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.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  This computational analysis provides evidence that as many as 10% of human genes are targets for regulation by small RNA molecules called 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 . More relin-4   . While cThe 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 . Recent lin-41, a 27-nucleotide (nt) intervening sequence between two consecutive let-7 sites is necessary for its regulation  complex, RNA-induced silencing complex (RISC) . Every RAmong 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  and bindDrosophila homolog of FMRP (FXR) and the Vasa intronic gene were identified as components of RISC , 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 (Danio rerio) and fugu (Fugu rubripes) fish genomes for potential targets using known and predicted miRNAs  from the human e Tables  and S3 ad miRNAs  and S5 aIn 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  nonuniform distribution of the number of sequence-complementary target sites for different miRNAs; (2) 5′–3′ asymmetry  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.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, let-7 and lin-4 miRNAs in Drosophila to develop the target prediction method and check for consistency . The surviving two target sites have high alignment scores, S = 115 and S = 110, while the other four sites are below threshold  and passes the conservation filter.We previously used known miRNA sites for the sistency . More re elegans  has narrhreshold , fully cD. 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 , first discovered in worm , let-7b , 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  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)  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.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.T > c,T where cT 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 > cS for match score, |ΔG| > |ΔcG| for free energy of duplex formation, and C > cC 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  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 equences . In addi(S) and energy (ΔG) 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  should be similar to the distribution of incorrect (non-meaningful) hits from real trials . 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  target sites—a difference of 20%. This difference may be indicative of a biological signal in the raw score Dicer, and the mammalian homologs of AGO1 and AGO2 –associated (SAPAP4), a neuron-specific protein, is regulated by many miRNAs highly expressed in rat brain primary cortical neurons .In summary, the three validation approaches  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) 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 let-7b), but the distribution is nonuniform  terms see  and domaHomeobox domain, 5.3-fold; KH domain, 4.0-fold; and Guanine-nucleotide dissociation stimulator CDC25 domain, 3.4-fold . The semaphorins and plexins (semaphorin receptors) are involved in axon guidance, angiogenesis, cell migration, the immune system, and the adult nervous system . 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 , 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  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.Do specific miRNAs target particular functional groups? We analyzed each miRNA individually for GO term and domain enrichment . The tar miR-135 , and themiR-181a and miR-142–3p are key components of a molecular circuitry that modulates hematopoietic lineage (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  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  is an FMRP-bound protein that is translationally regulated. The ing FMRP . In addiAPP mRNA . We predt region , and ano, 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.Other APP-interacting proteins, APP-binding family B member 1 miR-125, miR-135, miR-320, and miR-327, all of which are either exclusively expressed in brain or enriched in brain tissue (miR-125b are found copurified with polyribosomes in rat neurons in (PSD95 has one reported G-quartet in its 3′ UTR at position 648 (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 , SAP90, and CamKII are predicted miRNA targets   . The genDicer 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  are expressed fairly widely, while others  have lower and more restricted expression.Our data suggest that the RNAi–miRNA machinery itself is under miRNA regulation; for example nd Ago-1  will accrebellum . The tar message . Ago-3 iElavl1 (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 , contain three RNA-recognition motifs, which bind AU-rich elements in 3′ UTRs of a subset of target mRNAs . These Acadherin . Three ocadherin . This isCPEB2 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 , a member of the Argonaute family  known in mammals as miRNA targets ranked within the top 170 target genes with 6–20 sites in their UTRs . Indeed, oocytes . It also oocytes  and dend oocytes  and faci oocytes . CPEB bi oocytes , thereby oocytes . In addind CPEB3 . Second,nogaster . The CPEe family  that is HuB, HuR, Eif-4 gamma, DAZ associated protein 2, VAMP-2 , 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.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 . Our premiR-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  CNOT7, a gene expressed in colorectal cell lines and primary tumors ((miR-15a); (2) LASS2, a tumor metastasis suppressor ((miR-15b); (3) ING4, a homolog of the tumor suppressor p33 ING1b, which stimulates cell cycle arrest, repair, and apoptosis ((miR-143); (4) Gab1, encoding multivalent Grb2-associated docking protein, which is involved in cell proliferation and survival ((miR-155); and (5) COL3A1, a gene up-regulated in advanced carcinoma ((miR-145).Our method predicted cancer-specific (by annotation) gene targets of y tumors   and Hox-A5, both known to be dysregulated at the level of protein expression in colon cancers (miR-16 and the target genes (indicating possible cleavage). Both of these target genes are also targets for another cancer related miRNA, miR-15.ntegrity . The fir cancers . Both of.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 informationHox 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.The full set of conserved target genes between fish and mammals indicates a wide functional range of conserved targets . Many Hohox 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  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.To investigate this issue we computed the average density of target sites for high-scoring targets  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.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 e strand . Of the 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  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  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.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 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 . 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 In the third method (this work), we used 218 mammalian miRNAs and 29,785 transcripts derived from Ensembl  and, as 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 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.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  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.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  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  at some (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).Regulation by miRNAs is obviously not as simple as one miRNA–one target gene, as perhaps the early examples 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 lin-4 (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 in lin-4 .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  depend oWe 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 . We are,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?miR-196 and miR-138; see Hox-B8, cleavage has been experimentally shown in mammalian cells  details of strand bias as deduced from siRNA experiments , (2) conAlthough the predicted targets are subject to error  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  to a subtle modulation of protein dosage in a cell through low-level translational repression .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 , but stiThe 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.Mature human and mouse miRNA sequences were obtained from the RFAM miRNA registry . To coveMature miRNA sequences for zebrafish and fugu were predicted starting from known human and mouse miRNA precursor sequences . Each prThe Ensembl database  served aOrthology mappings between genes from different genomes were obtained using “orthologue tables” from the EnsMart  feature 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  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  that contained target sites with more than one G:U wobble in the 5′ end are flagged in the S > 90 and ΔG < −17 kcal/mol, where S is the sum of single-residue-pair match scores over the alignment trace and ΔG is the free energy of duplex formation from a completely dissociated state, calculated using the Vienna package as in The miRanda algorithm  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.class,F using the log-odds ratio of the fraction of annotated target genes with the same class 1):,.Here, 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.We compiled a list of 464 gene identifiers of FMRP-associated mRNAs from five different publications . Among tWe 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 .Figure S1(347 KB PDF).Click here for additional data file.Table S1(25 KB XLS).Click here for additional data file.Table S2(8.0 MB XLS).Click here for additional data file.Table S3(17.0 MB XLS).Click here for additional data file.Table S4(5.6 MB XLS).Click here for additional data file.Table S5(9.8 MB XLS).Click here for additional data file.Table S6(601 KB XLS).Click here for additional data file.Table S7(512 KB XLS).Click here for additional data file.Table S8(23 KB XLS).Click here for additional data file.Table S9(678 KB XLS).Click here for additional data file.Table S10(357 KB XLS).Click here for additional data file.Table S11(529 KB XLS).Click here for additional data file.Table S12(621 KB XLS).Click here for additional data file.Table S13(68 KB XLS).Click here for additional data file.


Phytophthora species blight potatoes and tomatoes, devastate soybean, rot cacao, and are the cause of sudden oak death. Understanding this versatile genus will be key to its control Phytophthora ramorum, it took less than four years to produce a draft sequence of its genome, one of the fastest-ever discovery-to-sequence stories for a complex pathogen. This achievement was a United States initiative, facilitated by the injection of federal and state funding into Phytophthora research. But the US is not alone in the battle against this genus. Phytophthora species cause thousands of millions of dollars of damage to the world's commercial crops every year: they blight potatoes and tomatoes, devastate the lucrative soybean, and rot cacao, threatening the world's supply of chocolate  in tens of thousands of oak trees across the coastal counties of California, is now present in at least three other US states and is threatening to take on the native flora of the entire North American continent. It is also lurking in Europe, although apparently with less devastating consequences.The most infamous of the Phytophthora. This year, the US federal government will channel US$7,400,000 toward research into SOD. A major focus of this funding is genomics. Sequencing the genomes of several Phytophthora will help clarify the phylogeny and evolution of these enigmatic organisms , it's about twice the size of the Arabidopsis genome. His latest research, published in the Journal ofBiological Chemistry (P. infestans protease inhibitor—extracellular protease inhibitor 1 (EPI1)—that could be one of a unique class of suppressor proteins that Phytophthora deploy to infect and counteract host defences. The pathogen seems to upregulate the epi1 gene during colonisation of its host. EPI1 inhibits plant apoplastic proteases—extracellular enzymes that are part of the host's defensive armoury that have evolved to prevent foreign proteins entering cells.The hemistry , describP. infestans colonisation of host apoplast,’ Kamoun and his colleagues report  and the US Department of Energy's Joint Genome Project based in Walnut Creek, California. The focus here is the soybean pathogen P. sojae and the SOD pathogen P. ramorum, for which draft sequences are now complete (www.jgi.doe.gov/). The genomes are much smaller than that of the 250-Mb P. infestans—P. sojae is about 90 Mb and P. ramorum is just 55 Mb. This, in part, explains the comparative speed of these sequencing efforts. But another factor is undeniably the fear of the unknown P. ramorum, which in 2002 netted the Virginia–California initiative US$3,800,000 in federal funding to describe its genome. Ultimately, however, the sequence of one species will help to inform on the sequence of other related species.Even more advanced than the P. sojae to infect its host. He agrees with Kamoun that understanding this machinery is the way to devise new control measures that could give plants the upper hand in the evolutionary arms race against their Phytophthora pests. At present, most strategies to limit the damage caused by species like P. infestans and P. sojae rely on developing resistant cultivars by selective breeding of varieties with major resistance genes—single genes that can block a pathogen. However, Phytophthora seem able to find ways to overcome these efforts. ‘P. infestans is absolutely notorious for its ability to genetically change in response to a major resistance gene,’ says Tyler. ‘Typically major resistance genes in potato barely last a single season.’Brett Tyler, Research Professor at the Virginia Bioinformatics Institute, is focusing on the molecular tools used by P. sojae for five to ten years. However, this resistance is starting to break down, and breeders are running out of major resistance genes with which to conjure new varieties. The alternative, says Tyler, is quantitative or multigenic resistance, which relies on getting plants to express several resistance genes at once, each of which makes a small contribution to the plant's overall resistance. It should be much harder for P. sojae to evolve a new attack against this kind of robust defence. The search is also on for new genes that could be used to encourage quantitative resistance to host species. One particularly promising approach is to pit pathogen against host to see which genes are switched on. The host should upregulate genes that defend it against infection and the pathogen should upregulate genes that it needs to attack. The discovery that plants produce proteases and that Phytophthora have responded by secreting protease inhibitors to disable them is important if long-lasting solutions are to be found. ‘We just need to find a way to introduce new protein-degrading enzymes into the plant that the pathogen doesn't know how to block,’ says Tyler. ‘With these genomic tools we can really accelerate the pace at which we can evaluate different possible protective measures.’Things look better for the soybean. New cultivars containing major resistance genes show resilience to Phytophthora can be mistaken for a fungus, so DNA profiling of isolates is crucial if species and strains of species are to be identified correctly so that action appropriate to each infection can be taken. Since 2000, Matteo Garbelotto, Adjunct Professor of Mycology and Forest Pathology at the University of California at Berkeley , has spent a significant part of his working life tracking the spread of P. ramorum, the Phytophthora that has killed off vast tracts of oak trees in native Californian forest  and viburnum (Viburnum spp.), which seem to act as carriers for the pathogen. ‘What we're seeing is a parallel to what has been happening throughout Europe, where the infection has basically moved using the commercial routes of the ornamental plant industry,’ Garbelotto says.DNA analysis has been crucial to confirm suspected cases of P. ramorum does not appear to have the same devastating consequences in Europe as it does in the US—at least not yet. It's not entirely clear why, but it could have something to do with the structure of the bark of different host species, suggests Garbelotto. ‘We normally see more infection where we have more corrugation, and that's because water accumulates in the fissures … where the zoospores have a chance to infect the bark.’ However, he notes, European beech (Fagus sylvatica) appears to be extremely susceptible to P. ramorum. ‘If it reaches areas where there are a lot of beeches, it could potentially mirror what's happening in California,’ he warns.P. ramorum infection were first described on rhododendrons in The Netherlands in 1993. Once this was confirmed to be the same species as the pathogen responsible for Californian SOD, there was speculation that P. ramorum had either been introduced to the US from Europe or vice versa. However, in December 2002, it emerged that these two populations are of different mating types—A1 in Europe and A2 in the US. The latest research from Garbelotto and his colleagues, due to be published in Mycological Research (Camellia spp.) and rhododendrons in a nursery in Washington state showed that the A1 and A2 strains were living side by side. ‘That was a big surprise for us,’ recalls Garbelotto. ‘We had no knowledge at that point that both the European and the North American type could be present in the same nursery.’In Europe, symptoms characteristic of Research , supportPhytophthora can produce a new species with different properties from those of either parent. One of the best cases comes from Europe, where a new Phytophthora emerged in 1993 that began to attack alder trees (Alnus spp.). Research carried out by scientists in the United Kingdom demonstrated that the alder Phytophthora was a product of a hybridisation event between P. cambivora and an unknown species similar to P. fragariae, neither of which attacks alder.Hybridisation between different species of Phytophthora species to hybridise and new phenotypes to emerge, there is legitimate concern that sexual recombination between the A1 and A2 mating types could produce something more devastating than either form. Within the controlled confines of his laboratory, Garbelotto has been exploring whether the two types of P. ramorum can get it together. Initial findings are that oospores—the product of sexual recombination—are being produced, although most of them abort before they reach maturity. However, 30% progress further, and  look like they could be functional. ‘They'll germinate,’ he predicts.Given this propensity for Phytophthora was isolated from two sites in England. Although the new species—currently referred to as Phytophthora taxon C sp. nov. (P. taxon C)— appears to cause relatively mild damage to its beech and rhododendron hosts, the UK's Department for Environment, Food and Rural Affairs (DEFRA) acknowledges that hybridisation of P. taxon C with P. ramorum could have serious consequences. ‘The potential for the pathogen to adapt further to its putative new environment intrinsically or via hybridisation is not known,’ note the authors of a DEFRA report on the mystery species (www.defra.gov.uk/planth/pra/forest.pdf). ‘Long-distance spread could easily occur through the movement of infected stock of rhododendron or beech and possibly other (as yet unknown) hosts,’ they warn.The threat that hybridisation could create a novel strain with a different host range is a concern that the UK is also taking seriously. In December 2003, an entirely new P. taxon C and P. ramorum are only distantly related, making hybridisation unlikely, says Joan Webber, Head of Pathology at the UK government's Forestry Commission. The closest known relative of P. taxon C is P. boehmeriae, a pathogen that has been recorded on several species of tree in China and Australia, and on cotton in China and Greece, suggesting possible origins for the newly described species. But, says Webber, the sequence match between P. taxon C and P. boehmeriae is only 92%—not especially close. Much more evidence is needed to build a strong case for the origin of this new Phytophthora, she says.DEFRA is monitoring the situation closely . HoweverP. infestans strain that caused the Irish potato famine. Jean Beagle Ristaino of North Carolina State University  is due to publish in Mycological Research an analysis of DNA extracted from diseased potato plants preserved from the nineteenth-century Irish epidemic  and forest dieback (P. cinnamomi), the chlamydospores play a crucial role, allowing the pathogen to survive from season to season. The formation of sexual structures is relatively rare, but oospores—the product of sexual recombination—occur in many species  and the true oaks coast live oak (Quercus agrifolia), California black oak (Quercus kellogii) and Shreve's oak (Quercus parvula var. shrevei). These species develop large wounds or cankers in their woody tissue, which disrupt physiology and in extreme cases lead to death in a matter of months.In 1995, trees in oak forests in the coastal counties of California started to show a range of alarming and lethal symptoms. Since then, the disease has reached epidemic proportions, with tens of thousands of trees dying along approximately 300 km of the central Californian coastline . By 2000P. ramorum does not appear to cause the same damage, leaving hosts like the Californian bay laurel  and rhododendrons (Rhododendron spp.) with the relatively mild symptoms of leaf blight and occasional branch dieback. However, these non-oak hosts probably act as reservoirs of disease and may even help it to spread. The sporangia and chlamydospores that are thought to be the main asexual propagules of the pathogen (In non-oak species, pathogen  are readP. ramorum was found at a large wholesale horticultural nursery in Los Angeles County. The California Department of Food and Agriculture, the US Department of Agriculture's Animal and Plant Health Inspection Service, and state agriculture departments around the country are tracing all plants shipped from this nursery over the past year in an effort to identify and destroy any infected material, and hence prevent any further spread of the disease. However, the rot has already set in at nurseries in three states outside California: Oregon, Washington, and Florida. A National SOD Nursery Survey is underway to try to assess how far the disease might have spread.In March 2004,


The exploitation of information extraction (IE), a technology aiming to provide instances of structured representations from free-form text, has been rapidly growing within the molecular biology (MB) research community to keep track of the latest results reported in literature. IE systems have traditionally used shallow syntactic patterns for matching facts in sentences but such approaches appear inadequate to achieve high accuracy in MB event extraction due to complex sentence structure. A consensus in the IE community is emerging on the necessity for exploiting deeper knowledge structures such as through the relations between a verb and its arguments shown by predicate-argument structure (PAS). PAS is of interest as structures typically correspond to events of interest and their participating entities. For this to be realized within IE a key knowledge component is the definition of PAS frames. PAS frames for non-technical domains such as newswire are already being constructed in several projects such as PropBank, VerbNet, and FrameNet. Knowledge from PAS should enable more accurate applications in several areas where sentence understanding is required like machine translation and text summarization. In this article, we explore the need to adapt PAS for the MB domain and specify PAS frames to support IE, as well as outlining the major issues that require consideration in their construction.PASBio by extending a model based on PropBank to the MB domain. The hypothesis we explore is that PAS holds the key for understanding relationships describing the roles of genes and gene products in mediating their biological functions. We chose predicates describing gene expression, molecular interactions and signal transduction events with the aim of covering a number of research areas in MB. Analysis was performed on sentences containing a set of verbal predicates from MEDLINE and full text journals. Results confirm the necessity to analyze PAS specifically for MB domain.We introduce PASBio contains the analyzed PAS of over 30 verbs, publicly available on the Internet for use in advanced applications. In the future we aim to expand the knowledge base to cover more verbs and the nominal form of each predicate.At present  We are now in an era where full genomes, data from high throughput experimental methods (e.g. micro-arrays) and electronic versions of scientific literature are easily available to every researcher over the Internet. These advances have made it possible to work on more than one gene at a time, ask complex questions and increase the pace of biological discovery. However, the progress made in scientific research until now has been recorded in the form of free-text articles readable only by humans and accessible by machine mostly through shallow keyword-based search engines. For improved methods of information access and knowledge discovery it is necessary to automatically map from the unstructured text representation into partially structured forms that provide discovered facts to databases.The large-scale data generated from the experiments in molecular biology needs to be assessed and integrated into the scientific communities' knowledge stores. This has created a need for various kinds of specialized databases. While some existing databases contain only molecular level information  othersPrevious research in IE for biology has focused intensively on the recognition of named entities (NE) from scientific texts -9, i.e. catalyst and reaction being catalyzed in the case of the first and second arguments to the predicate catalyze. Alternatively, functional roles can be employed such as thematic relations that try to express some linguistically motivated aspect of the argument's behavior such as agent, location or experiencer.Predicate-argument structure (PAS) analysis seeks to formally describe 'frames' for predicates  and the roles of their arguments (parts of the sentence surrounding it). Such roles usually need to be specified according to several factors including meaning and obligation. Meaning can be determined in several ways such as a domain or predicate-specific fashion such as Traditional IE systems that use regular expressions based on shallow chunking at the phrase level  capture weak notions of 'argument' for event predicates and their linear precedence. Such approaches seem to be inadequate to the goal of achieving high completeness and accuracy in event extraction. In recognition of this several major projects -24 have For PAS to be practically realized within IE three major knowledge components will be required: (1) a hierarchy of concept categories for objects of interest; (2) a definition of predicate-argument frames and the semantic labels of their arguments; and (3) the mapping rules that define how to transform the relevant parts of a surface sentence to the arguments in the PAS frame. Currently (1) is already quite advanced with several controlled vocabularies such as MeSH  or Gene In this work we introduce the concept of semantic analysis of argument roles in biological texts and propose the construction of PAS for molecular biology (PASBio). We have analyzed and annotated sentences from MEDLINE abstracts and full-text journal articles for building PASBio. The working scheme is similar to the PropBank project ,23. ResuIn this article we first give a short introduction to IE and PAS. Next, we describe the approach taken in the PropBank project. Then, we discuss and exemplify how the specification of predicate-argument frames needs to be extended to meet the requirements for extracting molecular events. The second half of the paper is devoted to explaining the methodology used to define the PAS and discussing results of our analysis and its comparison with those of PropBank. Finally, we describe how the PAS frames can be exploited by showing their place in the IE system for molecular biology and discussing existing IE systems used for event extraction in molecular biology.company outlook) then patterns such as "np (stock index) + vp (driven up) + integer (number %)" and "np (company) + vp (bid) + np (stock)" could be developed as a template. Sentences in documents which (1) contain a noun phrase (np) describing stock index, together with a verb phrase (vp) driven up, and followed by a number; or (2) contain a noun phrase representing a company name, followed by a verb phrase with bid, plus a noun phrase of stock index should be extracted. The difficulties are compounded because a single event can nearly always be written in a variety of syntactic forms due to linguistic processes such as passive voice, (pro-) nominalization, raising, etc.IE systems aim to provide instances of structured knowledge representations from unstructured free-form text. IE, based on the Message Understanding Conference (MUC) tradition of task segmentation  works fuspray alternation by Levin  blocked [MaryArg1] from [completing her dissertationArg2] with [his constant pesteringArg3]." is annotated by PropBank's PAS frame. An argument Arg2-secondary predication is annotated for "completing her dissertation" because this contains in itself the PAS of the verb complete. From this PropBank example, the meaning of the event denoted by block cannot be completely understood if the sentence just states as " [JohnArg0] blocked [MaryArg1]." as it is necessary to mention the action being stopped. In contrast in the biology domain, by mentioning only the entity being stopped (Sentence 3.1–3.3), the expert reader can understand that the event which applies to that entity is being stopped without providing an explanation of the action being stopped at the position of secondary predication. Similarly, an instrument used to block is encoded in the nature of an agent or causer. The structure of block and its examples are given in Figure MAN is used to label "specifically" as this adverb plays the role of a manner modifier.Verb generate is similar to that of block. Only Arg0-agent and Arg1-entity created are expressed in all observed sentences from our biology corpus.In Figure Verbs in this group have been used in biological documents with the same semantic sense as in PropBank. Moreover, their usage in both the biology corpus and PropBank indicates that their PAS frames are identical. Specialization of domain does not seem to affect verbs in this group.confer and lead. In both biology and newswire corpora, confer is used with semantic "to give (as a property or characteristic) to someone or something" and lead to is used in the sense of "to tend toward or have a result".In Figure express and transform are presented in Figures Verbs in this group have been used in biology documents with a different semantic sense compared to PropBank, or PAS frames for them are not found in PropBank. More than one semantic sense is found in our corpus for some verbs. PAS frames for express is used in the biology domain with the meaning "to manifest the existence of a gene or gene product" (or detection of the same by the experimenter) unlike its normal usage with the meaning of "give an opinion or send quickly". The PAS of express is given as Figure The verb transform, two senses are used in biology papers: "to cause (a cell) to undergo genetic (or neoplasmic) transformation" as shown in Figure transform found in our corpus is similar to the sense of "change" found by PropBank, there is still a huge gap between them. In the biological literature, illustrated by examples in sentences 8.1–8.3, this genetic transformation mentions only the agent or causer, what entity is getting transformed, and what will be the effect after transformation. It will not mention the start state of the entity undergoing transformation because it is known from the expert reader's domain 'common sense' knowledge that the start state refers to a normal condition of that entity. Transform in the second sense always occurs in a sentence connected by preposition into, and in the passive voice form in which no mention is made with regard to the agent.In the case of In the discussion so far we have assumed that the predicate is the center of semantic information. Here we intend to show that the argument contents can change the event description specified by the predicate, by examining sentences that describe an 'alternative splicing' event. Alternative splicing is used to generate multiple transcripts from a single gene and hence is a helpful event for increasing the functional complexity of eukaryotic systems.express: (a) "Northern blot analysis with mRNA from eight different human tissues demonstrated that [the enzymeArg1] was expressed exclusively [in brainArg3], [with two mRNA isoforms of 2.4 and 4.0 kbArg2]." and (b) "[A complementary DNA cloneArg1] encoding the large subunit of the essential mammalian pre-messenger RNA splicing component 2 snRNP auxiliary factor(U2AF65) has been isolated and expressed [in vitroArg3]." Sentence (a) is considered as a sentence denoting the alternative splicing event but sentence (b) is considered as a negative  sentence, which talks about expression of an mRNA of a splicing factor.Consider the following example of a set of sentences that talk about the 'expression' of a single type of mature mRNA generated from 'splicing' of pre-mRNA and generation (and expression) of multiple mature mRNA transcripts with different properties from the single type of pre-mRNA. Sentences annotated follow PASBio's frame for It would be difficult, based on word contents or regular expression methods, to put these two examples into different 'bins' for alternative splicing events. But the discussion about the length of the two different transcripts in Arg2 (with two mRNA isoforms of 2.4 and 4.0 kb) in the first sentence can be helpful to understand it as a sentence discussing about alternative splicing. On the other hand, the later sentence contains all the interesting words  but misses Arg2, hence describes just an expression event.Construction of PAS frames by expert introspection may be considered as a time-consuming process, however domain-specific PAS frame definitions have valuable uses in several applications as discussed below.Each PAS frame in PASBio provides a set of semantic relationships between arguments participating in an event and a verb conveying the event. Although we focus on applying PASBio for event extraction in the molecular biology domain, information processing applications that require semantic understanding of a sentence will be able to take advantage of this knowledge. For example, machine translation (MT) requires encoding a surface sentence of a source language into a language independent logical form of clause meaning, and then generating from this logical representation a surface sentence in a target language. PAS would be one practical choice to be used as such a logical representation in MT ,30. In tWith respect to our molecular event extraction system, as we discussed in the introduction, PASBio takes on the role of a reference source in the stage of corpus annotation for creating training examples for machine learning. The planned IE system is composed of 4 activities: (1) construction of PASBio semantic lexicon; (2) annotation of full-text journal in terms of semantic represented in PASBio's frames; (3) construction of the module for automatically transforming an unseen sentence into a logical form of semantic relationships drawn within each particular PASBio frame; (4) integration of the resultant automatic semantic interpretation module within the event extraction system. So far, manual annotation and machine learning have not been completed yet and will be reported elsewhere. For a description of an IE system that can make use of such an annotated corpus we refer readers to the work of for example Surdeanu et al.  who usesApart from our corpus-based semantic interpretation approach, several other research groups have proposed systems for event extraction from the biological literature, especially about the interaction information between genes and genes product. Related work so far can be summarized into two sets. The first set of methods use regular expressions and rely on syntactic patterns. These methods may use statistical models of the surface words ,13, ruleThe only work in the second set, that has taken into account a large number of linguistic and deeper semantic aspects is, that of Novichkova et al. . The appAs mentioned above, most of the approaches, whether a deep notion of predicate-argument relations is taken  or a shaRecently, another research group  reportedWith the explosion of molecular data, tools developed by computer scientists are gradually being applied and integrated in the domain of biology to aid in information access and knowledge discovery. Text data appearing as reports about biological discoveries demands automated IE methods for faster knowledge discovery. Traditional IE systems that use regular expressions based on shallow chunking at the phrase level  capture weak notions of 'argument' for event predicates and their linear precedence. Such approaches seem to be inadequate to the goal of achieving high accuracy in event extraction in molecular biology. PAS which is used as a representation of the semantic relationship between a verb and its arguments participating in the event has the potential to support deep knowledge acquisition from a sentence within the extended system framework that is now being proposed within the IE community.Due to the importance of PAS and the lack of a specific PAS frame resource for the domain of molecular biology, we have proposed the analysis of PAS for molecular biology in this article. We have analyzed sentences for 30 verbs (and different frames per senses of the verb) from MEDLINE abstracts and full-text journal articles where the sentences contain each verb in its verbal form and its participial modified form for building PASBio. Our analysis suggests in some cases a significant difference in the predicate frames compared to those obtained from analyzing news articles by the PropBank project. In addition to the significance of verb senses used in the molecular biology domain, syntactic constructions also differ markedly; such as the use of passives allowing the semantic subject to be omitted where they are part of the common-sense understanding in the domain. Human readers are required to have domain knowledge in order to understand the texts. Our result frames and examples are available to the wider research community as a knowledge base at PASBio's webpage.In the future, we intend to utilize knowledge from the PASBio frames for annotating a corpus to be used as training examples to achieve automatic annotation of PAS semantics into sentences. Furthermore, we aim to complete analyzing PAS for more verbs related to molecular events and afterwards to extend our analysis to sentences containing the nominal forms of verbs.The English language used in research articles of biological and biomedical sciences is a sublanguage of written natural language. While most of its vocabulary is similar to that of general English, some verbs are domain-specific in nature. Our main focus here is the verbs that are used for describing molecular events in biology. Various researchers have different areas of interest and new concepts are added in the literature continuously. However, the areas of cellular signaling, gene expression, regulation and disruption of expression events are very important for the larger community of investigators involved in basic biomedical research and those involved in high throughput analysis. They are discussed throughout different parts of papers as possible cause of normal and disease states of different organisms. Hence, ignoring the normal distribution (frequency) of different verbs in the literature we choose the verbs from those involved in the above-mentioned processes (events). Most of the verbs are shown in Figure Information extraction work is still largely carried out using PubMed abstracts. Using abstracts is advantageous because they contain the highest density of keywords compared to other section of research articles but our intuition is that bio-text mining should scale-up to analyze full journal articles where the most detailed results are contained along with supporting evidence, comparisons to others work and background information, etc.  Recent iSentences were carefully chosen to cover a broad usage of each verb under study from the MEDLINE and full text journal corpora as described before. We tried to choose equal numbers of sentences containing a particular verb in its verbal format and its participial modifier format. Before starting an analysis on each sentence, a sentence was parsed using Connexor Parser  that useThis work was directed by NC. TW carried out the analysis of the predicate-argument structures with technical support from NC and biological knowledge from PKS. PKS chose the predicates and the sentences analyzed from the MedLine corpus. Sentences from other corpuses were complemented by TW. TW prepared the figures (except fig


While the molecular pathway that mediates cellular intake of iron through the TfR is known, it was not clear just how TfR assists in iron release to the cell and how it binds HFE and transferrin. By introducing multiple mutations in human TfRs, Pamela Bjorkman and colleagues identified functional binding sites for transferrin in both its iron-loaded and iron-free (apo-Tf) forms and for HFE. From these data, the researchers mapped out a scenario of the dynamic interactions between receptor and ligands (the bound molecule) and worked out a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.Iron is an essential nutrient for sustaining life-forms as diverse as plankton and humans. But too much iron, or too little, can spell trouble. Mammalian cells maintain the proper balance partly with the help of a specialized cell surface protein called the transferrin receptor (TfR). TfRs bind to the iron-carrying transferrin protein (Fe-Tf) and escort their cargo to the cell's interior.  that support Fe-Tf but not apo-Tf binding, confirming that the receptor binding footprints of the two metal-binding states of transferrin are indeed different. With a structural model showing By suggesting a mechanism through which TfR binding regulates iron release, this structural model of the transferrin–TfR complex will bolster efforts to elucidate the molecular details of this process. Confirmation that transferrin and HFE do indeed compete for docking privileges reveals a possible role for HFE in maintaining iron homeostasis and will provide valuable insights into the dysregulation that leads to the warehousing of iron and resulting tissue and organ damage associated with hemochromatosis.


XRCC1 Arg399Gln, XRCC3 Thr241Met and XPD Lys751Gln, and investigate their role as susceptibility markers for colorectal cancer.Recent studies relating to the association between DNA repair-gene polymorphisms and colorectal cancer risk would, to the best of our knowledge, appear to be very limited. This study was designed to examine the polymorphisms associated with three DNA repair genes, namely: XRCC1, XRCC3 and XPD) in the context of colorectal cancer risk for the Taiwanese population. Genomic DNA isolated from 10 ml whole blood was used to genotype XRCC1 Arg399Gln, XRCC3 Thr241Met and XPD Lys751Gln by means of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis.We conducted a case-control study including 727 cases of cancer and 736 hospital-based age- and sex-matched healthy controls to examine the role of genetic polymorphisms of three DNA-repair genes , the XRCC3 241Thr/Thr genotype  or the XPD 751Gln allele , although individuals featuring a greater number of risk genotypes (genotype with OR greater than 1) did experience a higher risk for colorectal cancer when compared to those who didn't feature any risk genotypes (Trend test P = 0.03). Compared with those individuals who didn't express any putative risk genotypes, individuals featuring all of the putative risk genotypes did experience a significantly greater cancer risk , particularly for individuals suffering tumors located in the rectum  and diagnosed prior to the age of 60 years .The risk for colorectal cancer did not appear to differ significantly amongst individuals featuring the Our results suggest that DNA-repair pathways may simultaneously modulate the risk of colorectal cancer for the Taiwanese population, and, particularly for rectal cancer and younger patients. These cA damage . The intA damage . Since tA damage -6, the pA damage -11.XPD gene encodes a helicase that is a component of the transcription factor TFIIH [XPD 751Gln/Gln did demonstrate suboptimal DNA-repair capacity (DRC) in regard to its ability to remove UV photoproducts when compared to the XPD 751Lys/Lys and Lys/Gln genotypes. The XRCC1 protein is a scaffolding protein directly associated with polymerase beta, DNA ligase III and poly (ADP-ribose) polymerase (PARP) and functions in a complex to facilitate the base-excision repair (BER) and single-strand break-repair processes [XRCC1 399Gln allele were associated, more significantly, with higher levels of both aflatoxin B1-DNA adducts and glycophorin A variants when compared to individuals who exhibited the Arg/Arg genotype [Amongst the known genetic polymorphisms of the DNA-repair genes , the xeror TFIIH , this faor TFIIH . In 2002or TFIIH  reportedrocesses -19. In 1genotype . XRCC3 pXRCC1 399Gln allele, compared to the XRCC1 399Arg/Arg genotype, was associated with an increased risk for developing colorectal cancer, especially amongst young urban residents, although in 2003, Mort et al. [XPD, XPF, XPG, ERCC1) or XRCC1. In the present paper, we conducted a hospital-based case-controlled study to examine the role of genetic polymorphisms of three DNA-repair genes  in the context of colorectal cancer risk for the Taiwanese population.To the best of our knowledge, studies pertaining to these DNA-repair genes focusing on colorectal cancer risk would appear to be limited and controversial. In 2000, Abdel-Rahman et al.  observedt et al.  failed tn = 776) participating in this study were newly diagnosed and histologically confirmed. Patients suffering from familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, or inflammatory bowel disease and other related malignancies were excluded from study participation. Seven hundred and twenty-seven of the original 776 cases (94%) were finally included in this study. Seven hundred and forty-seven age (same age) and sex-matched controls were recruited from the Physical Check-Up Department during the same period. All the participating controls had received comprehensive health examinations including colonoscopies. After excluding individuals diagnosed with other colorectal diseases, a history of other cancers or the existence of a family history of colorectal cancer, 736 controls  were finally included in this study. With informed consent, the socio-demographic characteristics of study participants were ascertained by means of the application of a structured questionnaire, at which time 10 ml of venous blood was collected.Detailed descriptions of the specific characteristics of the study participants have been published previously . In brieXRCC1 Arg399Gln, XRCC3 Thr241Met and XPD Lys751Gln by means of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. All of the PCR reactions were carried out by a Mastercycler gradient thermocycler  in a final volume of 25 ul containing 200 ng of each primer, 50 ng genomic DNA, 1.5 mM MgCl2, 200 ul dNTPs and 1.0 unit of Taq DNA Polymerase in the buffer provided by the manufacturer. In addition, all laboratory genotyping personnel were blind to the case-control status of the samples.Genomic DNA isolated from 10 ml whole blood was used to genotype XRCC1 PCR products were amplified with the primers 5'-TTGTGCTTTCTCTGTGTCCA-3' (sense) and 5'-TCCTCCAGCCTTTTCTGATA-3' (antisense) and digested with MspI . The Arg allele revealed 374 and 221 bp fragments following digestion and polyacrylamide gel electrophoresis, whilst the Gln allele was not digested by MspI [XRCC3 PCR products were amplified with the primers 5'-GCCTGGTGGTCATCGACTC-3' (sense) and 5'-ACAGGGCTCTGGAAGGCACTGCTCAGCTCACGCACC-3' (antisense) and digested with NcoI (New England BioLabs). The Met allele revealed 97 and 39 bp fragments following digestion and polyacrylamide gel electrophoresis, while the Thr allele was not digested by NcoI [XPD PCR products were amplified with the primers 5'-CCTCTCCCTTTCCTCTGTTC-3' (sense) and 5'-CAGGTGAGGGGGACATCT-3' (antisense) and digested with PstI . The Gln allele revealed 646 and 88 bp fragments following digestion and polyacrylamide gel electrophoresis, whilst the Lys allele was not able to be digested by PstI [The 615 bp  by NcoI . The 734 by PstI .t-test. Multivariate unconditional logistic regressions were used to examine the association between the XRCC1, XRCC3 and XPD genotypes and the risk for colorectal cancer. Since the differences in the estimated risks between conditional logistical regression and unconditional logistical regression were small, unconditional logistical regression was used to estimate odds ratio (OR) and 95% confidence interval (CI) with the matching factors (age and gender) included in the model for estimation [Tests for Hardy-Weinberg equilibrium amongst controls were conducted using observed genotype frequencies and a chi-square test featuring one degree of freedom. Baseline sociodemographic characteristics between cases and controls were analyzed using the chi-square test and two-sample Students' timation . Other pXRCC1, XRCC3 and XPD genes on the risk for the colorectal cancer. Since the associations between polymorphisms of DNA-repair gene, cancer susceptibility and DNA repair capacity are inconsistent [Based on the multiplicative scale, the likelihood ratio test was further used to evaluate the interaction between nsistent , we defiMore men (56%) than women participated in this study Table . The meaXRCC1 399Gln, XRCC3 241Met and XPD 751Gln allele amongst the controls were, respectively, 0.27, 0.05 and 0.07, these genotype frequencies being in Hardy-Weinberg equilibrium. Due to the relatively low frequencies of variant alleles for these genes in the present study, any genotype featuring one or more variant alleles was combined for further analyses. The risk for colorectal cancer was not significantly different for individuals featuring the XRCC1 399Arg/Arg genotype , the XRCC3 241Thr/Thr genotype  or the XPD 751Gln allele .The genotypic distributions of the three DNA-repair genes for both cancer cases and controls are shown in Table P = 0.03). Subjects who demonstrated two or three of the putative risk genotypes did reveal a significantly greater risk for colorectal cancer  as compared to those individuals who did not feature any putative risk genotypes, although it appears that no gene-gene interactions arose amongst these three genes . When stratified by tumor site and age at diagnosis, these combined gene effects upon cancer risk were observed for individuals who revealed that their tumor was located in the rectum (Trend test P = 0.03) and those individuals for whom their tumor was diagnosed prior to their being 60 years of age . In 2003, Mort et al. [XRCC3 241Thr allele  and only slightly increased for those individuals who revealed the XRCC1 399Gln and XPD 751Gln alleles. In 2001, Park et al. [XPD 751Gln/Gln genotype featured a poorer (positive) response rate to chemotherapy and also a shorter survival period compared with colorectal cancer-suffering individuals who belonged to either the 751Lys/Lys or the 751Lys/Gln group.Although we didn't find any significant independent associations between these DNA-repair genes and colorectal cancer risk, the risk appeared to be slightly increased for individuals who featured the  reports ,22, the k et al.  reportedXRCC1 399Gln, XRCC3 241Met and XPD 751Gln alleles amongst the healthy controls in this study  were similar to the results for other studies conducted in Taiwan [XRCC1 399Gln, 0.45 for XRCC3 241Met and 0.36 for XPD 751Gln) for a British population. On the other hand, the XRCC1 399Gln allele frequency observed in the present study is greater than that observed by Abdel-Rahman et al. in 2000 (0.14 for XRCC1 399Gln) [XRCC1 399Gln allele [The apparent divergence between these studies and ours might be due to one of two reasons. Firstly, it may simply be that ethnic differences in allele frequency for the polymorphism might explain the controversial findings. The frequencies for the n Taiwan ,29 and Cn Taiwan ,31, but n Taiwan  (0.42 fo 399Gln) . Abdel-Rn allele . TherefoXRCC1 Arg399Gln, XRCC3 Thr241Met and XPD Lys751Gln genes in regard to colorectal cancer risk were observed in the present study. With the complexity of detail of environmental exposures to various carcinogens, it is plausible that the effective repair of DNA damaged by chemical mixtures necessitates multiple DNA-repair pathways . The failure of, or the presence of deficient DNA repair capacity for each specific DNA-repair pathway may contribute to an increase in cancer risk. Additive or multiplicative effects of combined genetic variants for different DNA-repair pathways have been previously reported for lung cancer [XRCC1 and XPD genes. Hu et al. [APE1 and XRCC1 genes that were present. It would therefore appear reasonable to hypothesize that genetic polymorphism(s) for DNA-repair genes may simultaneously contribute to colorectal cancer susceptibility.Combined effects of polymorphisms of the g cancer , melanomg cancer  and breag cancer . In 2003g cancer  found thu et al.  also obsXRCC1 399Gln allele appeared to be significantly associated with detectable DNA adducts in their blood mononuclear cells when compared to younger subjects who featured the 399Arg/Arg genotype. In 2001, Hemminki et al. [XPD 751Gln/Gln genotype exhibited a decreased DNA-repair rate for, specifically, UV-specific cyclobutane pyrimidine dimers in the skin. Therefore, it is possible that individuals who demonstrate a less-efficient DNA-repair capacity might develop tumors at a younger age than individuals who reveal an efficient DNA-repair capacity.In this study, we found that the combined effect of multiple DNA-repair genes upon colorectal cancer risk was significant for our younger age group, but not so for the older age group. This finding appears to be similar to the results of most of the previous studies pertaining to colorectal cancer , basal-ci et al.  also fouIn our study, we also found that the combined genetic effect of these three DNA-repair genes in regard to cancer risk was more pronounced for the rectum than for the colon. This difference may reflect certain etiological differences between colon and rectal carcinogenesis. In fact, different epidemiological characteristics, etiology, pathogenesis and clinical behavior have been reported for different anatomical sites of colorectal cancer ,50, thisXRCC1, XRCC3 and XPD polymorphisms in relation to the risk of colorectal cancer for the Taiwanese population. Our results suggest that genetic polymorphisms of the XRCC1, XRCC3 and XPD genes, particularly in combination, may be associated with an individual's susceptibility to colorectal cancer. Acknowledging the relatively limited sample size in the subgroups for the low allelic frequencies, further studies incorporating a larger sample size and/or another ethnic population are needed to confirm the genetic role of DNA-repair mechanisms as regards colorectal cancer susceptibility.To the best of our knowledge, this study is the first to report on Our results suggest that DNA-repair pathways may simultaneously modulate the risk of colorectal cancer for the Taiwanese population, and, particularly for rectal cancer and younger patients.The author(s) declare that they have no competing interests.CCY carried out the genotyping analysis, performed the statistical analysis and drafted the manuscript. FCS participated in the design of the study. RT and CRCC participated in the design of the study and provided clinical biospecimens. LLH conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Australia and New Zealand Health Policy is a new journal which aims to promote debate and understanding about contemporary health policy developments in Australia and New Zealand. Although there are other international journals focussing on health policy, there are no Australian or New Zealand journals with this focus.Australia and New Zealand Health Policy is to focus on contemporary critiques and contemporary developments. Accordingly an e-journal format is particularly appropriate. Australian and New Zealand Health Policy is an open access journal which means that all articles will be freely and universally accessible online which, amongst other things, means that all articles will be freely and universally accessible online without any barriers to access, which increases their visibility.One of the aims of  Australia and New Zealand Health Policy a new journal which aims to promote debate and understanding about contemporary health policy developments in Australia and New Zealand. Health policy is regularly in the media and is a high profile issue at election times. In Australia the health system has been characterised by conflicts over values and policy choices over the decades. So pervasive is this conflict that Sax entitled his 1984 book about health services, "A Strife of Interests" [Australia and New Zealand Health Policy aims to provide a prestigious venue for analysis and critique of health policy in the two countries.Welcome to terests" ,3. HealtAlthough there are other international journals focussing on health policy, there are no Australian or New Zealand journals with this focus. Other related-area local journals are medical, public health or hospital-related. Although the local journals publish occasional policy articles, this area is not their principal interest, nor are they necessarily the journals which policy-oriented academics or policy practitioners scan to keep abreast of developments.Australia and New Zealand Health Policy will address this by publishing annual reviews of policy developments.The absence of a health policy journal serving Australia and New Zealand has long-term consequences for the development of systematic analysis of and research into health policy. One consequence is that there is no forum where health policy developments are documented and tracked, a lacuna which precludes cumulative analyses of trends. Australia and New Zealand Health Policy is to focus on contemporary critiques and contemporary developments, an e-journal format is particularly appropriate. Debate will also be stimulated by providing for 'Comment' on published articles, in a way analogous to a letters column.As one of the aims of Australia and New Zealand Health Policy is a peer reviewed journal. In keeping with its policy-applied focus, articles will be refereed by two referees, preferably one with a strong practitioner background, such as currently or recently employed in a policy role in a health authority, and one from an academic background. At least one of the referees will have substantive content knowledge relating to the article. Articles published in Australia and New Zealand Health Policy will be listed in PubMed and permanently archived in PubMed Central as well as certain other national archives.Australia and New Zealand Health Policy contributes to understanding of health policy development and practice with a particular focus on Australia and New Zealand. It welcomes submissions which:• Review and critique contemporary health policy issues;• Identify major trends in health policy and emerging policy issues, including new evidence about the effect of policy changes;• Identify impacts of health services research on new policies;• Identify major public policy and governance trends and their application to health policy;• Analyse contemporary health policy themes which cut across a range of policy areas;• Report on international policy developments and new international comparisons of health policy involving Australia and/or New Zealand; or• Critique contemporary health policy developments.Australia and New Zealand Health Policy is an Open Access journal, which means:• All articles will be freely and universally accessible online without any barriers to access, which increases their visibility.BioMed Central copyright and license agreement.• You and your peers will be free to print out copies of your article, email it to colleagues, and post it on the web because of the Open access journals are funded by article processing charges rather than journal subscriptions. The costs are therefore borne by the authors, their institutions of from their grants. That is, all access to journals is free to readers via the web . Authors from institutional supporters are exempt from authorship charges. The institutional supporters pay a sliding scale based on the number of staff and postgraduate students in biomedical sciences.Institutional subscription has a number of benefits. In addition to the direct benefits in terms of waived author fees, there are public policy benefits in supporting an open access journal regime such as Biomed Central. Open access journals are one mechanism for putting pressure on regular journal publishers to moderate their price increases.Unfortunately there are no New Zealand institutional subscribers to BioMed Central at present, which means that New Zealand authors will face article processing charges, although no article processing charges will be payable on manuscripts submitted in the first six months following the launch of the journal. Article processing charges are also usually regarded as a legitimate charge against research grants. In the medium term, alternative arrangements, such as institutional support, should be encouraged, although after this time the editor-in-chief will be able to grant a limited number of discretionary processing charge waivers.Australia and New Zealand Health Policy commences its publication program with a series of articles which describe and evaluate health policy developments in Australia in 2003. Responses or commentaries on these articles would be welcome. It is the aim of the Editorial Board to encourage a cluster of articles at the start of each year about health policy developments in Australia and New Zealand in the previous calendar year. Authors who have an interest in reviewing contemporary developments are encouraged to submit manuscripts on these themes early in the New Year so that the articles can contribute in a timely way to health policy debate in the two countries.S.J.DuckettEditor-in-Chief