wet/dry cough and agreement between the 2 groups was good. When clinicians detected presence of a brassy cough, tracheomalacia was usually present. Inter-rater clinician agreement for cough qualities of dry/wet and brassy/non brassy was good. Accuracy and reliability of symptoms are important in clinical and research settings. Cane and colleagues [ 25 , 26 ]. found that parental reports of wheeze and stridor are often not accurately reported in a clinic setting. There is no data on the validity of cough quality in spite its use in management and diagnostic guidelines [ 11 , 27 , 28 ] and cough being the most common symptom seen by general practitioners [ 1 - 3 ]. The level of agreement recommended for symptoms and signs to be used in clinical prediction rules is kappa value of ≥ 0.6 [ 29 ]. The kappa values we obtained in this study well exceeded 0.6. Specifically, intra and inter-clinician evaluation was very good and parental reporting of cough quality (wet/dry) also related well to clinicians' evaluation. When compared to bronchoscopic findings, this study showed that a wet cough is always associated with BS grades of 3 or more. Dry cough is less valid; the presence of dry cough does not necessary indicate absence of secretions. However BS grades are less in dry cough as shown in the ROC curve. The generation of cough sounds and some factors that influence cough sounds have been examined in the laboratory [ 16 , 30 ]. Using cough sound analysis (spectrogram and time-expanded waveform), productive and non-productive cough can be differentiated in the laboratory [ 30 ]. However to date there is no data on its clinical reliability and its relationship to quantification of airway secretions. In humans, it is not known how much mucus is required and where it has to be located for the human ear to detect presence of a moist cough. It is likely that mucus in the large airways is required for detectable difference in cough quality as the sound of cough is generated from vibration of larger airways and laryngeal structures during turbulent flow in expiration [ 16 , 17 ]. Laminar airflow, which occurs in smaller airways, is inaudible [ 31 ]. In an animal model, Korpas and colleagues showed that a certain amount of mucus is required to alter cough sound; 0.5 ml of mucus instilled into the trachea of cats altered cough sound, too little mucin had no effect on cough quality whilst too much mucin impaired breathing [ 32 ]. Our study findings support this and it is not surprising that when the cough is dry, BS grades were less. The rheological properties of airway mucus also influence cough sound [ 17 ]. It is not known how airway secretions in the more peripheral airways influences the sound of cough. One possible limiting factor of our study is the choice of cut offs for BS grades in determining presence or absence of significant secretions. We chose to use a cut off of 3 as a minor amount of bubbles in the airways can be present from trickling of lignocaine into the airways or spillage from the upper airways. BS cut-off at grade 4 resulted in improved specificity but decreased sensitivity. Children grouped by both BS cut-offs (3 and 4) had significantly different airway cellular profile. The clinical significance of minimal BS grades and appropriate cut-offs can only be determined in a prospective follow-up study which is not an aim of this study. This study did determine that our BS scoring method was easy to use (most done in real time) and had very good inter-rater agreement. The clinical outcomes of wet and dry cough were not the aims of this study and thus cannot be determined here. To relate clinical outcomes to cough descriptors would ideally require a randomised controlled trial with dry and wet cough as entry criteria. A follow-up cohort study with strict clinical diagnostic categories would be useful and we have shown in a preliminary study that dry cough was significantly more likely to naturally resolve than wet cough [ 33 ]. In addition to the limitation of quantifying airway secretions using a bronchoscopic method, this study is also limited by a number of factors. Firstly, clinical repeatability or agreement of cough sounds was assessed by doctors in a tertiary setting. Whether or not these findings can be extrapolated to the secondary and primary setting can only be speculated. Hay and colleagues showed that inter-observer agreement for clinical signs of fever, tachypnoea and chest signs were poor to fair (kappa of 0.12–0.39) in the primary care setting but these signs are known to have good agreement in secondary care settings [ 34 ]. However as parents were almost as good as clinicians in our study and are 'untrained' compared to medical practitioners, we would expect that this data can be extrapolated to most primary and secondary settings. Secondly, anaesthesia and atropine could possibly influence mucus quantity and properties. However this influence, if any, is likely to be small as both bronchoscopists (ABC, IBM) are experienced (our recorded average total theatre time is relatively short at 22 mins) [ 22 ], and atropine is given just immediately prior to commencement of bronchoscopy. Determining the validity of cough quality in children is important not only because of the commonality of the clinical problem of cough but also its use in guidelines and research studies [ 11 , 27 , 28 ]. A particularly important finding is the presence of small amounts of secretions in children with dry cough which may have implications in the management of suppurative lung disease; a dry cough may represent early disease process where only a small amount of mucous is present. Conclusion We conclude that the description of a cough as wet or dry cough as determined by clinicians and parents has good clinical validity as it has good agreement with, and relates to, quantification of airway secretions. However as minimal amount of secretions may be present in children with dry cough, clinicians should be cognisant that a dry cough may eventually become wet if airway secretions increase. Thus it should not be assumed that airway secretions are absent in children with chronic dry cough and cough quality in these children should be reviewed. We also conclude that the brassy cough determined by respiratory physicians is highly specific for presence of tracheomalacia. List of Abbreviations BAL Bronchoalveolar lavage BS Bronchoscopic secretion K Kappa NPV Negative predictive value PPV Positive predictive value ROC receiver operating characteristic Authors' contributions AC conceived the idea, designed the study, performed the data analysis and drafted the manuscript. JG participated in data acquisition and coordination of project. ME participated in electronic acquisition of data and software for sound recordings. JF and NC designed the microbiology and cytological components respectively and both helped draft the manuscript. IBM helped in formulation of overall study design, data acquisition and drafting of the manuscript. All authors read and approved the manuscript. Supplementary Material Additional File 1 Figure 3: ROC curve ROC curve with 95%CI relating cough quality (wet/dry) to bronchoscopic secretion (BS) grades from 1–6 in children grouped according into age (a) ≤ 2 years and (b) > 2 years. Click here for file Additional File 2 Table 4: Assessment of cough quality vs bronchoscopic findings in children grouped by indication for bronchoscopy 4a: Assessment of cough quality vs bronchoscopic findings in children whose indication for bronchoscopy was cough 4b: Assessment of cough quality vs bronchoscopic findings in children whose indication for bronchoscopy was others (ie not cough) Table 5: Assessment of cough quality vs bronchoscopic findings in children grouped by age 5a: Assessment of cough quality vs bronchoscopic findings in children aged ≤ 2 years 5b: Assessment of cough quality vs bronchoscopic findings in children aged > 2 years Click here for file

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535547

Using GIS to establish a public library consumer health collection

Background Learning the exact demographic characteristics of a neighborhood in which a public library serves, assists the collection development librarian in building an appropriate collection. Gathering that demographic information can be a lengthy process, and then formatting the information for the neighborhood in question becomes arduous. As society ages and the methods for health care evolve, people may take charge of their own health. With this prospectus, public libraries should consider creating a consumer health collection to assist the public in their health care needs. Using neighborhood demographic information can inform the collection development librarians as to the dominant age groups, sex, and races within the neighborhood. With this information, appropriate consumer health materials may be assembled in the public library. Methods In order to visualize the demographics of a neighborhood, the computer program ArcView GIS (geographic information systems) was used to create maps for specified areas. The neighborhood data was taken from the U.S. Census Department's annual census and library addresses were accumulated through a free database. After downloading the census block information from the data was manipulated with ArcView GIS and queried to produce maps displaying the requested neighborhood demographics to view in respect to libraries. Results ArcView GIS produced maps displaying public libraries and requested demographics. After viewing the maps the collection development librarian can see exactly what populations are served by the library and adjust the library's collection accordingly. Conclusions ArcView GIS can be used to produce maps displaying the communities that libraries serve, spot boundaries, be it "man-made or natural," that exist prohibiting customer service, and assist collection development librarians in justifying their purchases for a dedicated consumer health collection or resources in general.

Background Libraries have the objective to build collections that support the communities they serve. To build a viable collection the collection development librarian, outreach/marketing librarian, and others, must determine exactly what populations reside in the neighborhood, in respect to race, spoken language, educational level, and age groups. One method collection development librarians use to gather demographic information is to physically visit the communities and integrate into the neighborhoods. They may attend events within the community in order to analyze the attendees or walk around the neighborhood to get a feel for the community. Another method for collection development is to perform an informal survey with people that visit the library and learn about their preferences and/or what they like to read or look for on the Internet. Using census information can be a third way to gather community/neighborhood information. The US Census Bureau takes a census of the United States every 10 years and publishes the results on the Internet. Taking the Census information and transforming it into a graphical format provides an objective view of the communities surrounding a library. One way to visualize the census data is through the use of GIS, Geographic Information Systems. GIS are databases arranged by spatial coordinates that, when programmed, can produce maps [ 1 ]. The US Geological Survey office defines GIS as "a computer system capable of capturing, storing, analyzing, and displaying geographically referenced information; that is, data identified according to location [ 2 ]." Many disciplines have used GIS and many large academic libraries even support GIS by establishing a GIS department and employing a librarian to specialize in GIS information. In the field of library science articles have been published describing the establishment of GIS departments in libraries and what GIS librarians do [ 3 - 7 ], but little has been published describing how a library has used GIS. Governments use GIS to visualize land use planning, tax appraisal, utility and infrastructure planning and more [ 8 ]. Businesses have used GIS for real estate analysis, marketing, and demographic analysis [ 8 ]. Based on this approach, a research project was developed to test the use of GIS in collection development for a large metropolitan public library system. Building a collection for a library has become a sophisticated art. Johnson states that "collection building consists of four steps: identifying the relevant items, assessing the item to decide if it is appropriate for the collection and evaluating its quality, deciding to purchase, and preparing an order [ 9 ]." Each step in the process of collection development produces a layer of difficulties. In order to get to these steps a collection development librarian must be aware of their audiences. To name a few necessary pieces of information, the librarian must know the predominate age of their clients, their reading genre, their reading level, their culture, and their preferred language [ 9 ]. After one has gathered community information then the decision of what to purchase for use by the community, can be initiated and carried out [ 10 ]. This paper reports the steps taken to use GIS in assisting a large metropolitan public library system in visualizing neighborhoods that surround branch libraries in order to make an educated decision on whether a dedicated consumer health collection should be established to support the community. The objective of the project was to determine if GIS could be used to improve collection development. Methods After comparing large library systems in major metropolitan areas, (Chicago, Los Angeles, and New York City) Chicago was selected as a convenient sample to be mapped with GIS because of its size, number of public libraries, their geographic distribution within the urban area, and ease of access to data. Compared to other large cities, the New York Public Library system has 86 libraries and Los Angeles has 67 public libraries [ 11 ]. The city of Chicago has 77 public libraries with one central library. With the selection of the city of Chicago, the next step was to create a map of the metropolitan area. ESRI ArcGIS v9.0 geographic information software (GIS) [ 12 ] was selected as the GIS application. The software permits data manipulation and mapping capabilities. There are four components of ArcGIS that work together to give a high level of functionality to the program: ArcReader, ArcView, ArcEditor and ArcInfo. By inserting data files into ArcEditor the datum may convert into a graphical representation. Once a base map has been created with geographic boundaries, locations may be established to show distances within the map. In order to create the city map of Chicago, data files were obtained from the U.S. Census Bureau website. The Census Bureau produces TIGER (Topologically Integrated Geographic Encoding and Referencing) Files available for free to the public [ 13 ]. These files contain geographic structures, such as streets, highways, and addresses, for tabulation and dissemination. To set the boundary lines of metropolitan Chicago, Cook County (the county in which the city of Chicago resides) was selected as the source for the data file [ 14 ]. This TIGER file contained the street map for the city of Chicago. The next step required mapping distances between neighborhoods and neighborhood demographics. In order to achieve this, files were obtained from the U.S. Census Bureau website [ 15 ] which distributes the 2000 Census statistics in TIGER File format. Downloading the files for Census blocks in Cook County provided the necessary data to build a visual representation of neighborhoods. The final piece of information needed to build a map showing neighborhoods in respect to public library locations, was to map the public libraries within Chicago. Because the base map of Cook County was a street map, addresses of public libraries were entered into ArvView to mark all the public libraries within the city of Chicago. The state of Illinois provides a database available on the Internet that permits searching types of libraries and locations of libraries, . The output of this search was imported into Excel, cleaned, and converted into a dBase file. The dBase file was imported into ArcView to coordinate the library addresses to the street addresses of the base map. This enabled the software to place an image for each public library in the city map of Chicago. With the city of Chicago and its public libraries now in a GIS application, the location of libraries was displayed in respect to neighborhoods and distances from library to library (See Figure 1 ). Because the Census data was used to build the map, ArcView can be queried to show demographics of the city. This allows useful data manipulation in relation to points of interest, in this case, public libraries. Thus, ArcView was queried to show the distribution of all men and women within the city of Chicago as well as the breakdown of their age groups. The software application produced a graphic which used color schemes with a map legend. If a librarian wanted to compare the number of men in their 40s to women in their 40s, ArcView can map those fields with a well-structured query. ArcGIS was also capable of creating maps that display census ethnic groups. For example, Figure 2 shows a high percentage of African Americans in the southern neighborhoods of the city as compared to the northern part of the city. With maps such as these, a collection development librarian can graphically see the demographic breakdown of neighborhoods and build collections according to its clientele [ 16 ]. Figure 1 Location of public libraries in the city of Chicago Figure 2 Density of African American population in southern part of Chicago Results Fifteen maps were created showing a breakdown of each demographic with the city's public libraries. Each map provided information regarding gender, age, and race of the clientele surrounding the libraries. These maps used a combination of color schemes to graphically represent differences between groups. The maps displayed different age groups divided by ranges of age (30–39, 40–49, 50–64, 65 and up). The age ranges presented in the maps varied by query and census information. The groups analyzed were: women, men, white women (group age 40–49), African American women (group age 40–49), Asian (men/women, Latinos (men/women). The color schemes used represent the number of people that live within the census block as to the specified GIS query. In Figure 2 , the numbers, 0–992 in the legend are the African Americans residing within the census block. Hence the red blocks have a large population (403–992) of African Americans as compared to the dark green areas. Figure 3 represents a query for African American women in their 40s. The results produced a map showing that 26–59 African American women in their 40s live within the shaded red blocks. Figure 3 African American women in their 40s in proximity to public libraries In this project ArcGIS was used to build maps of neighborhoods in the city of Chicago, display public libraries and spatially represent distances from libraries to populations. The US Census demographic information was incorporated into ArcGIS and queried to produce 15 different maps. Queries were built to display neighborhood demographics through colors showing the percentages of the population(s). Each query produced a map for a specific age or race. Once the map was produced the public libraries were added to the map and analysis was made regarding collection needs according to the neighborhood population surrounding the library. To decide if a public library should establish a consumer health collection, a map showing the breakdown of women in their 40s was built. As in Figure 4 , the map shows a very high percentage of women 40–49 close to the Uptown Branch Library. The collection development librarian can now assess the libraries existing collection for health related materials. If there are very few resources then the collection development librarian should begin to purchase consumer health materials. If the library has a strong consumer health collection already then building maps to show the racial breakdown of the neighborhood could lead the collection development librarian to purchase culturally sensitive health materials. Figure 4 High density neighborhoods with all women in their 40s in proximity to public libraries Discussion By creating maps showing the demographic breakdown of the city, a public library can build, adjust, or verify its collection in respect to the neighborhoods it supports [ 17 ]. It is easier to define the characteristics of the library's service area clientele by making visual representations (maps) of neighborhoods surrounding a library. The neighborhood information may then be used to justify additional resource purchases, a change in collection policy and even a change in library services to fit the culture of the neighborhood. By using GIS, libraries will be capable of finding specific populations within the library's neighborhood to support or serve thus taking some of the guesswork out of collection development [ 17 , 18 ]. Collections may be tailored specifically to the neighborhood by using the subjective data provided with GIS. Many studies have been conducted analyzing what people search for on the Internet and who is searching. Studies have found that women in their 40s search for and utilize health information from the Internet more than men [ 19 - 24 ]. Using this information to assist in collection development, a librarian would need to know how large the population of women in their 40s is in the neighborhood the library serves. Using the map created by ArcGIS showing the distribution of women in their 40s in respect to library locations can assist the collection development librarian in purchasing appropriate materials. Using the maps showing the race of women in their 40s would also assist the librarian in purchasing appropriate materials. For instance, if most women are African American then the collection development librarian would purchase materials supporting race-related health needs such as Sickle Cell Anemia, Coronary Heart Disease, Diabetes and others (See Figure 2 and 3 ). Recommendations Using ArcGIS can assist in defining the exact make-up of the population that a library serves. By turning the census information into a graphic the library is provided a chance to see how far (physical distance) their services may reach. Library systems that have branch libraries may have one collection development librarian who buys for each library. By using ArcGIS the central library can analyze the communities surrounding each library to purchase materials appropriately without having to spend time in each library learning the environment. Purchase decisions may now be justified with hard data from ArcGIS. For example, if ArcGIS shows that the largest population surrounding a library speaks a minority language, materials should be purchased in the dominant language to best meet the needs of the community. Limitations There is a fairly high learning curve to use ESRI Arcview GIS. There are five programs in ESRI Arcview and each program has separate features that must be used within the specific program to be imported into the primary map. Learning what each program does and then how to incorporate the data into the map requires hours of reading. Acquiring the necessary data to build a map requires background study of the geographic area in order to assure that the map is configured correctly. Upon attaining all necessary data for the map, one must build queries to manipulate the data according to the information need, and then configure the map accordingly. While GIS presents a new technique to use in performing collection development, there was not a way to test the differences from a collection built using GIS information and the current methods of collection development. In addition, research has discovered that knowing the service area radius of the library doesn't mean that people within the service area will actually use or belong to the neighborhood library [ 17 , 25 ]. When using GIS one must take into account geographic barriers to library access such as major highways, and railways. People are often reluctant to cross these barriers and may elect to visit another library further away with no geographic obstructions [ 17 , 25 , 26 ]. These barriers must be represented in the GIS map to fully display the areas libraries serve. The TIGER files contain some geographic structures but most will have to be hand coded. Thus, all neighborhoods should be examined in order to assure the marking of geographic barriers. While GIS can take the census information and demonstrate the population surrounding a library, the census does not describe the specific users of the library [ 25 ]. To build a map showing a library's service area, patron addresses (information now protected by the Patriot Act) would need to be coded into GIS. Using patron zip codes of residence encompasses too much or too little area and there is no correlation between US Postal Service ZIP Codes and US Census Bureau Geography [ 27 ]. Thus, mapping or defining areas through zip codes is extremely difficult. Without patron address information, there can be no clear realization of exactly how far people will travel to a library, and if people cross-neighborhoods to use a certain library [ 18 , 25 ]. Conclusions Budget constraints will add a need for justification of purchases for a library's collection. Using GIS may provide one method to justify additions to collections. As health care changes and individuals take more control of their health, libraries need to have materials that provide valid information in an age and language appropriate format and be culturally sensitive. Branch libraries respond to the demands of the neighborhood [ 25 ] and knowing what those demands are will make an effective library [ 17 ]. GIS can assist collection development librarians located in a central library buy for branch libraries, by saving them time, and answering demographic questions about the population a library serves. Knowing details about its communities will make a more functional library within a neighborhood. To verify the use of GIS for collection development, an analysis of a library's collection should be conducted and compared with information provided from a GIS application to see if the collection satisfies the library's neighborhood. List of Abbreviations GIS = geographical information systems

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Cyclic nucleotide binding proteins in the Arabidopsis thaliana and Oryza sativa genomes

Background Cyclic nucleotides are ubiquitous intracellular messengers. Until recently, the roles of cyclic nucleotides in plant cells have proven difficult to uncover. With an understanding of the protein domains which can bind cyclic nucleotides (CNB and GAF domains) we scanned the completed genomes of the higher plants Arabidopsis thaliana (mustard weed) and Oryza sativa (rice) for the effectors of these signalling molecules. Results Our analysis found that several ion channels and a class of thioesterases constitute the possible cyclic nucleotide binding proteins in plants. Contrary to some reports, we found no biochemical or bioinformatic evidence for a plant cyclic nucleotide regulated protein kinase, suggesting that cyclic nucleotide functions in plants have evolved differently than in mammals. Conclusion This paper provides a molecular framework for the discussion of cyclic nucleotide function in plants, and resolves a longstanding debate about the presence of a cyclic nucleotide dependent kinase in plants.

Background The discovery of cyclic 3'5'-adenosine monophosphate (cAMP) by Earl Sutherland in the late 1950s was one of the most significant paradigm shifts in biochemistry [ 1 ]. This breakthrough ushered in the concept of second messengers: intracellular molecules which transmit signals in cells and are derived from an extracellular signal. In the past half century, cyclic nucleotides (both cAMP and cGMP) have been implicated in a vast array of biological phenomena in all kingdoms of life [ 2 ]. The ubiquitous presence of cyclic nucleotides may be due to several characteristics which make it an ideal second messenger. Cyclic nucleotides are derived in a energetically favourable reaction from common metabolites (ATP and GTP), and can be broken down into non-toxic products (inorganic phosphate and AMP/GMP). The synthesis and degradation of cyclic nucleotides are controlled by enzymes termed adenylate (or guanylate) cyclases and cyclic nucleotide phosphodiesterases, respectively [ 3 , 4 ]. In plants, cyclic nucleotides have endured a checkered research history fraught with complications and setbacks. Despite this, recent work has shown unequivocally that cyclic nucleotides are present in plant cells [ 5 , 6 ], and that they play key roles in the regulation of plant physiology [ 7 - 9 ]. Furthermore, the recent identification and cloning of adenylate and guanylate cyclases in plants [ 7 , 10 ] may eventually give clues as to what signals the synthesis and degradation of these molecules in plants. Cyclic nucleotides are able to bind to two distinct protein domains, CNB domains and GAF domains. CNB domains were first identified in the regulatory subunit of mammalian cAMP-dependent protein kinase (RI and RII). Since several CNB domain containing plant proteins have been shown to be directly modulated by cyclic nucleotides, this indicates that the CNB domain in plants is functionally similar to CNB domains in other organisms [ 11 - 15 ]. GAF domains were initially identified as conserved domains in light sensing molecules but are known as small molecule binding domains in cyclic nucleotide regulated phosphodiesterases, the Anabaena cyclic nucleotide stimulated adenylate cyclase and several other proteins [ 16 ]. GAF domains have been shown to bind both cAMP [ 17 ] and cGMP [ 18 , 19 ]. Recent crystal structures of the GAF domains of human PDE2 [pdb:1MC0] and the yeast protein YKG9 [pdb:1F5M] have shown that this domain is an alpha/beta two layer sandwich with no structural or sequence homology to the CNB domain [ 18 , 20 ]. Therefore, GAF and CNB domains have evolved independently to bind cyclic nucleotides. In order to further explore the roles of cyclic nucleotides in plants, we performed a bioinformatics based analysis of the completed Arabidopsis thaliana and Orzya sativa genomes [ 21 - 23 ] in order to elucidate the potential targets of cyclic nucleotides in plants. Results and discussion GAF domains Based on the PDE2 crystal structure 11 residues were proposed to be involved in cyclic nucleotide binding [ 18 ], but comparison to the cAMP binding GAF domain of the Anabaena adenylate cyclase shows that these residues may only be strictly conserved in mammals. Further complicating our analysis is the fact that GAF domains are known to bind other small molecules such as 2-oxoglutarate, formate and bilins [ 24 - 26 ]. GAF domains form a structural scaffold which can be utilized to bind several possible small molecules depending on the functional groups on that scaffold. Therefore, their role in cyclic nucleotide signalling must be verified by biochemical means rather than strictly by sequence analysis. Our analysis indicated that in plants there are two types of proteins which contain GAF domains. These are the phytochrome proteins and the ethylene receptor proteins. Phytochromes Phytochromes are light sensing signal transduction molecules which function to control several aspects of plant biology. Interestingly phytochromes were found to function upstream of cyclic nucleotides in their signal transduction pathways since their functions can be mimicked by cGMP and calcium in phytochrome knockout cells [ 27 - 29 ]. The light sensing portion of the phytochrome molecule is a covalently linked bilin molecule which is known to be bound by the GAF domain. Therefore it is unlikely that the GAF domain of the phytochrome is also able to bind cyclic nucleotides directly, although it is clear that cyclic nucleotides are somehow involved in this signalling pathway. Ethylene receptors Ethylene responses have been documented for nearly a century in plants. This gaseous hormone is involved in many aspects of plant physiology, including fruit ripening, organ development, germination, seedling growth, flowering and response to challenges such as pathogens and stress [ 30 ]. There are five putative ethylene receptor isoforms in both Arabidopsis and rice as determined by genome sequencing [ 31 ]. All known ethylene receptors contain a GAF domain in a cytoplasmic region amino-terminal to the kinase domain. It has been speculated that this domain may be involved in cyclic nucleotide signalling but examination of heterologously expressed, functional ETR1 [Swiss-Prot: P49333] showed no detectable cyclic nucleotide binding (G. E. Schaller, personal communication). There are other ethylene receptors which have GAF domains and which to our knowledge have not been tested for cNMP binding, however, to date there is no evidence of cNMP regulation of ethylene receptors. Currently the function and ligands of the GAF domain in ethylene receptors is unknown. CNB domains From the alignment of CNB domains in animals, bacteria and plants, it was apparent that there are some strong similarities, as well as some significant differences (Figure 1A ). In order to visualize whether plant CNB domains could fold in a similar manner to the other well characterized CNB domains, we generated an in silico model. We chose the plant protein which showed highest similarity to known crystal structures ( Arabidopsis thaliana CNTE1) and based our model on the solved crystal structures of RIα, RIIβ, HCN2, CAP and Epac2 (Figure 1B and see additional file 5 ). We then examined our model's overall topology as well as its cyclic nucleotide binding site. The basic fold of the domain is two anti-parallel beta sheets consisting of four strands forming a sandwich, ending with an alpha helix (the hinge region). Connecting these sheets are exposed loops, the most important of which is the phosphate binding cassette [ 32 ]. It is important to note that our structure models very well against all CNB domains with excellent conservation of all secondary structure and most loops. We calculated the backbone root mean square deviation for our model versus each of the templates as: RIIβ domain 1: 0.76 Å; RIIβ domain 2: 0.83 Å; Epac1 domain 1: 1.08 Å; RIα domain 1: 0.85 Å; RIα domain 2: 0.94 Å; HCN2: 0.82 Å and CAP: 1.12 Å. Our model agrees in general with a previously reported model for the Arabidopsis CNGC2 [ 33 ], although a detailed comparison between the two models was not performed. The use of a less distant target (atCNTE1) as well as several templates (seven compared to one) adds to the reliability of our structure. In all mammalian cAMP-binding structures solved, there is a key arginine residue (Arg 209 in RIα) which forms a salt bridge with the cNMP's phosphate group. This residue is absent in some CNB domains, despite evidence that at least some of these domains do in fact bind cyclic nucleotides. For example, this residue is absent in the Drosophila ether-a-go-go channel, which is known to be modulated by cyclic nucleotides [ 34 ]. Examination of our model shows that in the region near the phosphate, there are two residues which may functionally replace the arginine, Tyr 91 and Ser 92 (Figure 1B ). In bacterial CAP, hydrogen bonding is provided to the phosphate by the Arg 82 sidechain, Ser 83 amide nitrogen atom and sidechain hydroxyl group, as well as a water molecule. In some mammalian isoforms, the serine residue is changed to an alanine and therefore is only able to provide backbone hydrogen-bonding. In our plant atCNTE1 model, the serine residue is conserved, but the arginine residue is missing. Since there was no good template to model the phosphate binding cassette onto, our model only approximates the position of this loop, and will require verification by other structural studies. The hydroxyl and amide groups of Ser 92, as well as the hydroxyl group of Tyr 91 are all within proximity of the cNMP phosphate and could play a role in stabilizing the cyclic nucleotide (see blue residues in Figure 1 ). Examination of the region which contacts the base, indicates that our model is most similar to the structure of CAP in this region, so it is likely that the base moeity of a cNMP is bound in a syn orientation as in CAP. This is in agreement with a previously reported atCNGC2 model [ 33 ]. Further analysis of the binding site for the nucleotide indicates that it is likely cGMP which binds to atCNTE1. This conclusion is based upon the presence of three residues (Tyr 80, Ser 92 and Ser 109) which could potentially differentiate between cyclic nucleotides, each of which has a preference for cGMP (Figure 1 and [ 35 , 36 ]). Other conserved structural features of our model are the hydrophobic pocket forming residues Tyr 36, Val 42, Val 43, Tyr 53, Leu 55, Ala 60, Phe 82, Ala 93 and Val 95 and Leu 105 (see green residues in Figure 1 ) as well as several conserved glycine residues which are involved in turns between the beta strands (39, 58 and 83) and the helix capping Asp 109. This residue signals the end of the hinge region alpha helix and is present in most CNB domains examined. Phylogenetic analysis indicates that the plant CNB domains segregate into three subfamilies (Figures 2 and 3 ). The phylogenetic distribution of the CNB domain matches their domain context in that CNGC, shaker-type and CNTE proteins form separate groups. Furthermore, for each of the three protein classes, the CNB phylogeny matches the phylogeny of the full-length protein, implying that these proteins obtained the CNB domain prior to isoform duplication (Figure 3 , [ Additional file 1 ], [ 37 ]). Since all three branches have been detected in both Arabidopsis and Oryza, it is likely that the specific plant cyclic nucleotide responses developed prior to monocot-dicot divergence. We did not find CNB domains in any protein kinases, transcription factors or guanine nucleotide exchange factors in our analysis. Each of the three classes of CNB domain containing proteins will be discussed below. Cyclic nucleotide gated ion channels Plant CNGC ion channels were first identified in a screen for calmodulin binding partners in barley [ 38 ]. There are now known to be 20 CNGC proteins in Arabidopsis likely indicating a high level of channel redundancy [ 37 , 39 ]. We also detected 16 CNGC proteins in rice by examination of the TIGR Rice Genome Annotaion Resource [ 40 ]. Electrophysiological studies have shown the CNGC channels to be permeable to potassium, sodium and calcium [ 13 - 15 , 41 - 43 ]. Cyclic nucleotides have been shown to activate channel opening in all CNGC proteins examined thus far leading to an influx of cations into the cell [ 13 - 15 , 33 ]. Mutagenic screens have shown that mutations in atCNGC2 and atCNGC4 create faulty pathogenic reactions [ 13 , 44 ]. When taken together with data showing that cyclic nucleotides are necessary for pathogen responses and that calcium and potassium influxes are characteristic of early phases of plant pathogen responses [ 45 ], this seems to imply that cyclic nucleotides may play a role in controlling plant immune responses. Finally, work by Maathuis and Sanders [ 8 ] has shown that cyclic nucleotides can modulate sodium uptake in Arabidopsis plants, implying that there is a cyclic nucleotide controlled channel which plays a role in salinity tolerance. They showed that cyclic nucleotides are required for limiting sodium uptake in root protoplasts, but the exact molecule (cAMP or cGMP) responsible for this effect has not been pinpointed. Shaker-type potassium channels Plant potassium channels fall into two classes, the KCO channels and the shaker-type channels [ 37 ]. In addition to the 9 shaker-type channels described in Arabidopsis thaliana , we have found 10 channels in Oryza sativa . A variety of mutational studies have implicated the shaker-type channels in several key processes involving the movement of potassium including: from the soil (AKT1, KAT3), long distance transport (AKT2), transport into growing pollen tube (AKT6), secretion into xylem sap (SKOR) and transport during guard cell opening either into the cell (KAT1, KAT2) or out of the cell (GORK) [ 37 ]. Shaker-type channels are voltage dependent outward (GORK, SKOR) or inward (KAT and AKT) rectifying channels. Analysis of heterologously expressed channels have shown that cyclic nucleotides function to adjust the activation potential of these channels [ 11 , 12 ]. Since cyclic nucleotides have already been implicated in some of the processes controlled by shaker-type channels [ 7 - 9 , 46 ], it is reasonable to believe that cyclic nucleotides are physiological regulators of shaker-type potassium channels. Cyclic nucleotide regulated thioesterases Initially we detected a short CNB containing protein which was only slightly larger than the domain itself. Sequencing of the EST provided by the Arabidopsis Biological Resource Center [ 47 ] showed the protein was actually mis-annotated by the automated gene-finding algorithm. Further analysis indicated that there are two isoforms of this protein in Arabidopsis and one in rice. Each protein contains an amino-terminal CNB domain and a carboxy-terminal acyl-CoA thioesterase domain. Searches of other partially sequenced plant genomes and EST databases indicated that these proteins are present in several plant species, but not in any other division of life and thus represents a novel plant-specific cyclic nucleotide target. Comparison of these protein sequences indicates a high level of conservation, including residues conserved for both catalysis and cyclic nucleotide binding domain structure. Arabidopsis CNTE1 had previously been partially characterized as a thioesterase and shown to have activity versus both 16:0-CoA and 18:1-CoA when over-expressed and partially purified from E. coli [ 48 ]. Fatty acid synthesis requires the use of acyl-CoA's as building blocks for incorporation into lipids. It is therefore possible, that these thioesterases function as scavengers which remove "irregular" fatty acids from the pool of available building blocks [ 48 ]. Furthermore, the thioesterases could divert fatty acids away from biosynthetic pathways and β-oxidation during germination or during stressful conditions. In most cases when a small molecule binding domain is connected to a catalytic domain on the same polypeptide, the catalytic domain is regulated by the small molecule [ 49 ]. The conservation of this protein across planta indicates that the CNB domain likely has a role in controlling the thioesterase activity of this enzyme, but it is unknown at this time exactly what role cyclic nucleotides play in this process. In order to address this we cloned and tried to express the atCNTE1 protein in E. coli , however after extensive trials we were unable to express and purify soluble protein. A cyclic nucleotide dependent protein kinase in plants? We found no PKG or PKA regulatory subunit homologs in the Arabidopsis genome. There has been a long standing controversy in the plant field as to the existence of a plant cyclic nucleotide dependent kinase [ 50 - 52 ]. As PKA is the major cAMP target in mammalian cells we chose a biochemical approach to further explore the possibility that a PKA-like enzyme may be present in Arabidopsis. We performed protein kinase assays with extracts of Arabidopsis thaliana using the PKA substrate Kemptide. Kemptide is a peptide which has a motif which was confirmed as the optimal substrate for PKA [ 53 , 54 ] and is routinely used in mammalian PKA assays. As Figure 4A shows, there was no detectable increase in kinase activity in the plant cell extracts when cAMP or cGMP are added. Fractionation of extracts, as well as testing a range of cyclic nucleotide concentrations also did not allow us to detect any differences in kinase activity with addition of cyclic nucleotides (data not shown). For comparison, adipocyte extracts (a cAMP responsive mammalian tissue) were assayed as well, illustrating the large increase in protein kinase activity in these cells when cAMP is added. Furthermore, blotting of A. thaliana extracts with polyclonal antibodies raised against mammalian PKA subunits (both the catalytic and the RIIα subunit) reveals that no structurally similar proteins are present in this extract (Figure 4B ). Blotting with a monoclonal antibody to the RIIβ subunit gave similar results (not shown). Although there is a weak band present in the Arabidopsis extract which cross-reacted with the catalytic subunit polyclonal antibody, it is likely unrelated to cyclic nucleotide signalling. Protein kinase catalytic domains are very highly conserved [ 55 , 56 ] and therefore a minor amount of cross-reactivity is not unexpected. Further adding to the validity of the western blotting experiment is the observation that several studies have shown that true PKA-like enzymes in non-mammalian eukaryotes do cross react with antibodies raised against the regulatory subunits of mammalian PKA [ 57 , 58 ], whereas this is not detected in our plant extracts. The lack of evidence for kinase activity could be attributed to substrate specificity, differences in binding affinity or expression levels in plant extracts relative to mammalian extracts, so our experimental approach does not exhaustively rule out the possibility of a cNMP dependent kinase activity. However, we feel that these data in concert with the genomic and blotting data strongly suggest that there is no cNMP dependent kinase in plants. Finally, our data imply that if such a protein exists, it would bear little or no sequence, structural or biochemical similarity to the classically studied mammalian enzyme. Conclusions As the understanding of cyclic nucleotide signalling in a variety of systems has progressed, it has been increasingly difficult to describe a general role for cyclic nucleotides in biology. They control 'well-fed' gene transcription in bacteria, and modulate signal transduction and ion currents in mammals, resulting in a large number of possible physiological responses. This analysis is potentially limited in that it only analyses cNMP domains which have already been previously identified and characterized in other systems. However, conservation of CNB and GAF domains as the only known cyclic nucleotide binding domains present over a wide cross-section of life indicates that these domains are likely to control most, if not all cyclic nucleotide responses. It is possible however, that plants have evolved entirely novel domains which can be modulated by these second messengers. It will be interesting to compare this in silico analysis with future biochemical data regarding the direct effectors of cyclic nucleotide signalling in plants. It is interesting that no homologous proteins in the CNGC, shaker-type or type II acyl-CoA thioesterase families have been found which lack CNB domains. This implies that cyclic nucleotide binding is indispensable to their cellular role. Although it would have been interesting if this analysis revealed more novel classes of plant cyclic nucleotide binding proteins, the fact that (with the exception of CNTE) all cyclic nucleotide binding proteins had been previously identified indicates that the previously attained biochemical data agrees with our bioinformatic evidence. The identification of no transcription factors or protein signal transduction molecules with CNB domains implies that cyclic nucleotides may be unable to directly modify the proteome of plant cells. This is in stark contrast to bacterial, yeast and mammalian systems. The only common domain context of CNB domains in animals and plants is the CNGC channels, however, even these channels appear to have evolved independently [ 39 , 59 ]. Therefore it is clear that the roles of cyclic nucleotides in prokaryotic and eukaryotic, as well as plant and animal systems differ and that evolutionarily distant branches of life have evolved different mechanisms by which these molecules are utilized. It is worth pointing out that the ubiquitous presence of cyclic nucleotides in all forms of life may indicate that although the means by which this particular biochemical tool is used differ, it is still an indispensable component of biology's toolbox. Methods Bioinformatics In order to identify the proteins which contain CNB or GAF domains, we initially used the Simple Modular Architecture Research Tool (SMART at smart.embl.heidelberg.de; [ 60 - 62 ]) to scan all predicted Arabidopsis proteins for CNB and GAF domains in the EMBL, TIGR or NCBI databases. Once redundancies were removed, a list of proteins was generated [see additional file 2 ]. In order to ensure broad coverage of possible variants, we also examined the Interpro collection of protein sequence analysis algorithms, all of which use slightly different methods [ 63 ]. As an additional method, the predicted proteins of the Arabidopsis genome were searched using the BLAST algorithm [ 64 ]. As search bait, we used several known cyclic nucleotide binding domains including those from GAF domains (human PDE2A [Swiss-Prot: O00408] and Anabaena cyaB1 [Trembl: P94181]) as well as CNB domains (human CGK2 [Swiss-Prot: Q13237], human RIIβ [Swiss-Prot: P31323], human Epac2 [Swiss-Prot: Q8WZA2], human rod CNGC [Swiss-Prot: P29973] and E. coli CAP [pir: E86000]). This yielded no new inclusions to our list of proteins, but did confirm each of our previous entries. For examination of the Oryza sativa spp . Japonica genome we performed BLAST searches using the aforementioned baits, as well as each of the Arabidopsis proteins. This search was performed using the Blast utility of the TIGR rice database [ 40 ]. The criterion for inclusion was that the CNB or GAF domain had to match the consensus motif with an E-value of less than 0.5 over the entire domain as determined by SMART. For newly identified proteins from the Orzya sativa , we named them so that they agreed best with the nomenclature of Maser et al . [ 37 ] [see additional files 1 , 2 , 3 ]. Sequence alignments were performed using the ClustalX [ 65 ] or T-COFFEE algorithms [ 66 ]and then inspected visually. Neighbor-joining trees were generated by ClustalX or PHYLIP [ 67 ], then were visualized with TreeView [ 68 ]. Trees generated using a variety of analysis methods (parsimony, distance and maximum likelihood) yielded similar results to the neighbor-joining trees. Sequencing of atCNTE1 One protein, which appeared to contain only a cyclic nucleotide binding domain and no other motifs was found in the Arabidopsis database. We obtained the clone corresponding to this putative gene from the Arabidopsis Biological Resource Center and sequenced it. Sequencing was performed at the University of Calgary Core Sequencing Facility. We determined that the gene prediction algorithm which scanned the genome improperly predicted the intron/exon structure of this gene. The new gene, which we named cyclic nucleotide regulated thioesterase 1 (atCNTE1) was deposited in the NCBI database [GenBank: AY874170]. A subsequent BLAST search using this gene found another isoform of this gene in Arabidopsis (atCNTE2) and one isoform in Rice (osCNTE1) which we also included in our analysis. Theoretical model of atCNTE1 We generated a model of the atCNTE1 cyclic nucleotide binding domain (residues 28–117) based on an alignment of atCNTE1 with the CNB domains of RIIβ [pdb: 1CX4] [ 69 ], RIα [pdb: 1RGS] [ 70 ], CAP [pdb: 1CGP] [ 71 ], HCN2 [pdb: 1Q3E] [ 72 ] and Epac1 [pdb: 1O7F] [ 32 ]. This was submitted to the SWISS-MODEL server via the DeepView program [ 73 ]. The alignment was iteratively refined to allow for best agreement of sequence and structural similarity. Cyclic nucleotide dependent protein kinase assays Unless otherwise indicated all chemicals were purchased from Sigma-Aldrich. Assays were performed on extracts of Arabidopsis cells grown in suspension culture [ 74 ] or isolated male Wistar rat adipocytes from epididymal fat pads [ 75 ]. Both cell types were homogenized in 50 mM Tris pH 7.5, 5% (v/v) glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine and 0.1% (v/v) 2-mercaptoethanol. Adipocytes were lysed by 10 strokes of a dounce homogenizer while plant cells were lysed by two passages through a french press cell at 15 000 psi. The extracts were clarified by centrifugation for 15 min at 4000 RPM in a SS34 rotor at 4°C. These extracts were assayed for kinase activity using 32 P-γ-ATP (Amersham-Pharmacia), 30 μM Kemptide substrate, 50 mM HEPES pH 7.4, 1 mM dithiothreitol and 10 μM cyclic nucleotide as specified. Reactions were allowed to occur for 10 minutes at 30°C and assays were terminated by spotting onto squares of P81 paper followed by extensive washing with 75 mM phosphoric acid [ 76 ]. Assays were performed in duplicate on three separate preparations with error bars indicating the standard error between preparations (n = 3). Protein concentration was determined by the method of Bradford with bovine serum albumin (ICN Biomedicals) as a standard [ 77 ]. Western blotting of extracts Bovine heart PKA catalytic subunit and RII were purified to homogeneity [ 78 , 79 ]. Purified protein was injected into rabbits and serum was obtained according to standard methods [ 80 ]. Extracts of adipose and plant cells were prepared as described above. Samples were boiled into SDS-PAGE buffer and separated on a 10% denaturing gel [ 81 ]. The proteins were then transferred to nitrocellulose for 2 h at 100V and blocked overnight with 5% (w/v) skim milk powder. Blots were probed with antibodies for 1 h and visualized by enhanced chemiluminesence. The PKA catalytic subunit antibody was affinity purified according to [ 82 ] and used at 0.5 μg/mL while RII was used as crude immune serum at a 5000X dilution. For the RII western blots, both a polyclonal and a monoclonal antibody (anti-RIIβ BD Transduction Laboratories) gave identical results. List of abbreviations CAP catabolite activator protein cAMP 3'5'-cyclic adenosine monophosphate CGK2 cGMP dependent protein kinase 2 cGMP 3'5'-cyclic guanosine monophosphate cNMP 3'5'-cyclic nucleotide (cAMP or cGMP) CNB cyclic nucleotide binding CNGC cyclic nucleotide gated channel CNTE cyclic nucleotide dependent thioesterase Epac exchange protein directly activated by cAMP GAF domain found in c G MP-phosphodiesterases, a denylyl cyclases and F hlA GORK guard cell outward rectifying potassium channel PKA protein kinase A SKOR stellar potassium outward rectifying channel SMART simple modular architecture research tool Authors' contributions DB performed the bioinformatic analysis, modeling and biochemical studies and drafted the manuscript. MEF participated in the modeling. GBG conceived of the study and participated in its design and co-ordination. All authors have read and approved the final manuscript. Supplementary Material Additional File 5 Co-ordinate file of model generated for Figure 1b . Click here for file Additional File 1 Phylogenetic analysis of CNGC and shaker-type channels from Arabidopsis thaliana and Oryza sativa . Un-rooted neighbor-joining trees were constructed for (A) shaker-type and (B) CNGC channels using full-length sequences. For the shaker-type channels, numbers at nodes indicate number of trees out of 100 in which the node occurred. These are omitted in (B) for clarity. Scale indicates number of differences per residue. Trees were generated using ClustalX [ 65 ] and visualized with TreeView [ 68 ]. Click here for file Additional File 4 Relevant Sequence Alignments Protein sequence alignments upon which phylogenetic analyses in Figure 2 , and additional file 2 are based. Click here for file Additional File 2 Cyclic nucleotide binding proteins in Arabidopsis thaliana and Oryza sativa . Proteins containing CNB domains including species, name, aliases, accession number, other domains present (AR indicates ankyrin repeat, CaM indicates calmodulin binding domain and TE indicates type II acyl CoA thioesterase domain), sequence length, residues encompassing the CNB domain and E-value for the CNB domain as determined by SMART [ 62 ]. All accession numbers are from NCBI except osCNGC5b, osCNGC5c and osCNGC18 which were obtained from the MIPS Oryza sativa database . Click here for file Additional File 3 Protein sequences of all sequences analysed in this manuscript. Sequences are named according to details in the text and are in FASTA format. Click here for file

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522749

Comparison of glucose tolerance in renal transplant recipients and hemodialysis patients

Background Impaired glucose tolerance is a risk factor for atherosclerosis in hemodialysis patients and renal transplant recipients. Methods To check the relationship of impaired glucose tolerance with the other atherosclerotic risk factors, fasting blood sugar and the standard two hour glucose tolerance test, serum tryglyceride, serum cholesterol, cyclosporine through level (in renal tranpslant recipients) and hemoglobin A1C were measured in 55 stable renal transplant recipients, 55 hemodialysis patients and 55 healthy controls with similar demographic characteristics. Patients with diabetes mellitus and propranolol consumers were excluded. The mean age and female to male ratio were 39 +/- 7 years and 23/22, respectively. Results Four of the renal transplant recipients and twelve of the hemodialysis patients had impaired glucose tolerance. Significant linear correlation was observed with body mass index and IGT only in hemodialysis patients (r = 0.4, p = 0.05). Glucose tolerance also had a significant correlation with triglyceride levels (217.2 +/- 55 mg/dl in hemodialysis patients vs. 214.3 +/- 13 mg/dl in renal transplant recipients and 100.2 +/- 18 mg/dl in control groups, p = 0.001). The glucose tolerance had significant relationship with higher serum cholesterol levels only in the renal transplant recipients (269.7 +/- 54 in renal transplant recipients vs. 199.2 +/- 36.6 mg/dl in hemodialysis and 190.5 +/- 34 mg/dl in control groups, p = 0.0001). In the renal transplant recipients, a linear correlation was observed with glucose tolerance and both the serum cyclosporine level (r = 0.9, p = 0.001) and the hemoglobin A1C concentration (6.2 +/- 0.9 g/dl). The later correlation was also observed in the hemodialysis patients (6.4 +/- 0.7 g/dl; r = 67, p = 0.001). Conclusions We conclude that although fasting blood sugar is normal in non-diabetic renal transplant and hemodialysis patients, impaired glucose tolerance could be associated with the other atherosclerotic risk factors.

Background Mortality and morbidity due to cardiovascular diseases are frequent in patients with diabetes mellitus and high prevalence of diabetes and cardiovascular disease, also, are observed in patients with end-stage renal disease treated by renal replacement therapy, either renal transplantation (RT) and dialysis [ 1 ]. Although uremia is typically associated with impaired glucose metabolism via multiple mechanisms [ 2 - 4 ], hemodialysis improves, although not completely, the uremic induced glucose impairment [ 5 - 7 ]. Impaired glucose metabolism is also a common and an important problem after RT. By improvement of immunosuppression after RT, the incidence of post transplant diabetes (PTDM) has been decreased from 41% to 2.5% [ 8 , 9 ]. Although we routinely screen and treat only full-blown diabetes at the post transplant periods, an overlooked aspect is the impaired glucose tolerance, which may be a risk factor to induce atherosclerosis. Impaired glucose tolerance de novo , may be a risk factor of post-transplantation mortality and morbidity [ 10 ]. Although increased levels of glycosylated hemoglobin (HbA1C) and lipid concentrations have been shown in hemodialysis patients [ 11 ] and renal transplant recipients [ 12 ] with diabetes, their impairment is not clear in the both groups with impaired glucose tolerance without apparent diabetes mellitus. In this study we investigated glucose tolerance and lipid profiles in non-diabetic hemodialysis and renal transplant patients. Methods We selected fifty five RT recipients with more than one year of good renal allograft function (serum Cr < 1.5 mg/dl), under conventional triple therapy composed of cyclosporine A (CsA), azathiopurine and prednisolone. Their allograft sources were living donors. Fifty five stable HD patients and another fifty five healthy controls (C), were also enrolled in this study. The mean age (39 ± 7 years), sex (F/M ratio was 33/22), body mass index (BMI) 24.7 ± 1.28 kg/m 2 ) were similar in the three groups (see table 1 ). Patients with diabetes mellitus and propranolol consumers were excluded. Table 1 Demographical, biochemical, hematological and therapeutical factors in hemodialysis patients and renal transplant recipients. HD RT Control Age (years) 48 ± 3 46 ± 4 47 ± 4 Male/female ratio 33/22 33/22 33/22 BMI (Kg/m 2 ) 24.6 ± 1.4 23.8 ± 1.2 23.6 ± 1.3 Cholesterol (mg/dl) 199.2 ± 36.6 269.7 ± 54 190.5 ± 34 Triglycerides (mg/dl) 217.2 ± 55 214.3 ± 13 100.2 ± 18 HgA1C (g/dl) 6.42 ± 0.7 6.2 ± 0.9 5.7 ± 0.7 Hb level 10.9 ± 0.8 12.4 ± 1.1 13.3 ± 0.8 Therapy with vitamin D3 0.5 μg/day (number) 25 -- -- Impaired glucose tolerance (number) 12* 4** -- *Significant correlation with BMI, serum triglycerides and HgA1C ** Significant correlation with serum cholesterol, CysA concentration and HgA1C The levels of serum triglyceride, cholesterol (measured by enzymatic spectrophotometry)[ 13 ], CsA (measured by ELISA in whole blood, only in renal transplant recipients) and glycosylated haemoglobin concentration (Hb A1c) (measured by column chromatography) were measured after 10 hours fasting (in the hemodialysis group, in the early morning before hemodialysis). Fasting blood sugar and the standard 2 hours glucose tolerance test (after ingestion of 75 g of glucose) were detected in the three groups by spectrophotometry. Statistical analysis was performed by Kuruskal wallis, U-Mann Whitney, multiple comparison and regression correlation coefficient tests, using SPSS 10.05. Results On the basis of WHO classification [ 14 ], four of our (7.5%) renal transplant recipients and twelve (22%) of the hemodialysis patients had impaired glucose tolerance, i.e. the 2 hour of glucose tolerance test was between 140 and 200 mg/dl. It was more obvious at the end of the second hour of GTT. Although BMI was roughly similar in the three groups (Table 1 ), a significant linear correlation was observed between BMI and impaired glucose tolerance only in HD patients (r = 0.4, p = 0.05) (fig 1 ), but not in the RT recipients. The glucose tolerance (especially at the first hour) in the HD patients had a significant linear correlation with the level of serum triglycerides (r = 0.87, p = 0.001) (Fig 2 ). Serum triglyceride concentration was 217.2 ± 55 mg/dl in HD vs. 214.3 ± 13 mg/dl in RT and 100.2 ± 18 mg/dl in C groups, (p = 0.001). On the other hand the four RT recipients with IGTT (i.e. 100% of RT recipients with IGTT) had the higher serum cholesterol levels (308.4 ± 24.4 mg/dl)) compared with the remaining RT recipients with normal GTT (248.7 ± 55.6 mg/dl) with p = 0.031 (table 2 ). The mean of serum cholesterol was 269.7 ± 54 mg/dl in RT vs. 199.2 ± 36.6 mg/dl in HD and190.5 ± 34 mg/dl in C groups (p = 0.0001). A linear correlation was observed between impaired GTT and both of the serum Cyclosporine level (r = 0.9, p = 0.001) and HbA1c in RT recipients (Fig 3 ). The mean of HbA1c was 6.2 ± 0.6 gr/dl in the RT recipients with normal GTT vs. 4.34 ± 0.26 g/dl in the RT recipients with IGGT (p < 0.001, table 2 ). The later correlation was also observed in HD patients, in whom the mean of HbA1C level was 6.4 ± 0.7 gr/dl in the group (r = 67, p = 0.001). In contrast of a close relationship of IGTT and higher HbA1c, the gender, age, times after transplantation and BMI did not impact on IGTT in RT recipients. Although in logistic regression analysis higher serum level of cyclosporine was correlated with increased GTT impairment, we could not evaluate the implication of corticosteroids on this test, because all of the 55 RT recipients were received prednisolone at a doses of 5 to 10 mg/day. Figure 1 GTT has a linear relationship with BMI in hemodialysis patients. Impairment of GTT is more significant in the hemodialysis patients with higher BMI. Gtt2 = glucose tolerance test at the second hours of 75 gr oral glucose. bmih = body mass index in hemodialysis patients Figure 2 The glucose tolerance in the HD patients had a significant linear correlation with the level of serum triglycerides. gttd1= glucose tolerance test in dialysis patients, tgh= serum concentration of triglyceride in hemodialysis patients. Figure 3 Cyclosporine level and HbA1c have correlations with the IGTT in RT recipients ▲ = Serum Cyclosporine level ○ = HbA1c concentration Table 2 Impaired glucose tests in HD and RT recipients have higher values of serum triglyceride, serum cholesterol and cyclosporine concentration than patients with normal glucose tolerance tests. no. of cases Serum Triglyceride(mg/dl) Serum Cholesterol(mg/dl) HbA1c (gr/l) Cyclosporine (mg/dl) RT recipients with IGTT 4 231.4 ± 150 308.4 ± 24.4 7.34 ± 0.26 320.4 ± 36.6 RT Recipients With normal GTT 51 201 ± 75 248.7 ± 55.6 6.2 ± 0.6 295.1 ± 29 P = 0.59 P = 0.02 P = 0.001 P = 0.2 HD patients with IGTT 12 272.1 ± 41.3 201.1 ± 39 7 ± 1 HD patients with normal GTT 43 195.9 ± 45 198.5 ± 36.8 5.9 ± 0.7 P = 0.001 P = 0.87 P = 0.007 Controls 55 100.2 ± 18 190.5 ± 34 5.7 ± 0.7 Discussion Impaired glucose tolerance occurs in about 50% of patients with chronic renal failure (CRF) patients. It is due to multiple factors, which the two most important of them being insulin resistance at target organs and impaired insulin secretion from the pancreas [ 15 ]. Insulin sensitivity would be reduced by up to 60% in non-diabetic patients with CRF before dialysis [ 16 ]. Marked improvement in insulin sensitivity and consequently glucose tolerance has been reported in non-diabetic patients after 10 weeks of HD, although they are not completely returned to normal [ 15 ]. Thereby, impaired glucose tolerance during HD is secondary to non-effective removable toxins by HD compared with peritoneal dialysis. In the latter more effective removal of middle molecule toxins causes better glucose tolerance, although glucose rich dialyzet solution is used [ 16 ]. The other causes of impaired glucose tolerance in HD patients may be secondary to metabolic disturbances, such as anemia [ 17 ], malnutrition [ 18 ] and vitamin D3 deficiency [ 19 ]. Although all of our HD patients had normochromic-normocytic anemia, the severity was not proportionate with impaired glucose tolerance (The data has not been shown). The patients were well nourished and were under treatment with daily oral vitamin D3 (Rocaltrol), 0.5 micrograms per day. So malnutrition and vitamin D3 deficiency could not to contribute to impaired glucose tolerance in our HD patients. Impaired glucose tolerance was also observed in 7.5% of our RT recipients. All of the presumed risk factors for post transplant diabetes mellitus such as old age [ 18 ], family history of any known diabetes mellitus in their first relatives[ 21 ], cadaveric allografts [ 22 ] and obesity did not exist in the patients. Previously Boudreaux et al. [ 23 ] reported that those patients who weighed more than 70 kg had a higher incidence of post transplant diabetes mellitus (PTDM). A relative risk of 1.4 for developing PTDM for every 10 kg increase in body weight more than 60 kg has been shown [ 12 ]. Although in our study obese patients (BMI > 30 kg/m2) were not included in the both groups, a correlation was observed between impaired glucose tolerance and higher BMI in our HD patients. In RT recipients, the major risk factor for impaired glucose tolerance was immunosuppressive therapy. Through using higher doses of CsA and corticosteroids, PTDM was previously more common, but the complication has been decreased to 2–5% in FK506-based immunosuppressive protocols [ 24 , 25 ]. Although this relatively uncommon complication is a major cause of post-transplant mortality and morbidity, even minor glucose intolerance is associated with an increased long-term risk for cardiovascular disease [ 26 ]. The importance of impaired glucose tolerance should not be underestimated in these patients with high risk of atherosclerosis. Hyperlipidemia, another risk factor for atherosclerosis, on one hand accompanies the impaired glucose tolerance observed in the HD and RT patients and on the other hand increases the risk of atherosclerosis induced by impaired glucose tolerance. As reported previously, a tendency to higher pre-transplantation serum triglyceride concentration was associated with post-transplantation impaired glucose tolerance [ 27 ]. Hypertriglyceridemia is common complication in dialysis patients. In non-transplant populations it is regarded (along with low HDL cholesterol levels) as a prominent feature of insulin resistance syndrome, and also is a cardiovascular risk factor in organ transplant recipients [ 28 ]. Our study confirmed the relationship between impaired glucose tolerance and triglyceride levels in HD patients, and between impaired glucose tolerance and cholesterol levels in RT recipients. The latter was also accompanied by a higher level of HgA1C. Commonly used tests of HgA1C may be unreliable in patients with end-stage renal disease because of the presence of anemia, shortened red blood cell survival, and assay interferences from uremia. But HgA1C in the range of 6% to 7%, as was found in our study, estimates glycemic control within the range of patients without severe renal impairment [ 1 ]. So in the range of mild to moderate increased HgA1C in HD and uremic patients, it would be a reliable marker of impaired glucose tolerance. Conclusions There was increased HgA1C and impaired glucose tolerance in HD and RT patients. This was accompanied by hyperlipidemia in HD patients (with hypertriglyceridemia) and RT recipients (with hypercholesterolemia). The impact upon the progression of atherosclerosis needs more study in haemodialysis and renal transplant populations at a long term follow up. Competing interests None declared. Authors' contributions HA reviewed the literatures and wrote the manuscript and also helped to do statistical analysis, AN performed GTT and the other biochemical markers, MN participated as coordinator between laboratory and clinic, HTK selected the patients and collected data Pre-publication history The pre-publication history for this paper can be accessed here:

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514562

Intrathecal baclofen withdrawal syndrome- a life-threatening complication of baclofen pump: A case report

Background Intrathecal baclofen pump has been used effectively with increasing frequency in patients with severe spasticity, particularly for those patients who are unresponsive to conservative pharmacotherapy or develop intolerable side effects at therapeutic doses of oral baclofen. Drowsiness, nausea, headache, muscle weakness, light-headedness and return of pretreatment spasticity can be caused by intrathecal pump delivering an incorrect dose of baclofen. Intrathecal baclofen withdrawal syndrome is a very rare, potentially life-threatening complication of baclofen pump caused by an abrupt cessation of intrathecal baclofen. Case presentation A 24-year-old man with a past medical history of cerebral palsy and spastic quadriparesis developed hyperthermia, disseminated intravascular coagulation, rhabdomyolysis, acute renal failure and multisystem organ failure leading to a full-blown intrathecal baclofen withdrawal syndrome. Intrathecal baclofen pump analysis revealed that it was stopped due to some programming error. He was treated effectively with supportive care, high-dose benzodiazepines and reinstitution of baclofen pump. Conclusion The episodes of intrathecal baclofen withdrawal syndrome are mostly caused by preventable human errors or pump malfunction. Educating patients and their caregivers about the syndrome, and regular check-up of baclofen pump may decrease the incidence of intrathecal baclofen withdrawal syndrome. Oral baclofen replacement may not be an effective method to treat or prevent intrathecal baclofen withdrawal syndrome. Management includes an early recognition of syndrome, proper intensive care management, high-dose benzodiazepines and prompt analysis of intrathecal pump with reinstitution of baclofen.

Background Baclofen is a gamma-aminobutyric acid (GABA) analog that has inhibitory effects on spinal cord reflexes and brain. Intrathecal baclofen (ITB) therapy consists of long-term delivery of baclofen to the intrathecal space. Intrathecal baclofen has been used to treat spasticity due to cerebral palsy, brain or spinal cord injury, multiple sclerosis, dystonia, stroke and stiff-man syndrome, particularly for those patients who are unresponsive to conservative pharmacotherapy or develop intolerable side effects at therapeutic doses of oral baclofen [ 1 ]. Side effects such as drowsiness, nausea, headache, muscle weakness and light-headedness can occur as a result of the pump delivering an incorrect dose of baclofen. Sudden cessation of ITB administration can cause mild symptoms like reappearance of baseline level of spasticity associated with pruritis, anxiety and disorientation [ 2 ]. These mild symptoms represent "loss of drug effect". All patients experience "loss of drug effect" when ITB is discontinued, only a small (but unknown) proportion of patients develop a full-blown potentially life-threatening withdrawal syndrome. We report a case of ITB withdrawal syndrome developing hyperthermia, severe spasticity, disseminated intravascular coagulation, rhabdomyolysis, acute renal failure and multisystem organ failure. Case presentation A 24-year-old man was admitted to our intensive care unit (ICU) with a possible diagnosis of seizure disorder and sepsis. He had a past medical history of cerebral palsy and spastic quadriparesis. Three years ago, he had an ITB pump implanted for spasticity refractory to the high doses of oral baclofen. He had a significant improvement in spasticity, social and functional capacity in the past three years. Later, he developed some disorientation and increased spasticity. He was taken to a local physician who prescribed oral baclofen (120 mg daily in four divided doses) for his increased spasticity. He also advised him to have his ITB pump checked immediately. The following day, his spasticity increased even after taking oral baclofen. He developed multiple seizures and respiratory distress in the next 24-hour period. Subsequently, he was admitted in a local hospital where he was orally intubated and transferred to our ICU for aggressive management. On presentation, his temperature was 104.6°F (40.3°C), heart rate 127 beats per minute, and the blood pressure was 85/45 mm/Hg. His ventilator settings were: assist-control ventilation mode; respiratory rate, 15 breaths per minute; tidal volume, 650 mL; positive end expiratory pressure (PEEP), 5 cm H 2 O; and FiO 2 , 60%. His spontaneous respiratory rate was 18 breaths per minute and an oxygen saturation of 100% was noted on pulse oximetry. In the local hospital, he was documented to have a high fever of 107°F (41.6°C) and he had received intravenous lorazepam, phenytoin, pantoprazole, piperacillin/tazobactem and dopamine. On physical examination, neurologically he was unconscious with decerebrate posturing and his Glasgow coma scale was 6. He had an absent corneal and gag reflexes. He was moving all four limbs in response to noxious stimuli. He was also noted to have an extreme spasticity in all four limbs. Lung examination revealed decreased breath sounds in the left lower base. Cardiac examination was unremarkable. He had a palpable baclofen pump on abdominal wall and bowel sounds were heard. The differential diagnoses were septic shock, meningitis, neuroleptic malignant syndrome and malignant hyperthermia. The initial laboratory results showed serum creatinine phosphokinase (CPK) 5250 U/L (Normal, 25–235 U/L) and CPK-MB fraction 12.1 ng/ml (Normal, 0.5–6.3 U/L). Serum chemistry revealed sodium 142 mmol/L, potassium 5.1 mmol/L, chloride 120 mmol/L, bicarbonate 13 mmol/L, and creatinine 2.1 mg/dl. Hemogram showed white blood cell count 12.2 K/UL, hemoglobin 16.5 g/dl and platelet count 9 K/UL (Normal, 130–400 K/UL). Liver function test showed aspartate aminotransferase (ALT) 1128 U/L, alanine aminotransferase (AST) 1140 U/L, alkaline phophatase 90 U/L, total bilirubin 1.2 mg/dl, conjugated bilirubin 0.7 mg/dl, prothrombin time 20.2 seconds (Normal, 10–12.5 seconds), and INR 2.0 (Normal, 0.9–1.1). Blood and urine cultures were obtained. Chest radiograph was normal. A computed tomography (CT) scan of the chest revealed atelectasis of the left lung base. His CT scan of head did not show any acute infarct or bleeding. His initial management included intravenous fluids, norepinephrine, platelet transfusion, phenytoin, propofol and broad-spectrum antibiotics (vancomycin, ceftriaxone) for suspected meningitis and septic shock. He received intravenous lorazepam (4–8 mg every four hours) for his spasticity. Next day, his spasticity improved and an ITB specialist investigated his baclofen pump. His baclofen pump analysis revealed that it was stopped due to some programming error, which was restarted at a previously prescribed baclofen rate (260 μg/day). On third hospital day, his serum CPK was 15,878 U/L, AST was 2566 U/L, ALT was 2993 U/L, while CPK-MB fraction came down to 3.4 ng/ml. His urine output decreased (<400 ml/ day) and serum creatinine increased in the range of 5–6 mg/dl. Later, he was hemodialyzed few times during the course of hospitalization due to acute renal failure. His echocardiogram showed left ventricular ejection fraction of 20–25% and severe global hypokinesis. His electroencephalogram did not reveal any epileptogenic activity. He developed full-blown multisystem organ failure with an evidence of shock liver, renal failure, respiratory failure, disseminated intravascular coagulation and myocardial depression. His nutrition was started on nasogastric tube feedings, and proper ventilator care was taken through a tracheostomy tube. His serum baclofen obtained at the time of admission was less than 0.02 μg/ml (Expected values, 0.08–0.4 μg/ml). After a three-week course of aggressive management in ICU, he was weaned off from the ventilator and his multiple organ shock resolved. At a six-month follow-up, he was observed in a nursing home with his baseline functional, social, and family activities. Discussion Intrathecal baclofen provides an effective improvement in the spasticity of patients whose spasticity is not sufficiently managed by oral baclofen or other oral anti-spastic medications [ 1 ]. Regardless of the cause of spasticity (cerebral or spinal), anti-spastic effects of baclofen occur at spinal level. Poor response of oral baclofen in many patients can be explained by the fact that the spinal cord represents only about 2% mass of the brain, and receives proportionately a lower blood flow as a fraction of cardiac output. Therefore, cerebral side effects often occur before therapeutic anti-spastic effects of oral baclofen are observed. A programmable ITB pump provides a direct, pattern-controlled delivery of baclofen to the spinal cord. Precise delivery of the ITB pump yields better spasticity reduction at 1000 times lower than the doses of oral baclofen. In addition, the adverse effects are minimized as compared with oral baclofen. ITB provides reduced tone, spasms and pain, improves speech, mobility, sleep quality and bladder control, with a response rate up to 97% in adults and children [ 3 , 4 ]. The ITB pump is approximately 3 inches wide and 1 inch thick. It is surgically implanted in the subcutaneous tissue of anterior abdominal wall. Baclofen is delivered via a silicone rubber catheter into the lumbar subarachanoid space. The ITB pump delivers approximately 100–900 μg/day of baclofen, and it is titrated for the desired clinical response. It is also equipped with an alarm that signals low volume, low battery, or malfunction. Nine years ago, catheter or pump-related malfunction that leads to an overdose or withdrawal has been reported in 40 % of the patients with ITB pump [ 5 ]. Catheter system, operative techniques and programming system of the ITB pump have now been improved significantly to reduce the incidence of an overdose or withdrawal. The precise mechanism of action of baclofen as a muscle relaxant and anti-spasticity agent is not fully understood. Baclofen inhibits both monosynaptic and polysynaptic reflexes at the spinal cord level [ 6 ], possibly by decreasing excitatory neurotransmitter release from primary afferent terminals, although actions at supraspinal sites may also contribute to its clinical effects. Baclofen also causes enhancement of vagal tone and inhibition of mesolimbic and nigrostriatal dopamine neurons (directly or via inhibiting substance P) [ 7 ]. Baclofen is a structural analog of the inhibitory neurotransmitter GABA, and may exert its effects by stimulation of the GABA B receptor to cause muscle relaxation. Baclofen reduces increased muscle tone, Babinski sign, tendon reflexes, ankle clonus and sometimes decreases muscle force. Long-term ITB infusion causes down-regulation of GABA B receptors in the CNS and spinal cord. Down-regulation of GABA B receptors accounts for the decreased sensitivity to the baclofen over time. Although GABA B receptors are down-regulated, it is the baclofen itself that causes increased inhibitory tone in the CNS and spinal cord [ 8 ]. Therefore, abrupt ITB withdrawal results in a predominance of excitatory effects and simulates other conditions that are associated with CNS hyperexcitability and severe spasticity. Sudden cessation of ITB administration can cause mild symptoms like reappearance of baseline level of spasticity associated with pruritis, anxiety and disorientation. These mild symptoms represent "loss of drug effect". All patients experience "loss of drug effect" when ITB is discontinued. However, more severe symptoms like hyperthermia (109.4°F), myoclonus, seizures [ 9 - 12 ], rhabdomyolysis, disseminated intravascular coagulation, multisystem organ failure [ 10 ], cardiac arrest, coma and death [ 9 , 12 , 13 ] have been well reported, and represents a full-blown life-threatening ITB withdrawal syndrome. Food and drug administration (FDA) of USA has included a drug label warning for baclofen withdrawal syndrome in April 2002 [ 1 , 13 ]. Differential diagnoses include malignant hyperthermia, neuroleptic-malignant syndrome, autonomic dysreflexia, sepsis and meningitis. ITB withdrawal syndrome has been fatal in some cases. Six patients have died out of 27 cases reported to FDA [ 13 ]. Most reported episodes of ITB withdrawal were caused by preventable human errors or oversights. However, catheter dislodgement, catheter migration and kinks, and other catheter-related issues might be more common than pump-related malfunctions [ 1 , 14 ]. Close attention to pump refilling and programming procedures may reduce the incidence of ITB withdrawal syndrome. Benzodiazepines are helpful in controlling spasticity and seizures during ITB withdrawal syndrome [ 1 , 10 ]. Benzodiazepines activate central receptors and GABA A receptors of spinal cord by different mechanisms [ 1 ]. Therefore, ITB induced down-regulation of GABA B receptors do not interfere with benzodiazepine's mechanism of action. During a planned removal of ITB pump due to infection or other causes, premedication with high doses of benzodiazepines and augmented oral baclofen is usually administered in the hospitals to prevent spasticity. Similarly, high doses of oral baclofen is also tried in some cases of ITB withdrawal syndrome [ 15 , 16 ]. But failure of high doses of oral baclofen (80 mg three times daily) have been reported recently [ 17 ]. High doses of oral baclofen may not be adequate to treat or prevent ITB withdrawal because of down-regulation of central GABA B receptors due to chronic ITB administration. Moreover, it has been suggested that it may take many hundreds of grams of oral baclofen to achieve a therapeutic baclofen level in the cerebrospinal fluid, compared to the patients who had effective spasticity control with an ITB pump [ 17 ]. Although, our patient received oral baclofen (120 mg daily in four divided doses) initially but these doses may be low enough to prevent ITB withdrawal syndrome. Failure of high doses of oral baclofen suggests that resumption of GABA B receptor agonist by prompt restoration of ITB pump and proper supportive care might be the best treatment. Similarly, high-dose benzodiazepines may be effective because of similar mechanism of action on widespread CNS GABA A receptors. High-dose benzodiazepines could be an initial life saving strategy even before analysis and restoration of ITB pump is achieved, or in cases, where resumption of ITB administration is not as simple as correcting a programming error. Intrathecal baclofen bolus is appropriate, but due to the risk of inadvertent overdose, an experienced physician should immediately perform reinstitution of ITB pump. We had restarted ITB in our patient at a previously prescribed dose. However, a much higher dose of baclofen could have safely been given to overcome the spasticity since the patient was in an ICU. High-dose dantrolene (a direct muscle relaxant that acts on sarcoplasmic reticulum of skeletal muscle) has been tried to reduce spasticity and fever in ITB withdrawal syndrome [ 18 ]. Reduction in fever may be due to cessation of repetitive and thermogenic contractions of muscle fibers. It is unlikely that dantrolene has any GABA agonistic effects, and administration of dantrolene may not be accompanied by any correction in anomalies of CNS functions. Therefore, seizures, autonomic instability and death may occur in ITB withdrawal syndrome even after controlling spasticity with dantrolene. Cyproheptadine (a non-selective serotonin antagonist) has also been used postulating that ITB withdrawal may be a form of serotonergic syndrome that occurs from the loss of GABA B receptor-mediated presynaptic inhibition of serotonin [ 19 ]. We did not consider dantrolene or cyproheptadine in our patient due to lack of sufficient clinical support in the treatment of ITB withdrawal syndrome. There was a three-day period of delay in diagnosing the ITB withdrawal syndrome leading to deterioration and multisystem organ failure. However, our patient had a successful recovery in response to restoration of baclofen pump and adequate intensive care management. Conclusions Baclofen withdrawal syndrome is a potentially life-threatening complication of intrathecal baclofen pump. Empty pump reservoir, catheter leaks or displacement, pump malfunction, programming error and refill of pump with improper drug concentration are the possible mechanisms which could lead to an ITB withdrawal syndrome. Regular check-up of the ITB pump by a specialist, educating patients and their caregivers may decrease the incidence of ITB withdrawal syndrome. Oral baclofen replacement may not be an effective method to treat or prevent ITB withdrawal syndrome. Early recognition of syndrome, high-dose benzodiazepines, prompt analysis of the ITB pump with reinstitution of baclofen, and proper intensive care management are mainstays for the management of ITB withdrawal syndrome. List of abbreviations GABA -gamma amino butyric acid, ITB – intrathecal baclofen, CPK – creatinine phosphokinase, ALT – aspartate aminotransferase, AST – alanine aminotransferase, CT – computed tomography. Competing interests None declared. Authors' contributions IM: Direct patient care, article conception, critical and extensive revision of article for important intellectual content, review and drafting of the original article. AH: Literature search, case review and summary, drafting of the original article. Both authors read and approved the final manuscript and contributed equally to the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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534103

Lipoprotein lipase in hemodialysis patients: indications that low molecular weight heparin depletes functional stores, despite low plasma levels of the enzyme

Background Lipoprotein lipase (LPL) has a central role in the catabolism of triglyceride-rich lipoproteins. The enzyme is anchored to the vascular endothelium through interaction with heparan sulphate proteoglycans and is displaced from this interaction by heparin. When heparin is infused, there is a peak of LPL activity accompanied by a reduction in triglycerides (TG) during the first hour, followed by a decrease in LPL activity to a stable plateau during the remaining session while TG increase towards and beyond baseline. This suggests that tissue stores of LPL become depleted. It has been argued that low molecular weight (LMW) heparins cause less disturbance of the LPL system than conventional heparin does. Methods We have followed LPL activity and TG during a dialysis-session with a LMW heparin (dalteparin) using the same patients and regime as in a previous study with conventional heparin, i.e. a primed infusion. Results The shape of the curve for LPL activity resembled that during the earlier dialyses with conventional heparin, but the values were lower during dialysis with dalteparin. The area under the curve for LPL activity during the peak period (0–180 minutes) was only 27% and for the plateau period (180–240 minutes) it was only 36% of that observed with conventional heparin (p < 0.01). These remarkably low plasma LPL activities prompted us to re-analyze LPL activity and to measure LPL mass in frozen samples from our earlier studies. There was excellent correlation between the new and old values which rules out the possibility of assay variations as a confounding factor. TG increased from 2.14 mmol/L before, to 2.59 mmol/L after the dialysis (p < 0.01). From 30 minutes on, the TG values were significantly higher after dalteparin compared to conventional heparin (p < 0.05). Conclusion These results indicate that LMW heparins disturb the LPL system as much or more than conventional heparin does.

Background Lipoprotein lipase (LPL) hydrolyses triglycerides (TG) in circulating lipoproteins [ 1 , 2 ]. This is a necessary first step in catabolism of the TG-rich lipoproteins as evidenced by the massive hypertriglyceridemia in patients with genetic deficiency of the enzyme [ 3 ]. Fine-tuned regulation of LPL activity is an important aspect of energy homeostasis [ 4 ]. Patients on chronic hemodialysis (HD) often have reduced LPL activity and derangements of lipoprotein profiles [ 5 - 7 ]. During HD, conventional heparin is commonly used as anticoagulant and this releases LPL from its binding sites at the vascular endothelium into the circulation. It has been suggested that repeated heparinisation may induce release and subsequent degradation of LPL that exceeds the rate of enzyme synthesis and thereby causes a depletion of LPL stores [ 8 - 11 ]. In a previous study we followed the LPL activity and the TG changes during a dialysis-session using conventional heparin as anticoagulant [ 12 ]. There was a peak of LPL activity accompanied by a reduction in TG during the first hour, followed by a decrease in LPL activity to a stable plateau during the remaining session while TG increased towards and beyond the original baseline. When compared to a group of healthy control subjects, the peak LPL activity was only about 50 % in the HD-patients while the plateau activities were comparable. Our interpretation was that the functional pool of LPL, represented by the initial peak, was impaired in HD-patients, while the production of lipase molecules, reflected by the plateau, was only marginally reduced. In recent years, conventional heparin has increasingly been replaced by various low molecular weight (LMW) heparins. A major argument is the ease of administration [ 13 , 14 ]. A single injection of a LMW heparin can often replace a primed infusion of conventional heparin. The increase in plasma LPL activity is less pronounced after LMW compared to conventional heparin [ 15 ], and it has been suggested that this causes less disturbance of lipoprotein metabolism [ 10 ] although this conclusion has been questioned [ 16 ]. Direct studies of the lipase-heparin interaction have shown that a heparin decasaccharide is enough to fill the heparin-binding grove on the lipase molecule [ 17 , 18 ]. Decasaccharides fall in the middle or lower size range in preparations of LMW heparins [ 19 ]. Several lines of evidence indicate that also in biological systems, decasaccharides are sufficiently long to exert full effect on LPL. Chevreuil et al . found that on a weight basis, decasaccharides released more LPL from perfused rat hearts than conventional heparin did [ 20 ]. Several groups have reported that LMW heparins or decasaccharides release LPL from tissues in vitro or from cultured cells as efficiently as or even more efficiently than conventional heparin does. It is therefore unlikely that the lower plasma LPL activities after LMW heparin are due to less release of the lipase. More likely, LMW retards clearance of the lipase by the liver less efficiently than conventional heparin does. Two studies have directly demonstrated such a difference in liver perfusion experiments [ 20 , 21 ]. In a recent study we infused a LMW heparin (dalteparin) for eight hours to healthy volunteers to explore the influence on LPL activity and TG response [ 22 ]. The peak LPL activity was only about 30%, and the subsequent plateau activity only about 40%, compared to the activities observed during a similar infusion with conventional heparin. A bolus of conventional heparin given when the LPL activity had levelled off to a plateau brought out about the same amount of activity irrespective of if the subjects had been infused with dalteparin or conventional heparin. We concluded that dalteparin and conventional heparin had reduced the peripheral stores of LPL to a similar extent and that the difference in plasma levels of LPL activity was due to a more rapid hepatic clearance of the LPL-dalteparin complex. There was a tendency towards a more pronounced increase in TG after dalteparin compared to conventional heparin, indicating that lipoprotein metabolism might be more, rather than less, disturbed by the use of LMW heparin. As HD-patients are increasingly subjected to repeated treatment with LMW heparin during dialysis we have now followed LPL activity and TG during a dialysis-session with dalteparin using the same regime as in our previous study with conventional heparin, i.e. a primed infusion [ 12 ]. Methods Subjects and protocol The study design was based on the protocol used in a previous investigation in which nine HD-patients were studied during a dialysis-session using conventional heparin as anticoagulant [ 12 ]. The present study, on the same HD-patients, was performed three months later with a LMW heparin (dalteparin, Pharmacia, Stockholm, Sweden) as anticoagulant. In all other respects the dialysis regime, as well as medication and diet recommendations, was kept unchanged. The median age was 73 years and the median BMI was 24.7. The diagnoses were diabetes nephropathy (BE), chronic pyelonephritis (AJ), nephrosclerosis (HB), polycystic kidney disease (MK, CH), chronic glomerulonephritis (RH, RS, KL) and in one patient the origin was unclear (BV, not biopsied). The patients had been on maintenance hemodialysis for 5–38 months and were treated with bicarbonate hemodialysis either two (RS, HB) or three (MK, BE, KL, AJ, RH, CH, BV) times a week, depending on residual renal function. All dialyses were performed with hemophan dialysers (GFS+16, GAMBRO, Lund, Sweden) and Biosol dialysis solution (Pharmalink, Stockholm, Sweden). A central dialysis catheter was used as dialysis-access in five of the patients and an arteriovenous-fistula/graft in four. The patients were treated with antihypertensive drugs (ACE-inhibitors, beta-blockers, calcium channel inhibitors), diuretics, sodium bicarbonate and phosphate-binding drugs. The diabetic patient was non-insulin dependent and was treated only by diet recommendations. One patient (RS), having a rejecting renal transplant, was treated with low doses of corticosteroids and cyclosporine. No one was treated with lipid lowering drugs. The experiments were carried out after an overnight fast, and 48–96 hours had passed since the previous hemodialysis. A loading dose of 40 IU dalteparin per kg body weight was given, followed by a continuous infusion of 1000 IU/hour, in accordance with the manufacturer's recommendations. Blood samples were drawn before start and then regularly at 15, 30, 60, 120, 180 and 240 minutes. According to existing routines, the patients had a combined breakfast/lunch, containing 25 g fat, about two hours after the dialysis was started. The ethical committee approved the study and informed consent was obtained from all patients prior to participation. Handling of samples and assay methods Blood samples for measurement of LPL activity were collected in heparinized tubes. They were immediately chilled in ice water and centrifuged in a cooling centrifuge within 15 minutes. The plasma was frozen at -20°C and then stored at -70°C until analyses. LPL activity was measured as described [ 23 ] using an emulsion containing a trace amount of [ 3 H]-oleic acid-labelled triolein, 100 mg soybean TG and 10 mg egg yolk phospholipids per mL, prepared by Fresenius-Kabi, Uppsala, Sweden. Hepatic lipase was inhibited by pre-incubation of the plasma samples with immunoglobulins from a rabbit antiserum to human hepatic lipase. The assay medium contained a relatively high concentration of heparin, and possible differences in the heparin concentration or type in the sample would not affect the activity. All assays were made in triplicate and the mean value was used. A standard sample of human post-heparin plasma was run on each assay day and the value was used to calibrate for between-assay-variations. LPL protein mass was determined with an enzyme-linked immunosorbent assay, as previously described [ 24 ], using immunoaffinity-purified chicken antibodies raised against bovine LPL for capture and the monoclonal antibody 5D2, also raised against bovine LPL, for detection (a gift of Dr. J. Brunzell. Seattle, Washington, USA). Blood samples for lipid determination were drawn in tubes without anticoagulant, immediately chilled in ice water, centrifuged and frozen as described above. Total cholesterol, HDL-cholesterol and TG were determined by routine methods on a multianalyzer (Vitros 950 IRC; Johnson & Johnson, Clinical Diagnostics Inc, New York, NY, USA). Baseline LDL-cholesterol levels were calculated using the Friedewald formula [ 25 ]. Antifactor Xa activity was determined using a chromogenic substrate (COACUTE ® , Chromogenix AB, Mölndal, Sweden). Statistics The values are expressed in terms of median and range and were examined for significant differences by paired Wilcoxon signed-rank test. Simple linear regression and the Spearman rank correlation test were used to evaluate relationships between variables. Two-tailed P values below 0.05 were considered to be statistically significant. Results Baseline data Table 1 gives baseline data for the HD-patients at the time of the dialysis-session with dalteparin. There were no significant differences compared to the values at the time of the dialysis with conventional heparin three months earlier [ 12 ]. There was no significant difference between the ultrafiltration rates during the two dialyses. Table 1 Baseline data for the HD-patients at the time for the dialysis-session with dalteparin. Median values for the dialyses with dalteparin (D) and conventional heparin (H), respectively. There were no statistically significant differences between values on the two occasions. HD-patients Gender Age (years) Dbw* (kg) Ultra-filtration(L) BMI (kg/m 2 ) TG (mmol/L) Cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) MK F 53 71.5 3.0 24.7 3.41 4.5 0.65 2.3 BE F 64 81 2.2 34.2 2.55 5.4 1.24 3.1 KL F 79 76 2.8 28.3 2.14 6.3 0.76 4.5 CH M 55 62.5 3.5 19.5 1.21 3.6 0.99 2.1 BV M 70 77.5 1.3 26.2 1.47 4.6 0.99 2.9 RS M 73 65 2.7 23.3 2.86 7.1 0.96 4.9 RH M 78 62 2.7 21.5 1.09 4.6 1.26 2.9 AJ M 78 73 3.0 26.5 2.84 5.0 0.74 2.9 HB M 90 70 3.0 24.2 1.38 4.4 1.30 2.5 median D 73 71.5 2.8 24.7 2.14 4.6 0.99 2.9 median H 73 70.5 2.2 24.6 1.84 5.6 1.09 3.7 *Dry body weight Anticoagulation effect During the dialysis with dalteparin, the anti-Factor Xa activity was between 0.52 and 0.87 IU/mL. The target value recommended by the manufacturer is 0.5–1.0 IU/mL. Hence, the plasma dalteparin concentration remained well within the range for effective anticoagulation throughout the dialysis-session. LPL activity and mass The LPL-activity rose rapidly when dalteparin was administered. The highest value was at 15 minutes, median 15 mU/mL (range 9–32). The activity remained high at 30 minutes, but then decreased so that at 120 minutes the median was only 9 mU/mL (range 5–15) (Fig 1 ). The activity then remained essentially unchanged to the end of the dialysis at 240 minutes (6 mU/mL, range 5–11). The shape of the curve resembled that during the earlier dialysis with conventional heparin (Fig 1 inset), but the values were much lower during the dialysis with dalteparin (p < 0.01). The area under the curve (AUC) for LPL activity during the peak period (0–180 minutes) was 1774 mU/mL × minutes (range 1116–3001). This is only 27% of the AUC observed during the earlier study with conventional heparin [ 12 ] (p < 0.01). For the plateau period (180–240 minutes) the AUC was 390 mU/mL × minutes (range 308–618) which is 36% of the corresponding AUC during the dialysis with conventional heparin (p < 0.01). Figure 1 Plasma LPL activities during infusion of dalteparin. The figure shows individual curves for the nine subjects in the present study. The inset compares median values from the present study (□) with those from three earlier studies where the same protocol for infusion was used. Control subjects given conventional heparin [23] (●), control subjects given dalteparin [22] (○), HD patients given conventional heparin [12] (■). The inset in Fig. 1 compares the median values for LPL activities in the present study to the LPL activities observed in earlier studies with age and gender matched healthy subjects given conventional heparin [ 23 ], or dalteparin [ 22 ], and with HD patients given conventional heparin [ 12 ]. The values during dalteparin infusion were lower in both controls and in HD patients. Values in HD patients were lower than in controls both with dalteparin and with conventional heparin. Thus, the highest values were for controls given conventional heparin and the lowest values were those in the present study with dalteparin in HD patients. The differences were remarkably large. These studies have been carried out on separate occasions over several years, but duplicate samples had been saved frozen. To check the consistency of the values, the duplicate samples from the 0, 15 and 30 min time points were thawed and assayed for LPL activity and mass. There was good agreement between the activities from the earlier and the repeated assay for all four studies (Fig 2A ). Hence, the large differences between the LPL activities registered for controls and HD patients and for the two different heparin preparations were real. As an additional test of the consistency of the data, we plotted the increase in LPL activity and LPL mass from basal (before heparin) at the 30 min time points (Fig 2B ). The basis for this is that previous studies have indicated that heparin releases mainly the active form of the lipase [ 26 ]. For regression analysis, changes in LPL mass of less than 100 ng/mL were excluded because heparin may release some inactive LPL [ 26 ] and because of the uncertainty in calculating small differences between pre- and post-heparin mass. The analysis returned a slope of 0.46 ± 0.05 mU/ng (r = 0.94, p < 0.001), consistent with the expected specific activity of human LPL (around 0,4 mU/ng under our assay conditions). Figure 2 Evaluation of the consistency of data from four separate studies by repeated assay of frozen samples. Samples had been obtained and treated as described in the methods section and then stored frozen at -70°C in three earlier studies of plasma LPL during infusion of conventional heparin or dalteparin in control subjects or in dialysis patients [12,22,23] and the present study. Samples from the 0, 15 and 30 min time points were thawed and assayed for LPL activity and mass. Panel A shows the LPL activities at 15 and 30 min recorded on the second assay, as a function of the value recorded on the original assay. Regression analysis gave a slope indicating that the repeated value was 113 % of the original (r = 0.94, p < 0.0001). Panel B shows the increase of LPL activity over the baseline samples plotted against the increase of LPL mass for the 30 min samples. For this, values from the second assay were used (LPL mass was not determined in some of the earlier studies). A regression analysis, excluding samples for which the increase in LPL mass was less than 100 μg/mL, returned a slope of 0.46 ± 0.05 mU/ng LPL (r = 0.94, p < 0.001). Same symbols as in Fig 1. Triglycerides TG remained essentially unchanged for the first two hours and then increased. The change from 2.14 mmol/L (range 1.09–3.41) at the start, to 2.59 mmol/L (range 1.49–5.04) at the end of the dialysis represents a 21 % increase (p < 0.01). Compared to the values during the earlier dialysis with conventional heparin, there was no statistically significant difference at the start of dialysis, but from 30 minutes and through the remaining session TG values were significantly higher during the dialysis with dalteparin (p < 0.05) (Fig 3 ). There was no drop in TG at 30 to 60 min like that found using conventional heparin. There was no correlation between LPL activity and changes in TG during any of the dialysis-sessions. To further illustrate the changes in TG concentrations we have set the baseline value for each individual to 100% and calculated the changes from this. Median values for the changes are plotted in Figure 4 which reinforces the conclusion that there was no significant decrease of TG during the first two hours during the dialysis with dalteparin, in contrast to the marked drop observed during dialysis with conventional heparin [ 12 ], and during infusion of either conventional heparin [ 23 ] or dalteparin in control subjects [ 22 ]. Figure 3 Plasma TG concentrations during infusion of dalteparin. The figure shows individual curves for the nine subjects in the present study. The inset compares median values from the present study (□) with those from three earlier studies where the same protocol for infusion was used. Control subjects given conventional heparin (●), control subjects given dalteparin (○), HD patients given conventional heparin (■). P < 0.05 for dalteparin compared to conventional heparin given to HD patients. Figure 4 Changes in plasma TG concentrations during infusion of dalteparin or conventional heparin to HD patients or matched controls. For this, the basal TG concentration for each individual was set to 100% and the concentrations observed at the subsequent time points were calculated relative to this. The figure shows median values from the present study (□) and from three earlier studies where the same protocol for infusion was used. Control subjects given conventional heparin (●), control subjects given dalteparin (○), HD patients given conventional heparin (■). Cholesterol Total cholesterol increased from 4.6 mmol/L (range 3.6–7.1) at start, to 6.1 mmol/L (range 3.8–8.4) at the end (32%, p < 0.05). This differs from the earlier dialysis with conventional heparin, when total cholesterol did not change from baseline [ 12 ]. HDL-cholesterol did not change from baseline. Again, this differs from the dialysis with conventional heparin when HDL-cholesterol increased from 1.09 mmol/L (range 0.67–2.06) at start to 1.19 mmol/L (range 0.67–2.31) at the end (p < 0.05). No correlation was found between LPL activity and total or HDL-cholesterol changes during any of the dialysis-sessions. The median LDL-cholesterol, calculated by the Friedewald formula, was 2.9 mmol/L (range 2.1–4.9) before the dialysis and increased to 3.75 (2.1–5.6) at 240 min. This corresponds to an increase of 29% (p < 0.05). Discussion This study shows the same pattern of plasma LPL activity in HD patients given dalteparin as observed in previous studies with control subjects given dalteparin [ 22 ] or conventional heparin [ 23 ] and in HD patients given conventional heparin [ 12 ]. The novel aspect is how remarkably low the plasma LPL activities were in the HD patients. This prompted us to re-analyze frozen samples from the earlier studies to rule out the possibility of assay variations as a confounding factor. The increase in LPL mass was also low in the HD patients given dalteparin, and corresponded well to the increase of LPL activity. This further supports the conclusion that infusion of dalteparin to HD patients resulted in low levels of LPL in the circulating blood, compared to what was seen when conventional heparin was infused [ 12 ] or when controls were given dalteparin or conventional heparin [ 22 , 23 ]. There are several earlier studies that show that administration of LMW heparin results in lower plasma levels of LPL than conventional heparin [ 27 - 30 ]. LMW heparin preparations differ considerably in their molecular characteristics and caution should be exercised when extrapolating from one preparation to another. The comparisons to our earlier study with conventional heparin [ 12 ] are based on clinically relevant doses during HD, as recommended from manufacturer's and clinical guidelines, not on a molecule-for-molecule basis. In a study with dalteparin Persson et al . found that the early LPL activity was only about half as high compared to values observed after conventional heparin [ 27 , 29 ]. This is similar to the difference between the AUCs for the early peak of plasma LPL activity observed here and in an earlier study with conventional heparin in HD patients [ 12 ]. The LPL activities were lower in HD patients than observed in controls given dalteparin [ 22 ]. A similar difference was earlier found for infusion of conventional heparin in HD patients [ 12 ] compared to controls [ 23 ]. One possible explanation is that the depletion of LPL stores during the dialysis-sessions is not fully restored between the sessions. Arnadottir et al . have, however, found that the amount of LPL released by a bolus of heparin is restored within 24 hours [ 30 ]. In rats, it takes about four hours after a single bolus of LMW heparin before the LPL stores are replenished [ 31 ] and chylomicron catabolism occurs at normal rate [ 32 ]. A more likely explanation is that the kidney dysfunction as such causes an impairment of the LPL system [ 33 ]. Yet another possibility, that has not been explored, is that the kidney itself makes an important contribution to overall LPL stores. This is the case in mink, where kidney has a higher LPL activity than any other tissue, and in mice [ 34 , 35 ]. Administration of heparin causes a temporary derangement of lipoprotein metabolism. In our studies with control subjects given conventional heparin or dalteparin the TG concentration decreased after heparin and then gradually increased again so that at the end of the study period TG exceeded the baseline level (see inset in Fig 3 ). This probably reflects that LPL first becomes more available for lipoprotein catabolism as it circulates in blood but then becomes less available when the lipase is removed from blood by the liver and the tissues stores become depleted. This is in accord with observations in animal experiments. Chevreuil et al . found that the clearance of injected radioactively labeled chylomicron TG was dramatically increased five minutes after rats had been given conventional heparin or LMW heparin [ 32 ]. This was associated with an increased appearance of label in plasma FFA, supporting the view that the rate of lipolysis was increased. In contrast, injection of chylomicrons one hour after the heparins resulted in substantially slower clearance compared to saline-treated controls. Appearance of label in plasma FFA was also decreased, suggesting that impaired lipolysis was responsible, at least in part, for the impeded chylomicron clearance. The decrease of plasma TG was small and statistically not significant in the HD patients given dalteparin. This may, at least in part, be explained by the relatively low plasma LPL activities. In addition, there are reports that in patients with nephrosis there are inhibitors of LPL in the circulating blood and that VLDL isolated from such patients are lipolyzed slowly by LPL [ 36 ]. On the other hand, the rise of TG from two hours was pronounced in the HD patients. This indicates that the LPL stores were depleted in these patients even though the plasma LPL activities were low. This is in accord with results from animal experiments. Chevreuil et al. injected decasaccharides to rats [ 20 ]. This resulted in only a small and short-lived increase of LPL activity in blood. Nonetheless, the decasaccharides had apparently removed most of the functional LPL from peripheral tissues. The LPL activity that could be released from isolated hearts by single-pass perfusion with heparin ("functional LPL") was decreased by 75% one hour after the rats had been injected with decasaccharides. The catabolism of chylomicron TG by perfused hearts was delayed to a similar extent. The clearance of labeled chylomicrons injected to rats was markedly delayed from 30 minutes to 2 hours after a decasaccharide injection. After one hour, the fractional catabolic rate was only one-third of the control value. All of this suggests that dalteparin causes a profound depletion of functional LPL even though the plasma levels of LPL activity are relatively low. Conclusions The peak level of LPL in plasma after injection of dalteparin is less than half of that after conventional heparin. This was shown here for a group of HD patients, but a similar difference has earlier been observed for healthy controls. The peak level of LPL is lower in HD patients than in controls both after dalteparin and after conventional heparin. This is probably a consequence of the kidney disease but the detailed mechanism is not known. These two effects compound to an almost ten-fold difference in peak LPL activity comparing the present HD patients given dalteparin to a group of healthy controls given conventional heparin. Analysis of LPL activity and mass in frozen samples from our earlier studies ruled out the possibility of assay variation as a confounding factor. Prior in vitro studies of the LPL-heparin interaction, and animal experiments, indicate that decasaccharides, or longer heparins, release the enzyme efficiently from its binding sites at the vascular endothelium. The difference in plasma levels of LPL is probably due mainly to a difference in how much the respective heparin retards the uptake of LPL by the liver. Immediately after heparin, plasma LPL activity is high and catabolism of TG-rich lipoproteins is accelerated. This acceleration was not evident during infusion of dalteparin to HD-patients. Then follows a period when tissue stores of LPL are depleted and lipoprotein metabolism is retarded. This depletion was at least as marked after dalteparin as after conventional heparin, despite the lower plasma LPL levels. In the present study, the TG level increased significantly more after dalteparin than after conventional heparin. Our results indicate that LMW heparins disturb the LPL system as much or more than conventional heparin does. Abbreviations AUC – area under the curve; HD – hemodialysis; HDL – high density lipoprotein; LMW heparin – low molecular weight heparin; LPL – lipoprotein lipase; TG – triglycerides; VLDL – very low density lipoprotein Competing interests The author(s) declare that they have no competing interests. Authors' contributions BN participated in the design of the study, carried out the patient studies, assembled the data, did the statistical analyses, and participated in writing of the manuscript; BS and GO conceived of the study and coordinated the work. TO participated in the design of the study and in writing of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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546230

Free does not mean affordable: maternity patient expenditures in a public hospital in Bangladesh

Objective This study investigated a) the amount and types of out-of-pocket expenditures by patients for nominally free services in a large public hospital in Bangladesh, b) the factors influencing these expenses, and c) the impact of these expenses on household income. Methods Eighty-one maternity patients were interviewed during their hospitalization in the Dhaka Medical College Hospital. Patients were selected by quota sample to match the distribution of maternity patient categories in the hospital. Patients were interviewed with a semi-structured, in-depth questionnaire. Results All interviewees incurred substantial out-of-pocket expenditures for travel, hospital admission fees, medicine, tests, food, and tips. Only two of the expenditures, travel expenses and admission fees, were not supposed to be provided free of charge by the hospital. The median total per-patient expenditure was $65 (range $2–$350), equivalent to 7% (range 0.04%–225%) of annual household income. Half of all patients reported that their families had to borrow to pay for care at interest rates of 5%–30% per month. A third of these families reported selling jewelry, land or household items to moneylenders. The rural patients reported more difficulty in paying for care than the urban patients. Factors increasing the expenditures were duration of hospitalization, rural residence, and necessary (e.g. C-section, hysterectomy) and unnecessary (e.g. episiotomy) medical procedures. Conclusion Free maternity services in Bangladesh impose large out-of-pocket expenditures on patients. Authorities could reduce the burden by reducing the duration of hospital stays, limiting use of medical procedures, eliminating tips, and moving routine services closer to potential users. Fee for service could reduce unofficial expenditures if the fee were lower than and replaced typical unofficial expenditures, otherwise adding service fees without reform of current hospital practices would lead to even more burdensome expenditures and inequities.

Background In developing countries governments often subsidize services at public health care facilities and provide them free of charge to users. However, evidence suggests that users still incur large expenditures using the 'free' services for such things that are supposedly provided without charge. Studies have found that patients incurred substantial out-of-pocket expenditures for medicine, food and travel from the use of 'free' public health facilities [ 1 - 3 ]. A study in Vietnam found that out-of-pocket payments can cause serious equity problems such as the poor becoming poorer without greatly affecting the non-poor [ 4 ]. Household difficulty in payment of health care expenses can result in the 'distress sale' of property, delay or abandonment of treatment, and sacrifice expenditures on food and education [ 3 , 5 ]. Other studies have found that introducing or increasing user fees negatively affect the utilization of public health facilities [ 6 - 9 ]. Three previous studies have explored issues related to patient expenditures in Bangladesh [ 3 , 10 , 11 ]. Nahar et al. enumerated the patient expenditures and affordability of free maternity services for normal delivery and caesarean section. Killingsworth et al. explored the linkage between official and unofficial fees in public health facilities, and concluded that these fees had income and equity effects. Stanton et al. reviewed literature on user fees and pointed out the need to further investigate the factors and practices causing patient expenses before institutional implementation of user fees. Thus, this study examined the type, amount and household financial results of out-of-pocket expenditures by patients for nominally free services in a large government hospital in Dhaka. The study also identified the factors and medical practices producing and influencing the out-of-pocket expenditures. Plans to begin fees for service in Bangladesh make it important to document the amount of money actually being paid by the patients under the present system. If current expenditures are large, fee for service may have serious negative impacts on utilization and on the economic well-being of Bangladeshi households. If current expenditures are modest, it is possible that such fees will have a lesser impact. Methods Study site The study was conducted in the Department of Obstetrics and Gynaecology (ObGyn) of the Dhaka Medical College Hospital (DMCH). DMCH is the largest teaching hospital in Bangladesh with 850 beds located in the capital city. DMCH is government funded and provides a wide range of out- and in-patient services. Public hospitals have two payment categories for in-patients: non-paying and paying. Patients first go to an out-patient unit for diagnosis where they are categorized as out- or in-patient. Those categorized as in-patient are then classified as paying or non-paying by observing the clothes and general appearance of the woman and any accompanying relatives. Non-paying patients pay only the hospital admission fee. Paying in-patients are charged the fees for hospital admission, bed, and surgery. The various fees are: hospital admission fee: $0.23, bed fee: $1.34–3.50 per day, surgery fee: $12.50–125. Taka was converted into US dollars using the 1994 exchange rate of US$1.00 = Taka 40.00. Neither patient category is supposed to pay for medicine, tests, food, nursing and other support services during hospitalization; these commodities and services are theoretically provided free by the hospital. Study population, sampling and sample size The study interviewed 81 non-paying in-patients hospitalized for reproductive health conditions (about two thirds were for maternity conditions). Patients were selected by quota sample matching the distribution of the patient categories in the hospital i.e. the selected medical conditions accounted for the greatest number of ObGyn admissions reported for the hospital, and also reflect the causes associated with high maternal mortality and morbidity in Bangladesh [ 12 ]. These included normal vaginal delivery (NVD), caesarean section (C-section), abortion, and hysterectomy. NVDs included cases with episiotomy, without episiotomy, and with eclampsia. C-sections included elective and eclamptic cases. Abortion included non-septic and septic abortions. Hysterectomies included abdominal and vaginal hysterectomies for treating fibroid, prolapsed uterus, and pelvic inflammatory disease. Table 1 illustrates the distribution of the selected cases for this study. Table 1 No. of in-patients surveyed by medical condition No. of patients Normal Vaginal Delivery 19 with episiotomy 5 without episiotomy 5 with eclampsia 9 Caesarean section 20 elective 10 eclamptic 10 Abortion 20 non-septic 10 septic 10 Hysterectomy 22 prolapsed uterus 10 fibroid 9 pelvic inflammatory disease 3 Total 81 Variables Information was collected on various characteristics of the study participants. Demographic characteristics included age, education, marital status, and residence. Socio-economic characteristics included occupation and annual household income. Information was also collected on underlying medical condition. Out-of-pocket expenditure related information included types and amounts of expenses incurred during hospitalization such as those for travel, medicine, food, fees, etc. Factors influencing expenditures included type of treatment received and duration of hospitalization. Sources of funds included amount borrowed and interest charged for borrowed amount. Data collection tools and technique Data were collected from patients and their relatives with semi-structured open-ended questionnaires between January – June 1994. The interviewers were physicians employed in DMCH. The interviewers selected the cases by diagnosis from patient admission records. To minimize possible selection bias the first case was selected randomly from the records and then every third case was selected. The selected patients were interviewed a minimum of three times to minimize recall error. Recall error was also minimized as information was collected while patients were still hospitalized. During the first interview demographic and socio-economic information was collected with structured questions. During the second and third interviews information related to expenditures was collected with open-ended questions. To illustrate the data collection process a description of an interview with a typical C-section patient follows. C-section patients are usually hospitalized for two weeks in DMCH. On the first day of hospitalization an interviewer collected information on patient's age, education, marital status, etc. On the eighth day of hospitalization the second interview collected information on treatment received, treatment related out-of pocket expenditures, annual household income, amount of money borrowed to pay for treatment, source of borrowed money, and interest rate charged. On the fourteenth day the third interview collected more monetary information on out-of-pocket expenditures, and on expected expenditures immediately after leaving the hospital. This survey did not cover the expenditures for the full course of the treatment. Expenditure estimates were derived for the duration of the current hospitalization only, i.e. from the day of admission until the day of discharge. Expenditures immediately before admission and after discharge from the hospital included only travel expenses to and from the hospital for the patient and her accompanying relatives. Results Socio-demographic characteristics of study participants The median age of the study participants was 26 years (range 15–60 years). The majority (88%) of the patients were married, the rest were separated (4%), divorced (2%), and unmarried (1%). Forty-four percent of the patients lived in rural areas. The median annual household income was $750 (range $3–$6000) per respondent. The annual household income was higher for the urban (median $900; range $150–$6000) than the rural (median $615; range $3–$6000) respondents. Patient out-of-pocket expenditures All 81 patients interviewed reported incurring substantial out-of-pocket expenditures during their hospitalization. These out-of-pocket expenditures were for travel, hospital admission fee, medicine, tests, food, tips, and other items. As expected there were expenditures related to travel and admission fees which the hospital is not supposed to subsidize. But there were also expenditures for medicine, tests, food, tips, and other items which were supposed to be provided free from the hospital but were not. The median total expenditure for hospitalization was $65 (range $2.15–$350) per patient. On average, 61% of these expenditures ($49) were for services and commodities that were supposed to be provided free from the hospital but were not. The per patient median expenditure for the various expense categories were: medicine $26, tests 0, tips $1.25, food $1.25, other items $4.38, travel $22.25, and hospital admission fee $0.25. On average, medicine constituted 42%, travel 38%, tests 5%, food 4%, tips 2%, admission fees <1%, and others 8% of the total expenditures. C-section and hysterectomy cases had the highest median expenditures. Table 2 illustrates the out-of-pocket expenditures by items not supposed to be provided free by the hospital and items supposed to be given free from the hospital. A description of the expenses follows. Table 2 Distribution of the out-of-pocket expenditures by medical condition (in US$) in 1994 Expenditures on items NOT supposed to be provided from hospital Expenditures on items supposed to be provided free from hospital Travel Fee Medicine Food Tips Other Tests Total NVD (n = 19) median 12.50 0.25 11.25 0.88 1.25 3.88 0.00 62.50 mean 29.72 0.23 18.35 2.24 2.11 5.99 3.42 62.04 range 1–127 0.10–0.33 1–70 0–23 0.75–5 0–25 0–23 6–225 C-section (n = 20) median 36.70 0.10 51.88 2.50 2.06 11.25 0.00 118.75 mean 44.32 0.20 63.52 4.25 2.56 16.79 1.88 133.50 range 5–150 0.10–0.75 25–160 0.50–16 0.25–7 2–75 0–25 41–350 Abortion (n = 20) median 3.08 0.18 12.06 0.84 0.63 0.00 0.00 15.56 mean 11.67 0.17 18.93 1.40 0.77 0.00 0.00 32.94 range 1–63 0–0.30 1–75 0–5 0–2 0.00 0.00 2–125 Hysterectomy (n = 22) median 23.25 0.25 36.25 2.50 1.25 5.00 0.00 75.50 mean 32.04 0.38 30.89 4.84 2.38 4.02 10.15 84.70 range 1–86 0.10–4 1–50 0–19 0–10 0–10 0–75 2–178 Total (N = 81) median 22.25 0.23 26.25 1.25 1.25 4.38 0.00 65.25 mean 29.50 0.24 33.05 3.23 1.96 6.64 4.02 78.65 range 1–150 0–4 1–160 0–23 0–10 0–75 0–75 2–350 NVD: normal vaginal delivery Expenditures on items supposed to be provided free from hospital Medicine All patients were supposed to be provided required medicines free from the hospital but were not. Medicines included antibiotics, analgesics, syringe, catheter, blood, and so forth. Medicine was usually bought when patients were admitted at night. The medicine required for treatment is ordered by the on-duty physician but it takes several hours for the hospital management to process the order. Thus, no free medicine is available immediately. To start the treatment, the on-duty physician requests the patient's relatives to buy the medicine which is purchased from nearby private pharmacies. Tests All tests (e.g. pathology, radiology) are supposed to be provided by the hospital but sometimes the patients had the tests done in a private laboratory because waiting time for tests is very long in the DMCH due to the high patient load. Food Food is provided by the hospital but the interviewees found the hospital food of poor quality or totally lacking (liquid food such as soup or horlicks had to be bought for patients who had undergone surgery since these were not provided by the hospital). Relatives usually stayed with the patient in the hospital because of lack of ayahs (cleaning ladies) or nurses to provide necessary services. Thus, food was usually bought from a vendor or brought from home for both patient and relatives. Tips Tips ( bakshish ) are payments made to ayahs and guards. Ayahs were given tips for routine services such as pushing the patient's trolley to and from the labour/operation room, shaving the patient before delivery/surgery, giving enemas, etc. Guards at the gates were tipped each time a relative came to visit the patient during non-visitor hours. However, ayahs and guards are salaried hospital employees and are supposed to provide these services free of charge. The patients were reluctant when talking about the tips probably because they were still hospitalized and depended on these employees for access to certain services. Other items The other expenditures included items for the patient (e.g. hot water, bucket for hot water) and the newborn baby (e.g. blanket) that were supposed to be provided by the hospital free of charge but were not. Expenditures on items not supposed to be provided by the hospital Travel Travel expenses are not supposed to be provided by the hospital. Travel expenses consisted of travel to and from the hospital by the patient and any accompanying relatives, and travel expenditures of relatives during hospitalization for purchasing medicine and food for the patient. The patients came to DMCH because they expected 'free' and 'affordable' services compared to private clinics, or they were referred from a primary/secondary level facility, or to get better treatment here. Patients from rural/peri-urban areas took longer to reach DMCH than those from urban Dhaka (range half an hour to two days). Hospital admission fee This expense is also not supposed to be covered by the hospital. The official price of admission was $0.23, but it was zero for two patients and more than the official price for half of the patients interviewed. The study could not elicit the reason the patients paid more than the official price. When probed the patients could not or would not elaborate beyond the amount paid. The patients were very reluctant when talking about paying more than the official price for the admission fee. Factors increasing out-of-pocket expenditures Duration of hospitalization and rural residence of the patients increased the out-of-pocket expenditures. Rural residence increased the travel expenses and thus the total expenditures. Longer duration of hospitalization increased virtually all expenditures. The median duration of hospitalization was 8 days (range 1–34 days) per patient. Duration of hospitalization was the longest for hysterectomies followed by C-sections. Duration of hospitalization was related to severity of medical condition (e.g. eclampsia), necessary medical procedures (e.g. hysterectomy), and unnecessary medical procedures (e.g. episiotomy). One day of extra hospitalization increased expenditures by $2.30 per patient. Choice of medical procedures increased the patient expenditures. Episiotomy increased expenditures as patients were hospitalized for a longer duration and resulted in the purchase of more medicine. Episiotomy increased expenditures for both uncomplicated NVD (by 37%) and eclamptic NVD (by 84%) compared to cases where no episiotomy was performed (data not shown). The medical reason for performing episiotomies is the prevention of perineal tearing but because of a high case load at DMCH physicians perform episiotomies to reduce the length of delivery time, effectively turning hospital expenditures into patient expenditures. Eclampsia increased the expenditures for NVD by 180% (data not shown). Eclampsia is not under the control of the health system or the patient, and procedures used for treating eclampsia are unavoidable. C-sections caused higher patient expenditures compared to NVDs (median $119 and $63 respectively) because C-sections had a longer duration of hospitalization and required more medicine. Elective C-sections and eclamptic C-sections incurred similar expenses because elective C-sections were hospitalized for a longer duration even though there were no complications. Vaginal hysterectomies were 25% less expensive than abdominal hysterectomies because they required less invasive procedures, used local anaesthesia, and had a shorter duration of hospitalization. Sources of funds for patient expenditures The respondents said that they were willing to pay for care. However, rural households reported more difficulty in paying for care than urban households. Difficulty was inferred from the number of households who borrowed to pay for care, and the ratio of the amount borrowed to the annual household income. The median patient expenditure was equivalent to 7% (range 0.04%–225%) of annual household income, and was higher for rural (median 10%; range 1%–225%) than urban (median 7%; range 0.04%–78%) respondents. Half (n = 40) of the households reported borrowing to pay for care. The patient who spent 225% of her annual household income was a rural patient who had a hysterectomy for prolapsed uterus. Surgical patients like her are usually hospitalized for a month as they require more tests than non-surgical patients. This patient's total expenditures were not higher than the others who also had a hysterectomy, however, her annual household income was much lower than that of the others. More urban (n = 23) households borrowed than rural (n = 17) households but the amount borrowed was higher for rural households. The median amount borrowed per household was $38 and was equivalent to 8% (range 0.58%–208%) of the annual household income. On average, the rural households (median 14%; range 2%–208%) borrowed almost double the amount than the urban households (median 6%; range 0.58%–28%). Most often (n = 30) money was borrowed from friends and relatives without interest. When borrowing was from money-lenders (n = 10), households reported interest rates of 5%–30% per month. Three households put up security such as jewelry, land and household goods when borrowing money from moneylenders. The highest reported percentage of money borrowed to income for a rural household was 208% compared to 28% for an urban household. Finally, greater amounts of money were borrowed by C-section and hysterectomy patients than the other categories of patients. Table 3 illustrates the duration of hospitalization, total patient expenditures, annual household income, and amount borrowed by type of residence. Table 3 Distribution of median duration of hospitalization, expenditures, income, and amount borrowed by residence Urban Rural % of patients 56% (n = 45) 44% (n = 36) Duration of hospitalization (day) 7 (1–33) 9 (1–34) Total patient expenditures (US$) 59.25 (2.15–350) 79.25 (2.40–250) Annual HH income (US$) 900 (150–6000) 615 (2.50–6000) Borrowed amount (US$) 37.50 (6.25–250) 52.50 (12.50–200) Parenthesis shows range HH: household Discussion and conclusions The study findings indicate that all the surveyed patients incurred substantial out-of-pocket expenditures for a one time hospitalization. The median per patient expenditure was $65, and two-thirds of these expenditures were for commodities and services that were supposed to be provided free by the hospital but were not. Half the households borrowed to pay for care since they did not have the ability to pay (a finding similar to those found by Nahar et al.). A third of these households sold jewelry, land or household items to moneylenders. The rural households reported more difficulty in paying for care than the urban households. The rural patients had lower income but incurred higher expenditures and borrowed larger amounts than the urban patients. The study data are a decade old but worth presenting because Bangladesh is only recently beginning health sector reform and fee for service. Also, the hospital practices with regard to providing maternity care remain the same, in the hospital studied and in other public hospitals in Bangladesh. Other limitations of the study are its small sample size and that all the interviewees came from only one hospital. The small sample size is the norm when doing in-depth interviews. When a paper is qualitative not quantitative no statistical tests are customarily done. The results are striking despite the limitations. The costs related to NVD and C-section were much higher in this study than that estimated by Nahar et al. (NVD: $62 and $32 respectively; C-section: $133 and $118 respectively). Possible reasons for the differences are: a) recall error higher in the Nahar et al. study as they interviewed post-partum mothers, whereas, this study interviewed patients during hospitalization; b) the Nahar et al. study included only uncomplicated cases, whereas, this study included both uncomplicated and complicated cases. The annual household income was lower in this study compared to the Nahar et al. study ($750 and $1476 respectively). This may be attributable to having rural patients in this study whose income is much lower than urban patients, whereas, Nahar et al. studied only urban patients. Changing current practices regarding the length of hospitalization and medical procedures (e.g. episiotomy) will reduce patient expenditures. This can be achieved by having shorter duration of hospitalization for elective C-section and elective hysterectomy, and limiting the use of episiotomy. Limiting these practices may also lead to lower provider expenditures. Medicine constituted almost half of the total patient expenditures. However, lack of access to medicine resulted from an inefficient management system not from unavailability. Taking less time to process orders would make medicine available quicker to patients and so reduce their expenditures. Hospital management needs to ensure that patients pay only the official rate of admission fees. Hospital management also needs to ensure that patients do not pay tips to salaried hospital staff for routine services. The hospital could arrange for liquid food and hot water for surgical patients in addition to the other services it already provides, i.e. provide a more comprehensive hotel service. Alternatively the hospital could subcontract out its hotel services to those employees who extract tips from patients to provide such services. This may act as an incentive for eliminating the unofficial fees from tips. Making some services such as eclampsia and hysterectomy available at secondary level facilities will benefit the rural patients by reducing the travel expenses. Government hospitals can generate revenue by introducing or increasing some fees for medicine and tests for the paying category of patients but clearly exempting the poorest. How would the system determine who was the poorest? One option would be to let all rural women use hospital for free and have all urban women pay some fee. For service fees not to become a serious barrier to use current systems will have to eliminate or reduce practices that already result in high user expenditures from unofficial payments. Otherwise the added fees may lead to more borrowing from moneylenders, putting lands and goods at risk, and potential impoverishment of more households. A fee for service system needs to be based on information, and more than just setting a price. This can be facilitated by knowing what the patients were already spending in the current system and who were the most at risk of impoverishment. The challenge is to focus on realistic, short term changes that can reduce patient expenditures and inequities. Most of the recommendations of this study depend more on the will of the physicians and the hospital administrators than on the infusion of resources per se. Free maternity services in Bangladesh impose large out-of-pocket expenditures on patients. Authorities could reduce the burden by reducing the duration of hospital stays, limiting use of medical procedures, eliminating tips, and moving routine services closer to potential users. Fee for service could reduce unofficial expenditures if the fee were lower than and replaced typical unofficial expenditures, otherwise adding service fees without reform of current hospital practices would lead to even more burdensome expenditures and inequities. Competing interests The author(s) declare that they have no competing interests.

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535553

AIDS-defining illnesses among patients with HIV in Singapore, 1985 to 2001: results from the Singapore HIV Observational Cohort Study (SHOCS)

Background The objective was to describe the causes of initial and overall AIDS-defining disease episodes among HIV patients in Singapore. Methods A retrospective observational cohort study was performed of all adult patients seen at the national HIV referral center between 1985 and 2001. Data were extracted from the patients' records by ten trained healthcare workers. AIDS-defining conditions were established using predefined criteria. Results Among 1504 patients, 834 had experienced one or more AIDS-defining diseases. The most frequent causes of the initial AIDS-defining episode were Pneumocystis carinii pneumonia (35.7%), Mycobacterium tuberculosis (22.7%) and herpes simplex (7.4%). In total 1742 AIDS-defining episodes occurred. The most frequent causes were Pneumocystis carinii pneumonia (25.1%), Mycobacterium tuberculosis (16.2%) and cytomegalovirus retinitis (9.5%). Conclusions The most frequent causes of AIDS-defining illnesses in Singapore are similar to those reported in the West, prior to the introduction of anti-retroviral therapy. Opportunistic infections remain the most frequent AIDS-defining illnesses.

Background Cohort studies have provided valuable information on the clinical course of HIV infection in patients from Europe [ 1 - 16 ], North America [ 3 , 17 - 21 ], South America [ 22 , 23 ] and Africa [ 24 - 32 ]. The introduction of antiretroviral therapy has dramatically altered the incidence of AIDS-defining illnesses in the West. In the EuroSIDA study there have been substantial falls in the incidence and total number of AIDS-defining episodes between 1994 and 1998 [ 10 ]. During this period the proportions of reported AIDS-defining illnesses due to cytomegalovirus retinitis and Mycobacterium avium have decreased from 9% to 2% and 8% to 3% respectively [ 10 ]. The proportion due to non-Hodgkin's lymphoma has increased from 4% to 16% [ 10 ]. Substantial decreases in the incidence of disease caused by cytomegalovirus, Pneumocystis carinii , Mycobacterium avium and other opportunistic infections have also been observed in the United States [ 20 , 21 ]. Relatively few data are available on the course of HIV infection in Asian populations. In Bangkok between 1987 and 1993 the most frequent AIDS-defining diagnoses were extrapulmonary tuberculosis (22.8%), Pneumocystis carinii pneumonia (7.0%) and cryptococcal meningitis (10.9%) [ 33 ]. Little is known regarding whether the pattern of HIV-related conditions has changed following the introduction of antiretroviral therapy in Asia. The Communicable Diseases Centre (CDC) in Singapore is the national reference centre for adult patients with HIV and AIDS and nationwide 94.9% of all Singaporean adult residents, who have ever been diagnosed, have been referred there. Underreporting of HIV does not occur as reporting occurs automatically from the reference laboratory. Records have been kept since the first case was identified in Singapore in 1985. Therefore the Singapore HIV Observational Cohort Study (SHOCS) contains almost the entire country's HIV experience. Nucleoside analogues have been available in Singapore since the early 1990s and protease inhibitors and non-nucleoside reverse transcriptase inhibitors since 1997. The usage of these drugs has been increasing annually and during 2000 and 2001 approximately 70–80% of patients regularly attending CDC were taking some form of anti-retroviral therapy. Methods The details of the SHOCS cohort have been described previously [ 34 ]. The study was approved by the Ethics Committee of Tan Tock Seng Hospital. Data were extracted from the case notes of all Singaporean residents who were seen at CDC on or before 31 st December 2001, by ten trained healthcare workers. Probable and confirmed criteria were developed for the diagnosis of category C [ 34 ] and category B (AIDS-related complex) conditions, based on the 1993 guidelines of the United States' Centers for Disease Control and Prevention [ 35 ]. All disease episodes were initially determined by the data extractors and then checked by an infectious disease physician (RB). Diagnoses were not included if they did not fulfill the specified criteria even if there was a strong clinical suspicion of a particular condition. If there was insufficient evidence to satisfy the criteria for a specific diagnosis, a more general diagnosis was assigned, for example cerebral lesion (cause unknown) would be used if there was insufficient evidence to support a diagnosis of toxoplasmosis of the brain or primary cerebral lymphoma. Median CD4 counts were calculated based on the sample taken nearest the time of diagnosis of the initial AIDS-defining condition. CD4 counts from 893 patients were used, as in 37 (4.0%) cases no suitable count was available. Data were entered into a computer database (Microsoft Access™) and checked for errors and inconsistencies. Statistical analyses were performed using Stata 7.0™. Each new or recurrent AIDS-defining condition counted as one event (i.e. a patient with three diagnoses would contribute three events). An AIDS-defining condition was counted as a separate additional event if it recurred six or more months after the initial diagnosis or in the case of tuberculosis, six or more months after treatment completion. To examine the effects of highly active anti-retroviral therapy (HAART) on the proportions of AIDS-defining illnesses due to each of the three most common infections ( Pneumocystis carinii pneumonia, Mycobacterium tuberculosis and cytomegalovirus retinitis), comparisons were made between the pre-HAART era (1986 to 1995) and the established HAART era (2000 to 2001) using χ 2 tests. Results Among the 1504 patients infected with HIV who were seen at CDC between 1985 and the end of 2001, 834 developed one or more AIDS-defining conditions. The most frequent initial AIDS-defining disease episodes were due to Pneumocystis carinii pneumonia (35.7% of total diagnoses), M. tuberculosis (22.7%), herpes simplex (chronic mucocutaneous) (7.4%) and candidiasis (esophageal or tracheobronchial) (6.9%) (table 1 ). The median CD4 count at which the initial AIDS-defining condition occurred was 27 (inter-quartile range 11 – 63). The median CD4 count was higher for M. tuberculosis infection (52, IQR = 18 – 110) and Kaposi's sarcoma (46.5, IQR = 23.5 – 227.5) than for Pneumocystis carinii pneumonia (16.5, IQR = 9 – 40), herpes simplex (25, IQR = 9 – 63) and extrapulmonary cryptococcosis (13, IQR = 6.5 – 39) (table 1 ). 1742 AIDS-defining disease episodes were recorded. The 10 most frequent AIDS-defining conditions were all of infectious etiology. There were 1658 first episodes of disease and 84 recurrences. The most frequent AIDS-defining diseases were Pneumocystis carinii pneumonia (25.1% of total diagnoses), M. tuberculosis (16.2%), cytomegalovirus retinitis (9.5%), candidiasis (7.8%), chronic mucocutaneous herpes simplex (8.2%) and disseminated M. avium (8.2%) (table 2 ). Several additional conditions could have included undiagnosed AIDS-defining diseases. The most common of these infections was pneumonia of unknown cause (117 cases), which is likely to include some patients with Pneumocystis carinii pneumonia (table 3 ). Weight loss of >10% body weight was a frequent problem and affected 383 (25.5%) patients (table 3 ). Between 1996 and 2001 there was a continuous increase in the total number of AIDS-defining disease episodes occurring each year, due to the increase in the number of patients being followed up. There was no significant change in the overall proportion of AIDS-defining episodes caused by infections. Pneumocystis carinii and M. tuberculosis remained the two commonest causes. Between 1986 and 1995 they respectively accounted for 26.7% and 10.8% of all AIDS-defining disease episodes. During 2000 to 2001 the percentage of episodes caused by Pneumocystis carinii was not significantly different at 26.3% (Yates' χ 2 = 0.01, 2df, P = 0.94), but that due to M. tuberculosis had increased to 18.0% (Yates' χ 2 = 8.10, 2df, P = 0.004). There was no significant change in the percentage of episodes due to cytomegalovirus retinitis (9.7% between 1986 and 1995 and 8.5% during 2000 and 2001; Yates' χ 2 = 0.25, 2df, P = 0.62) (table 4 ). Discussion Comparison of HIV-associated morbidity with other populations In the SHOCS cohort the proportion of conditions which were of infectious etiology remained very high throughout the study period. The distribution of conditions was similar to that seen in Western countries prior to the introduction of anti-retroviral therapy [ 1 ]. However the proportion of conditions caused by mycobacteria was higher than in Europe [ 1 , 12 ]. In Bangkok the most common AIDS-defining conditions were infections. Between 1986 and 1993 the most frequent AIDS-defining illnesses in Bangkok were extra-pulmonary tuberculosis (22.8%), cryptococcal meningitis (10.9%) and Pneumocystis carinii pneumonia (7%) [ 33 ]. Between 1993 and 1996 they were extrapulmonary cryptococcosis (38.4%), tuberculosis (37.4%), wasting syndrome (8.1%) and Pneumocystis carinii pneumonia (4.8%) [ 36 ]. Pneumocystis carinii pneumonia caused a much higher proportion of AIDS-defining diseases in Singapore than in Bangkok and cryptococcal meningitis occurred less frequently in Singapore. Potential sources of bias The SHOCS cohort provides data on the experience of 94.9% of Singaporean residents who have been diagnosed with HIV since the epidemic began. There are good diagnostic facilities in Singapore and patients with HIV-related illness receive extensive investigations. Therefore Singapore offers an ideal location for collecting information on the causes of morbidity and mortality in persons with HIV in Asia. Medical record keeping is of a high standard in our hospital and this allowed accurate diagnoses to be assigned for AIDS-defining conditions which occurred throughout the AIDS era. To ensure consistency, data was extracted by carefully trained healthcare workers and diagnoses were assigned using predefined criteria. All AIDS-defining diagnoses and all causes of death were reviewed by the same infectious diseases physician (RB). Follow-up rates in our cohort are very high with only 5.8% of patients lost to follow-up at 12 months and 9.3% after 3 years. The SHOCS cohort therefore provides a unique source of information on HIV infection in an Asian country since the start of the epidemic. In a retrospective study it is impossible to ensure that all patients received a full series of investigations in order to satisfy the study criteria for diagnosis of HIV-related conditions. As a consequence the criteria for making a probable diagnosis are unavoidably biased against certain conditions. For example if a patient undergoes a therapeutic trial of tuberculosis treatment he or she can be given the diagnosis of "probable Mycobacterium tuberculosis ". However if a patient receives a successful trial of treatment for disseminated M. avium , the diagnosis is "probable mycobacteriosis (species unidentified)". Differences in physicians' awareness of the link between HIV and opportunistic infections can influence diagnosis rates. For example, the increase in the proportion of AIDS-defining disease episodes caused by tuberculosis between 1986–1995 and 2000–2001 may have been due to greater awareness of the link between HIV and tuberculosis. Diagnosis rates for M. avium and M. tuberculosis may also have been affected by improvements in mycobacterial culture techniques. Conclusions Despite the availability of anti-retroviral medication, opportunistic infections remain a common problem among HIV patients in Singapore. This may partly be explained by the high proportion of Singaporean patients, who present with advanced disease and very low CD4 counts. These patients often already have one or more AIDS-defining opportunistic infections at first presentation. Opportunistic infections may also occur before adequate immune-reconstitution can occur when HAART is commenced when the patient has a very low CD4 count. Studies in the West have shown that AIDS-defining illnesses occur more frequently among patients who have a CD4 count below 50 when anti-retroviral therapy is commenced than among those with higher CD4 counts [ 37 , 38 ]. Immune-reconstitution may also be impaired by difficulties in maintaining high levels of adherence, sub-optimal treatment regimens and unplanned treatment interruptions (due to adverse effects, financial or logistical reasons). The current reductions in the cost of anti-retroviral therapy (Eg. due to generic manufacture) may enable high levels of anti-retroviral use to be achieved in many Asian countries. The experiences of the SHOCS cohort are likely to be repeated throughout Asia. Our results suggest that avoidable opportunistic infections may continue to occur even when high levels of anti-retroviral use are achieved. To reduce the burden of morbidity and mortality caused by these infections, efforts must be made to diagnose HIV infection earlier and to make the treatment of HIV and its associated opportunistic infections more affordable. List of abbreviations SHOCS = Singapore HIV observational cohort study, CDC = Communicable Diseases Centre, AIDS = acquired immunodeficiency syndrome, HIV = human immunodeficiency virus, HAART = highly active anti-retroviral therapy. Competing interests The authors declare that they have no competing interests. Authors' contributions RB participated in the design of the study, supervised data extraction, performed the statistical analysis and wrote the manuscript. SS designed the database and supervised data extraction. NP participated in the design of the study and contributed to the statistical analysis and production of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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544355

Intestinal parasites prevalence and related factors in school children, a western city sample-Turkey

Background Intestinal parasitic infections are amongst the most common infections worldwide. Epidemiological research carried out in different countries has shown that the social and economical situation of the individuals is an important cause in the prevalence of intestinal parasites. Previous studies in Turkey revealed a high prevalence of intestinal parasitic infection. The objectives of the current study were to determine the prevalence of intestinal parasitic infections in Aydin among 7–14 years old school children and to identify associated socio-demographic and environmental factors, behavioral habits and also related complaints. Methods Multistage sampling was used in the selection of the study sample. A questionnaire, cellulose adhesive and a stool specimen examination were done. Results A total of 456 stool specimens were collected. 145 students (31.8%) were infected with one or more intestinal parasites. 29 (6.4%) of the students were infected more than one parasite, 26 (5.7%) with two parasites and 3 (0.7%) with three parasites. The three most common were E. vermicularis , G. intestinalis and E. coli . Intestinal parasite prevalence was higher in rural area, in children with less than primary school educated mother, in children who use hands for washing anal area after defecation, and in children who use toilet paper sometimes or never. The relation between child health and mother education is well known. Children were traditionally taught to wash anal area by hand. Toiler paper usage was not common and might be due to low income or just a behavioral habit also. Most of the complaints of the study population were not significantly related with the intestinal parasitic infection. Conclusions Intestinal parasitic infection is an important public health problem in Aydin, Turkey. Rural residence, mother education less than primary school, sometimes or never usage of toilet paper, and washing anal area by hands after defecation were the significant associations. Interventions including health education on personal hygiene to the students and to the parents, especially to mothers are required. The ratio of uneducated women should be declined with specific programs. A multisectoral approach is needed.

Background Intestinal parasitic infections are amongst the most common infections worldwide. It is estimated that some 3.5 billion people are affected, and that 450 million are ill as a result of these infections, the majority being children. These infections are regarded as serious public health problem, as they cause iron deficiency anemia, growth retardation in children and other physical and mental health problems [ 1 ]. Epidemiological research carried out in different countries has shown that the social and economical situation of the individuals is an important cause in the prevalence of intestinal parasites. In addition, poor sanitary and environmental conditions are known to be relevant in the propagations of these infectious agents [ 2 - 4 ]. Previous studies at various institutions in Turkey revealed a high prevalence of intestinal parasitic infections among the following populations: 6–16 year olds (10.8%), 12–16 year olds (48.0%), 7–15 year olds (55.1%), and 6–12 year olds (88.0%) [ 5 - 8 ]. However, almost all of the studies were performed in isolated groups, such as collecting all samples from children attending the same school. Furthermore the majority of the studies were performed in the eastern part of Turkey. They have limitations in regards to giving an idea about all age groups in the region. Although there are studies on interactions between infection and socio-demographic, environmental factors, and behavioral habits from eastern regions, to our knowledge, there is lack of adequate information for the western part of Turkey [ 7 , 9 ]. The objectives of this current study were to determine the prevalence of intestinal parasitic infections in Aydin among 7–14 year old school children and its relation to socio-demographic factors, environmental factors, behavioral habits and complaints related to intestinal infections. Methods Study population The data for the present study was acquired from the primary schools in urban and rural areas of Aydin, a city in the western part of Turkey. The study design was cross-sectional. The sample size was calculated on a prevalence of 30%, d = 0.05 at a confidence level of 95%. A design effect of 2 was used to allow for multistage sampling [ 10 ]. The calculated study population size was 639. Multistage sampling was used in the selection of the study sample. Aydin was separated into four regions according to the socio-economic and health data taken from Directorate of Health. From these regions two schools were selected randomly, one from the urban communities and one from rural. The children were selected from the classes randomly based on the age, gender and weight for the population. First to eighth graders were included in the study, and the total numbers of the students enrolled in the classes were 74, 60, 70, 72, 72, 107, 104, and 81 students, respectively. Permission was obtained from Directorate of Education. The questionnaire and family information form The questionnaire contained four sections: 1. Socio-demographic data: age, gender, residence, education and occupation of parents, number of adults and children in the family and birth order of the child; 2. Environmental factors: housing conditions (ownership of the house, number of rooms and bathrooms) and water supply; 3. Behavior habits: type of toilet commonly used, hand washing (no washing/washing with only water/washing with soap), washing anal area by hands after defecation (yes/no), usage of toilet paper (always/sometimes/never); 4. Complaints: abdominal pain, nausea/vomiting, lack of appetite, abdominal distention, intestinal dismotility, salivation during sleeping, headache, irritability in sleeping, perianal itching, teeth grinding, and history of parasitic infections. An informational document about the study, including how to supply a stool specimen and a cellulose tape slide, was given to each participant for their family. Intestinal parasitic examination Mothers were asked to perform one cellulose tape test on their child who was participating in the study. Laboratory slides were provided with cellulose tape attached to them. The mother collected material for examination in the early morning prior to bathing or defecation. On the same morning a field worker collected these slides to be microscopically examined. The stool specimens (0.5–1.5 gr) were collected in labeled plastic vials without preservatives and transported to the laboratory within four hours after collection. They were examined for the presence of parasites by direct wet mount, Lugol's iodine solution and modified formaline-ethyl acetate sedimentation techniques. The presence of parasites was confirmed when observed by any of the methods above. Statistical analysis A computer program was used for data analysis. The descriptive data was given as a mean ± standard deviation (SD). The chi-squared test and Student t-test were used for the analytic assessment. The differences were considered to be statistically significant when the p-value obtained was less than 0.05. Results A total of 456 (71.4%) samples for both stool specimens and cellulose tape slides were collected. The response rate to the questionnaire was lower with 367 (57.4 %). The mean age was 10.34 ± 2.27, 10.17 ± 2.30 for girls and 10.51 ± 2.23 for boys. Important socio-demographic characteristics, housing conditions and hygienic habits of children are given in Table 1 . Table 1 Important socio-demographic characteristics, housing conditions and hygienic habits of children Characteristics No % Socio-demographic characteristics Residence Urban 258 56.6 Rural 198 43.4 Gender Female 232 50.9 Male 224 49.1 Education of mother No education/primary school incomplete 68 19.0 Primary/secondary school 269 75.4 High school and more 20 5.6 Education of father No education/primary school incomplete 21 5.8 Primary/secondary school 292 81.4 High school and more 46 12.8 Housing conditions Owner 239 66.0 3 rooms and less 154 43.5 4 rooms and more 200 56.5 1 toilet 180 49.9 2 and more toilet 181 50.1 5 and less people living in 267 74.4 6 and more people living in 92 25.6 Municipal water network 232 68.6 Hygienic habits Type of toilet commonly used Modern style 86 26.5 Traditional style 135 41.5 Both 104 32.0 Toilet paper Always 206 57.1 Sometimes 105 29.1 Never 50 13.9 Washing anal area by hands after defecation Yes 138 39.4 No 212 60.6 Washing hands with soap after toilet Always 308 85.3 Sometimes 51 14.1 Never 2 0.6 Taking a bath Once a day 18 5.2 Three times a week 161 46.1 Once a week or less 170 48.7 In all, 145 students (31.8%) were infected with one or more intestinal parasites. 29 (6.4%) of the students were infected with more than one parasite, 26 (5.7%) with two parasites and 3 (0.7%) with three parasites. The most common was Enterobius vermicularis (E. vermicularis ) with 63 (13.8%) pure and 20 (4.4%) with multiple infections, in total 83 (18.2%) infected children. The second was Giardia intestinalis ( G. intestinalis ) with 28 (6.1%) pure and 21 (4.6%) with multiple infections, in total 49 (10.7%) infected children. The third one was Entamoeba coli ( E. coli ) with 21(4.6%) pure and 15 (3.3%) multiple infections, in total 36 (7.9%) infected children. G. intestinalis was the most commonly found parasite in multiple infections. The distribution of parasites is given in Table 2 . Table 2 The parasites distribution of the study population Parasites No. % Single E. vermicularis 63 13.8 G. intestinalis 28 6.1 E. coli 21 4.6 H. nana 4 0.9 Total 116 25.4 Multiple E. vermicularis+G. intestinalis 11 2.4 E. vermicularis+E. coli 8 1.8 G. intestinalis+E. coli 4 0.9 G. intestinalis+H. nana 2 0.4 G. intestinalis+Taenia spp. 1 0.2 G. intestinalis+E. coli+H.nana 2 0.4 E. vermicularis+G. intestinalis+E. coli 1 0.2 Total 29 6.4 Overall total 145 31.8 No statistically significant difference was observed between presence of intestinal parasites and gender, (p = 0.805), and also age (p = 0.916). The prevalence of intestinal parasites were significantly higher (p = 0.042) in the rural area (36.9%) than in the urban area (27.9%). A summary of significant relations observed in overall intestinal parasitic infections for the study population are given in Table 3 . Table 3 Significant relations for the intestinal parasitic infection in the study population Risk Factor Overall infection χ 2 p n % Residence Urban 72 27.9 4.415 0.042 Rural 73 36.9 Mother education Less than primary school 29 42.6 4.436 0.035 Primary school and more 85 29.4 Toilet paper Always 50 24.3 13.596 0.000 Sometimes/never 66 42.6 Washing anal area by hands after defecation Yes 55 39.9 5.503 0.019 No 59 27.8 Parasite-specific significant relations were the following: The prevalence of E. coli infections was significantly higher (p = 0.010) in the rural area (11.1%) than in the urban area (5.0%). Mother education less than a primary school education (p = 0.012), washing anal area by hands after defecation (p = 0.013) were the significant relations for G. intestinalis , where sometimes or non-usage of toilet paper was significant for G. intestinalis (p = 0.008) and for E. vermicularis (p = 0.024) both. Family size was larger in the group infected with G. intestinalis (p = 0.029). The prevalence of intestinal parasitic infection was 31.0% in the group using a municipal water network and 34% in the group lacking a municipal system (p = 0.592). The prevalences of G. intestinalis in these two groups were 9.9% and 16.0% (p = 0.106) respectively. No further significant relationships were found between intestinal parasitic infection and environmental or behavioral factors. The most frequent complaint related with any parasite infection was intestinal dismotility (40.0%). Nausea/vomiting (37.7%) was second and abdominal distention (37.1%) was third. All of the complaints were seen in higher prevalence for E. vermicularis infections than G. intestinalis and E. coli . Discussion It was found that approximately one-third (31.8%) of the students, ages 7 to 14, in Aydin were infected by intestinal parasites. In a sample within the same age group in Izmir, a city also in the western part of Turkey, the prevalence for intestinal parasites was 22.4%, with E. vermicularis (16.0%) and G. intestinalis (11.9%) being the two most common infections, as was observed in Aydin [ 11 ]. In another study performed in Izmir, the prevalence of infection was 45.3% for E. vermicularis , 21% for G. intestinalis , 10% for H. nana , 4.3% for E. coli , 0.03% for Ascaris lumbricoides ( A. lumbricoides ), and 0.03% for Trichuris trichiura ( T. trichiura ). No Taenia was found [ 12 ]. In this current study; the most frequently observed parasites were E. vermicularis , G. intestinalis , and E. coli , 18.2%, 10.7% and 7.9%, respectively. Higher prevalence was found in the studies from the eastern part of Turkey, where the socio-economic and environmental conditions were lower. Additionally, it was observed that the types of parasites found in this study where different than those found in the eastern part of Turkey. A survey conducted among 1001 children in four elementary schools in Sanliurfa found parasites in 88% of the stool samples examined (50% A. lumbricoides , 53% T. trichiura , 22% G. intestinalis , and 11% E. coli ); unfortunately, no data on E. vermicularis was given, because samples with cellulose tape slides were not taken [ 7 ]. In an another study from the eastern region, in an elementary school age group, 48.12% A. lumbricoides , 4.43% T. trichiura and 15.35% G. intestinalis were found [ 13 ]. In the last study, samples with cellulose tape slides were not taken. Geohelmint infections ( Ascaris , Strongyloides and Trichuris ) were of lower prevalence in the western part of Turkey, but of higher prevalence in the eastern part. This difference may be due to improper toilet facilities which require individuals to defecate in areas around their homes as well as the use of fecal material for fertilizer in gardens in the eastern parts of Turkey [ 13 - 15 ]. In a study from the eastern region, 42.2% of children were found to be working in gardens watered with contaminated sewage and eating the vegetables of those gardens. There were 44.7% A. lumbricoides , 11.7% T. trichiura infections in these children while there was 12.2 % A. lumbricoides and 6.6% T. trichiura infections in the control groups [ 14 ]. Enterobiasis occurs worldwide, usually involving school-aged children [ 16 ]. In general E. vermicularis infection is transmitted by hand to mouth and/or person to person directly. High prevalence of E. vermicularis in the current study might be due to improper hygiene including not washing hands with soap after defecation, before eating and preparing foods. In the study area, there are two traditional methods of cleaning anal areas after defecation: (1) washing the anal area by hand with tap water (2) a piece of cloth is used to clean the anal area after defecation. The cloth is used multiple times until the person decides that it has become too dirty after which it is washed and reused. These improper cleaning practices after defecation could be the probable causes behind autoinfection. The higher prevalence of E. vermicularis could also be explained by the highly infectious nature of the parasites. G. intestinalis and E. coli were the most common intestinal protozoa among the study population. Both can be transmitted orally by drinking infected water and both are environmental contaminants of the water supply. The water supply is really an important risk factor for the Giardiasis, and several large outbreaks of giardiasis have resulted from the contamination of municipal water supplies with human waste [ 17 ]. The ingestion of contaminated water is a common problem in Turkey countrywide due to the lower quality of water and faulty sewage lines. The problem is greater in the rural areas that do not have a municipal water network or sewage system [ 18 ]. Contamination of drinking water with Giardia spp . has been increasingly recognized over the past 10 years as a cause of water-borne diseases in humans [ 19 ]. Giardia cysts have been isolated from water supplies in different parts of the world [ 20 , 21 ]. Epidemic giardiasis may be related to drinking water [ 22 ]. G. intestinalis and E. coli are most common in the western part of Turkey [ 11 ]. In a study assessing giardiasis cases in Turkey published within the last 15 years, the prevalence of G. intestinalis was found 11.6% in the western part of Turkey [ 23 ]. From the study, 68.6% of the study group uses municipal water, while the others either use a chlorinated collection tank with a crude water network or purchase bottled water. Although no statistically significant difference was observed, the prevalence of G. intestinalis was lower in the group using municipal water (9.9%) than the other group (16.0%) in the current study. It is thought that an in-depth assessment should be done on the ways that drinking water becomes contaminated. In this current study, there were no cases of Taenia spp . Since the consumption of pork and pork products are forbidden for Muslims this may account for the absence of T. solium cases in the population. It is also a common practice to eat uncooked meat in the eastern part of Turkey, but not in the western part. T. saginata infections are observed in the eastern studies, with prevalence of 13.8% and 12.9% [ 19 , 24 ]. However, in the western region infections by this parasite were not observed in this or in another previous study [ 12 ]. Differences due to gender were not observed in this current study. These results were similar to a study conducted in the central part of Turkey [ 24 ]. In this current study; the prevalence of intestinal parasitic infection was higher in the rural areas. A similar result was found in the central region of Turkey where the prevalence of intestinal parasites was higher in the rural area [ 25 ]. In this current study, most of the complaints by the study population were not significantly related with the intestinal parasitic infection. For example, perianal itching was noted in 15.8 % of the study population. There was no significant difference in the prevalence of this symptom in pinworm infected and non-infected children. Furthermore, no association was found between the prevalence of pinworm infection and a history of teeth grinding, colic, enuresis, and irritability. The complaints may not have been assessed effectively through the use of only a questionnaire without an interview, or they might not be useful for every individual's diagnosis. The prevalence of intestinal parasites was higher in groups where the mother in the household had less than a primary school education, where the hand is habitually used for the cleaning of the anal area and where toilet paper is seldom or never used. The relation between a child's health and the mother's education is well known. Health indicators of children whose mother's education level is lower are always worse [ 26 ]. In the last two groups, the habits of the children are a factor, along with a cultural dimension. They were taught to clean the anal area by washing with the hand. Toiler paper usage was not common, possibly due to low income. Usage of a piece of cloth instead of toilet paper was also common. The major limitation of the current study was a low response rate. The assessment would have been more valuable if a higher response rate could have been obtained. But, it was thought that the results were still important because there is little knowledge on the data of the region. Additionally, the current study was the first population-based study for the region. Although an important number of risk factors were discussed, a few risk factors (e.g.: shoe wearing) were not evaluated in the current study. This might have been another limitation of the study. The important risk factors for the region were evaluated in the study. Conclusion As a conclusion, intestinal parasitic infection is an important public health problem in Aydin, Turkey. Rural residence, households where the mother has less than a primary school education, periodic or non-usage of toilet paper, and the washing of the anal area by hand after defecation were the significant associations. Interventions including health education on personal hygiene to the students and to the parents, especially to mothers are required. The ratio of uneducated women should decline with specific programs. A multisectoral approach is needed. Competing interests The author(s) declare that they have no competing interests. Authors' contributions PO planned the research, performed the sampling and statistical analyzes and wrote draft and final version of the manuscript. SE contributed in planning the research, performed parasitic examinations and contributed discussing the results and writing manuscript. BG performed parasitic examinations and contributed discussing the results. OO organized the work in the schools and participated in its design. EB participated in initial study design, coordinated the study and revised the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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