The authors describe a powerful approach for discovering globally conserved regulatory elements between two genomes that does not require alignments. Its application to pairs of yeasts, worm, flies and mammals, yields a large number of known and novel putative regulatory elements, many of which show surprising conservation across large phylogenetic distances. We describe a powerful new approach for discovering globally conserved regulatory elements between two genomes. The method is fast, simple and comprehensive, without requiring alignments. Its application to pairs of yeasts, worms, flies and mammals yields a large number of known and novel putative regulatory elements. Many of these are validated by independent biological observations, have spatial and/or orientation biases, are co-conserved with other elements and show surprising conservation across large phylogenetic distances. One of the major challenges facing biology is to reconstruct the entire network of protein-DNA interactions within living cells. A large fraction of protein-DNA interactions corresponds to transcriptional regulators binding DNA in the neighborhood of protein-coding and RNA genes. By interacting with RNA polymerase or recruiting chromatin-modifying machinery, transcriptional regulators increase or decrease the transcription rate of these genes. Transcriptional regulators bind specific DNA sequences upstream, within or downstream of the genes they regulate, and a large number of experimental and computational studies are aimed at locating these sites and understanding their functions (for example ,2). The Here we describe a simple and efficient comparative approach for finding short noncoding DNA sequences that are globally conserved between two genomes, independently of their specific location within their respective promoter regions. Our method, which we call FastCompare, is based on a principle that we have termed 'network-level conservation' , accordiOur previous attempts at using network-level conservation relied on Gibbs sampling to find candidate regulatory elements . HoweverSaccharomyces cerevisiae and S. bayanus), worms (Caenorhabditis elegans and C. briggsae), flies (Drosophila melanogaster and D. pseudoobscura) and mammals (Homo sapiens and Mus musculus). For each phylogenetic group, we describe some of the most interesting, known and novel, predicted regulatory elements. For each of these regulatory elements, we perform independent validation using gene expression data, chromatin immunoprecipitation (IP) data, known motifs and data from several biological databases (Gene Ontology (GO)/MIPS, TRANSFAC), and show that the most globally conserved predicted regulatory elements are strongly supported by these independent sources.In the following sections, pairs of closely related species are termed phylogenetic groups. We applied FastCompare to the four following phylogenetic groups: yeasts . We chose to analyze 1 kb-long upstream regions, because most of the known transcription factor binding sites in S. cerevisiae are located within this range [The average nucleotide identity between tely 62%  (similartely 62% . The numis range ). On a tk-mers (with fixed k), and are relative measures of network-level conservation for these k-mers (the higher the conservation score, the more conserved the corresponding k-mer). We first describe the distribution of conservation scores for all 7-mers. As shown in Figure S. cerevisiae (see below for a detailed description of these sites). To verify that such high conservation scores could not be obtained by chance, we generated randomized sequences as described in Materials and methods and re-ran FastCompare on these sequences. The corresponding distribution of conservation scores is shown on Figure As described in Materials and methods, conservation scores are calculated for all k-mers with the highest conservation scores are likely to be functional. For a given k-mer, we define the 'conserved set' as the set of ORFs corresponding to the overlap between the two sets of orthologous ORFs containing at least one exact match to the k-mer in their upstream regions . We found that conserved sets defined for the highest-scoring 7-mers are significantly enriched with genes whose upstream regions contain occurrences of known motifs in yeast .Closer examination of Figure k-mers, we determine the optimal window within the initial 1 kb which maximizes the conservation score ; we then re-evaluate the functionality of each of the 398 k-mers with the independent biological information described above, using the new conserved sets. The full information for the 398 k-mers is available at [Then, for each of these 398 in vivo binding data, functional annotation and literature searches, we found at least 27 different known transcription factor binding sites among the 398 highest scoring k-mers. These regulatory elements, along with their support from independent biological data, are shown in Table k-mers, in the form of slightly distinct or overlapping sequences  are involved in regulating the yeast cell cycle. The other known sites are also involved in fundamental biological processes in yeast: amino-acid metabolism (Cbf1, Gcn4), meiosis (Ume6), rRNA transcription (PAC and RRPE), proteolytic degradation (Rpn4), stress response (Msn2/Msn4) and general activation/repression (Rap1, Reb1). As described in Materials and methods, our approach also handles gapped motifs. Thus, the binding sites for Abf1, a chromatin reorganizing transcription factor (CGTNNNNNNTGA), and Mcm1, a factor involved in cell-cycle regulation and pheromone response (CCCNNNNNGGA), were also identified as very high-scoring patterns and strongly supported by independent information (known motifs and chromatin immunoprecipitation).When we used the same independent biological data to evaluate the 400 highest-scoring 7-mers obtained on randomized data, we found only three known binding sites (RRPE, FKH1 and BAS1).k-mers, perhaps because their transcriptional network has undergone extensive rewiring since the speciation of the two yeasts, or because the corresponding transcription factors regulate few genes. In some cases, the presence of several known sites (clearly identified in terms of independent data) among the full set of 7-mers argues in favor of the rewiring hypothesis. For example, the binding site for the Rcs1 transcription factor, TGCACCC, only appears at the 1,883rd position within the list of ranked 7-mers. Despite its lack of conservation, this site is strongly backed by independent biological information: it is identified as a known motif, it is found in 33 microarray conditions, and its conserved set is significantly enriched in genes annotated with homeostasis of metal ions (p < 10-5), which is the known function for Rcs1 [k-mers.Several known binding sites are not found among the 398 top-scoring ions p < -5, whichk-mers and independently built the distribution of median distances to ATG for all 7-mers as described in Materials and methods  have their median distance below 350 (p < 10-52), while only seven have a median distance greater than 680. A closer examination reveals that a few known sites are particularly constrained. For example, the binding sites for Reb1, PAC, TATA, Swi4, Rpn4, RRPE and Mbp1 are found to be situated relatively close to the start of translation, with a median distance to ATG between 150 and 300 bp. Some of these constraints were also found to be good predictors of gene expression in a recent study [It is now known that functional regulatory elements can be positionally constrained, relative to other regulatory elements or to the start of transcription ,11,12. Tp-value < 10-7). However, neither of the two putative sites is supported by independent biological data. Additional expression data may help define their biological role. Other sites, such as CAGCCGC or GCGCCGC are found upstream of over- or underexpressed genes in many microarray conditions (15 and 6, respectively). While these two sites are similar to the canonical Ume6-binding site, the latter was not found in any microarray conditions , suggesting that the two sites are bound by other factors.We found many novel motifs among our highest-scoring predictions. For example, we found two strongly conserved motifs, AGGGTAA (rank 17) and TGTAAATA (rank 31), which are situated relatively close to ATG (with a median distance to ATG of 349 and 378.5 bp, respectively) and more often in upstream regions than in coding regions (with ratios of 1.95 and 1.83, respectively). Interestingly, TGTAAATA also has a statistically significant 5' to 3' orientation bias  and found 15 of the 27 known motifs we obtained when comparing S. cerevisiae and S. bayanus , is much less conserved between S. cerevisiae and S. castelli (rank 230). This argues for extensive rewiring in the Reb1 transcriptional network in the lineage that led to S. castelli.We repeated the same analysis on distinct pairs of yeast species other than lable at ). We alsvergence . We alsosults at ), althouD = 22 bp [D = 44.5) in S. cerevisiae. Indeed, it has been shown that the binding of Cbf1 in the vicinity of a very similar sequence (AAACTGTG) enhances the DNA-binding affinity of a Met4-Met28-Met31 complex for this sequence [To discover interactions between regulatory elements, we searched for co-conservation of pairs of high-scoring predicted regulatory elements, as described in Materials and methods. Not surprisingly, the most conserved interaction is between RRPE (AAAAATTTT) and PAC (CTCATCGC), with a median distance  = 22 bp ,13. We asequence , and thasequence .D = 38 and D = 63.5, respectively). The Reb1/RRPE interaction was also discovered independently as a good predictor of expression [D = 30). An interesting interaction between RRPE and an unknown motif, TGAAGAA, displays a conserved set strongly enriched in translation (p < 10-11), while RRPE alone is more strongly enriched in rRNA transcription (p < 10-14). The full sorted list of interactions is available at [Many of the predicted interactions have not yet been experimentally studied. For example, we found that the highest scoring Reb1 motif (CGGGTAA) is significantly co-conserved with both the highest scoring RRPE motif (AAAAATTTT) and the highest scoring PAC motif (CTCATCGC), with a short median distance between the two sites in both cases  using the procedure described in Materials and methods.The distribution of conservation scores for all 7-mers shows that high conservation scores are unlikely to be obtained by chance Figure . As showC. elegans and other metazoan organisms were identified among the 437 predicted regulatory elements.As shown in Table C. elegans, SKN-1 is known to initiate mesendodermal development by inducing expression of the GATA factors MED-1 and MED-2  [One of the most conserved is TGATAAG, the binding site for the GATA factors, a family of regulators controlling intestinal development (see  for revilineage) .C. elegans in at least two separate studies [D. melanogaster, and is assumed to create nucleosome-free regions of DNA, thus allowing additional transcription factors to bind those regions [C. elegans [The GAGA-factor binding site (AGAGAGA) was also found as a highly conserved pattern. GAGA repeats in upstream regions have been shown to be functional in  studies ,23. At l regions . However elegans .D. melanogaster and a C. elegans homolog has also been identified [C. elegans in response to insulin/insulin-like growth factor-1 signaling [We also found CAGCTGG, a site known to be bound by the myogenic basic helix-loop-helix (bHLH) family of transcription factors  and AP-4 transcription factors  ,26 (publentified . FastComentified ,31). DAFignaling .k-mers.Searching for gapped motifs found few strongly conserved sites. However, when searching for 8-mers with a 5-bp gap, we found that TGGCNNNNNGCCA, the known binding site for nuclear factor I (NFI) , had a sC. elegans sites returned by FastCompare and shown in Table Drosophila [Several of the osophila , mouse aosophila  for a rein vitro by the Drosophila homolog of Pbx-1, the extradenticle (exd) protein [C. elegans homolog of both Pbx-1 and exd. Other known sites discovered by FastCompare include CAGGTGA, similar to the known binding site for the Snail protein, a transcription factor involved in dorso-ventral pattern formation in Drosophila , and TTTTTCGCGC ). An E2Fgulation ,46.C. elegans appear to be constrained in terms of position. Using the distribution of median distances for all 7-mers , we found d0.025 = 690 and d0.975 = 1,135. Among the 437 highest-scoring k-mers, we found that 75 are located below the lower threshold, a proportion that is much higher than the expected 2.5% (p < 10-38). The binding sites for forkhead-related activator-4 (Freac-4), Sp1, E2F and AP-1 are particularly constrained , is found in the upstream regions of over- or underexpressed genes in 57 microarray conditions.As in yeast, several of the known binding sites in -90). This bias is confirmed by the fact that TCTCTCT alone (not taking into account its reverse complement) has a much higher conservation score (129.2) than AGAGAGA (34.3). We also found that several related motifs display a similar, albeit weaker, orientation bias, for example, GAAGAAG (p < 10-16), GGAGGAG (p < 10-10). It is interesting that all the GAGA repeats found to be necessary for correct expression of the ceh-24 and unc-54 genes are in fact TCTC repeats [p = 0.89). Interestingly, conserved GAGA repeats in D. melanogaster were also found to be constrained in terms of orientation, but at a much lower significance (p < 10-4, see below). Although it is possible that the TCTC repeats are bound at the 5' untranslated region (UTR) mRNA level, the positional distribution of the conserved AGAGAGA sites does not indicate a strong positional bias with respect to ATG (DATG = 893).Note that for a few predicted elements (for example, CAGGTGA, rank 111), the median distance falls outside of the optimal window; this is due to the fact that, for these elements, the median distance does not correspond to the peak of the distribution of distances to ATG. Hence, for these elements, the optimal window provides a better descriptor of the positional bias than the median distance. Additional analysis reveals that several of the known binding sites discovered in this study are constrained in term of orientation. For example, the binding site for the GATA-factor(s) (as shown in Table  repeats ,23. The k-mer found in yeast, the binding site for the Reb1 protein, had the same property. Moreover, this site displays a relatively strong orientation bias 5' to 3' (p < 10-10).FastCompare also returned many novel motifs; some of the most interesting ones are shown in Table k-mer, CGACACTCC, is one of the closest motifs to ATG, with a median distance of 234 bp, and its conserved set is strongly enriched in genes involved in positive regulation of growth  (p < 10-7). Another predicted regulatory element, CGAGACC (rank 20), is found upstream of downregulated genes in 23 microarray conditions. Interestingly, it is found upstream of downregulated genes in a study measuring gene-expression changes at several time points during worm aging [fer-15 and spe-9;fer-15) and at similar time points . In addition, the functional enrichment of its conserved set points at a potential role in embryonic development (p < 10-7). Another strongly conserved and novel motif, CTCCGCCC (rank 14), was independently found upstream of almost all transcribed worm microRNA genes in a recent study [Several of the other novel predicted regulatory elements in Table rm aging , in two nt study .k-mers found at the previous stage. For example, the most conserved k-mer, TCTGCGTCT, is very often co-conserved with AGAGAGA. The high-scoring interaction between the DRE-like motif, AATCGAT and the putative E2F-binding site, TTTTCGC, also appears interesting. Indeed, the conserved sets for both k-mers are separately enriched significantly with genes involved in embryonic development, according to GO (p < 10-8 and p < 10-7, respectively). However, the conserved set of genes having both elements in their upstream regions is even more enriched in this GO category (p < 10-9). TTTTCGC also seems to interact with the novel site CGACACTCC, and the corresponding conserved set is enriched with genes involved in modification-dependent protein catabolism (p < 10-5). The full list of motif interactions is available at [We found many interactions between the most conserved y p < 10-. TTTTCGCD. melanogaster and D. pseudoobscura, two species of Drosophila that diverged about 46 million years ago [We applied FastCompare to the genomes of ears ago . The numears ago ). It takThe distribution of conservation scores shown in Figure k-mers (k = 7, 8 or 9), which we further analyze below.As for the yeast and worm genomes, we used functional annotations (GO), expression data and known TRANSFAC sites to evaluate the FastCompare predictions. Unfortunately, expression data is often available for only a subset of genes and its analysis led to very few validations. However, Figure k-mers. The most conserved element, AACAGCTG, is similar to the site known to be bound by AP-4  and MyoD . One of the most interesting predictions is TATCGATA (rank 12); this palindromic motif, known as the DNA replication-related element (DRE), has been experimentally proved to be necessary for proper expression of several cell proliferation-related genes in D. melanogaster [ATG = 168), and the most over-represented k-mer (among the 469 highest scoring ones) within D. melanogaster upstream regions compared to exons, with a ratio of 5.39.As shown in Table nogaster  and, mornogaster  and catanogaster  in the sDrosophila transcription factors involved in development. For example, FastCompare predicts TTTATGGC (rank 14) and TAATTGA (rank 24), the binding sites for two homeodomain transcription factors. The first site matches the TRANSFAC consensus binding site for Abd-B ([CG]NTTTATGGC), while the second site is the known consensus binding site for the Antennapedia (Antp) class of homeodomain proteins [D. melanogaster. Also, FastCompare predicts CAGGTGC, the binding site for the Snail repressor/activator protein, a transcription factor required for proper mesodermal development [Several of the other predicted sites are known to be bound by proteins   as one of the most conserved putative regulatory elements between the two flies. This site is the binding site for the POU-domain family of transcription factors, and it is probably bound by one or several of the three POU-domain transcription factors in al cells , whereasal cells .Drosophila genomes were also found when analyzing the worm genomes. For example, GAGA repeats are found to be strongly conserved, slightly oriented 3' to 5' (p < 10-4), and very significantly found upstream of genes involved in morphogenesis (p < 10-23). GTAAACA (rank 147), the DAF16-binding site in C. elegans, is also one of the most conserved sites between the two Drosophila genomes. This site is probably bound by dFOXO, the unique homolog of the C. elegans DAF16 protein in D. melanogaster [Many of the known motifs found when comparing the two nogaster .k-mers are the DRE site (TATCGATA, DATG = 168) and the known AP-4/MyoD binding site (AACAGCTG, DATG = 373). However, both the optimal windows and the median distances in Table d0.025 = 798 and d0.975 = 1,126. Among the 469 highest scoring k-mers, 45 fall below 798 (p < 10-13) and 36 above 1,126 (p < 10-8), once again suggesting weaker positional constraints than in yeasts and worms, at least when considering the first 2,000 bp of 5' upstream sequences.As for both previous phylogenetic groups (yeasts and worms), the median distances to ATG for the conserved elements show that some of the predicted regulatory elements are severely constrained in terms of position. Among the most constrained Drosophila that to the best of our knowledge are unknown (Table Drosophila species [Sxl and sisterlessB) in a subsequent study . It is interesting to see that this particular site is significantly conserved upstream of genes involved in cell fate commitment (p < 10-8).FastCompare predicts many putative regulatory elements in wn Table . One of  species ; it was p < 10-9). The same site appears to be strongly oriented 5' to 3' (p < 10-12). Others, such as GTGTGACC or AAATGGCG, appear to be located closer to ATG than most other sites (DATG = 296 and 592, respectively).Some of these sites, such as the palindromic TTAATTA (rank 31), are found much more often in upstream regions than in exons (with an over-representation ratio of 3.07). Others, such as ACACACAC, are found to be significantly enriched upstream of genes in known functional categories (embryonic development, p < 10-12); ACACACAC and TAATTGC (an Antp variant site), embryonic development (p < 10-5). The full list of interactions is available at [We found many potential interactions between the most conserved sites discovered by FastCompare. For example, the POU-domain-binding site ATTTGCATA was found to be strongly co-conserved with TAATTGA, the Antp-binding site, and with many other potential homeodomain sites, such as AATAAAT and TAATTAA. The CACA repeats were also found to be co-conserved with several different sites, and in some cases, the set of genes having both sites simultaneously conserved in their upstream regions (conserved sets) was found to be enriched in certain functional categories, for example, ACACACAC and GAGAGAG, regulation of transcription (lable at .Alu) have colonized mammalian genomes and are likely to be conserved between closely related genomes. The distance between enhancers and the transcriptional start of the genes they regulate can be extremely large, reaching tens of kilobases. Finally, gene predictions and gene boundaries are still largely unverified experimentally for a large number of genes.The much larger noncoding regions of mammalian genomes present significant challenges for computational motif discovery. Also, many repeat elements . However, analysis of the FastCompare output yielded the same validations as for other species. Indeed, the distribution of conservation scores obtained on actual and randomized sequences shows that high conservation scores are very unlikely to be obtained by chance Figure . As withAlu repeats did not influence the output of FastCompare (data not shown). To overcome the overabundance of GC-rich sequences in the FastCompare output, we use longer k-mers as starting points, namely 8-mers instead of 7-mers. We started with the 600 highest-scoring 8-mers, and replaced each of these 8-mers by one of its substrings (7-mer) or one of its superstrings (9-mer), when their conservation score is higher. We then removed duplicates in the list and added the high-scoring 9-mers that have no substrings within the list. This procedure yielded 284 k-mers (k = 7, 8, 9). Subsequent validation was limited to this small set of high-scoring predictions.We found that masking k-mers. Among these are the well characterized sites for the Sp1, C/EBP, CREB and Myc/Max proteins or families of proteins. These four sites reside very close to ATG (their median distance to ATG is between 100 and 250 bp), suggesting that the four proteins (or families of proteins) may be involved in intimate interactions with the transcriptional complex. Sp1 is an ubiquitous transcription factor, involved in the basal expression of a large number of genes in mammals  involve Sp1-binding sites (CCCGCCC or CCGCCCC) with other known sites such as CACGTGAC (Myc/Max), TGACGTCA (CREB), CGCAGGCGC (unknown), GCCAATC (CCAAT-box) and ACTTCCG (Ets), and that the median distances between these sites are relatively small (138, 164, 200.5, 234 and 234, respectively).Interestingly, we found that some of the most conserved interactions between in vivo footprinting and electrophoretic mobility shift assay (EMSA) [D. melanogaster by the anterior morphogen Bicoid, and also recently shown to be bound in human by Goosecoid-like (GSCL) [p < 10-14). It is also the site with the strongest over-representation in upstream regions compared to exons that we observed, with a ratio of 7.06.Among the other predicted regulatory elements returned by FastCompare are CCGCCTC, a site known as the insulin response element ; CGGAAGTy (EMSA) ; TTTCGCGe (GSCL) . InteresFastCompare also predicts ATTTGCAT, the binding site for the POU-domain Oct-1 and Oct-2 proteins, known to bind the promoter and intronic enhancer of immunoglobulin genes ; it alsod0.025 = 342 and d0.975 = 1,185 and found that 83 k-mers among the 284 highest-scoring ones have a shorter median distance than 342 (p < 10-63) and only 11 have a larger median distance than 1,185. Indeed, a majority of the known sites identified by FastCompare are preferentially located near the 5' start of genes, with some elements being very close to ATG . Nonetheless, a few known motifs do not seem to show any positional constraints. For example, the Bicoid-like site TAATCCCAG has a median distance to ATG of 1,258.The distribution of distances to ATG for all 7-mers Figure  shows anp < 10-6).FastCompare identifies many putative regulatory elements which to the best of our knowledge are novel Table . Some ofTo gain a better understanding of the network-level conservation of regulatory elements between the different phylogenetic groups, we compared the results we obtained by applying FastCompare to yeasts, worms, flies and mammals in the previous sections. We calculated the overlap (and its significance) of the 400 highest-scoring 7-mers and 8-mers found for each phylogenetic group. As shown in Table k-mers in different phylogenetic groups , we found little overlap. The only significant overlap we found (after Bonferroni correction) was between the GATA sites (GATAAGA) in worm and fly (p = 2.5 × 10-4). As a control, we performed the same analysis within the yeast phylogenetic group, using the S. cerevisiae/S. bayanus and S. paradoxus/S. mikatae 400 most conserved 7-mers. One hundred and ninety-five sites were found in both groups of 7-mers, and for all of them, the overlaps between the conserved sets obtained separately in the S. cerevisiae/S. bayanus and S. paradoxus/S. mikatae analyses were highly significant, with hypergeometric p-values < 10-40. Therefore, our results strongly suggest that, while transcription factors have largely retained their ability to recognize specific DNA sites, their targets have largely changed through appearance or disappearance of those binding sites in promoters. This hypothesis is supported by recent analysis of the fission yeast cell cycle using microarrays, which showed that the role and the binding sites for several of the main transcription factors involved in regulating the yeast cell cycle (Swi4/Mbp1, Fkh1/Fkh2, Swi5/Ace2) are conserved between budding and fission yeasts (which diverged about 1 billion years ago), but the sets of genes that they regulate overlap much less than expected (only about 50 orthologous genes are cell-cycle-regulated in both species) [However, when we looked at the overlap between conserved sets for identical high-scoring species) .ATG = 230 for GCCACGCC) make it a promising candidate for experimental testing. Interestingly, the location constraints on these conserved sites can vary across phylogenetic groups. For example, the CRE appears weakly constrained in worms and flies in terms of distance to ATG (DATG = 708 and 825, respectively), but is very close to ATG in mammalian genomes (DATG = 107). However, the distances to ATG of the POU-domain-binding sites (862, 882 and 729, respectively) indicate that their positional constraints are shared among the phylogenetic groups. The same holds for the HRE binding site (845, 1,015.5 and 895, respectively).It is particularly interesting to consider the seven 8-mers that are top predictions for all three multicellular phylogenetic groups (note that many more 7-mers are conserved between these groups). These sites include the CRE (TGACGTCA, GACGTCAC and ATGACGTC), the POU-domain binding site (ATTTGCAT), and the HRE (CAAGGTCA). A fourth site is also shared (GCCACGCC, CCACGCCC), which to the best of our knowledge is a novel motif. Its strong over-representation in upstream regions compared to coding regions, and its closeness to ATG  clearly shows that our method is able to detect conserved and functional motifs in all the phylogenetic groups that we studied. In all analyses, we have shown that some of the discovered known or novel motifs were severely constrained, either in terms of position relative to the start of translation or in orientation. We also observed that some of the known or novel motifs are co-conserved within upstream regions, potentially revealing interactions between the (often unknown) transcription factors that bind them.Our results show that FastCompare can recover most of the known functional binding sites in k-mers discovered by FastCompare to user-supplied sequences or multiple alignments. An example is shown in Figure STE2 gene  from four different yeast species were aligned using ClustalW, and the most globally conserved k-mers are highlighted. All experimentally determined sites for STE2 were also predicted to be globally conserved by FastCompare. Moreover, several other sites also appear to be conserved, both at the global level (predicted by FastCompare) and the local level . In Figure We have created a set of web tools to superimpose the most globally conserved k-mer to be on different strands within pairs of orthlogous upstream regions. This flexibility substantially increases the number of k-mers that are supported by independent biological data (that is, true positives), at least for yeasts and worms (data not shown). However, it is difficult to evaluate whether this flexibility introduces more true positives than false positives. Also, transcription factors often bind several slightly distinct sites with different affinities, and it is widely acknowledged that binding-site degeneracy is better captured by using position-weight matrices (PWM) instead of k-mers or consensus patterns [k-mers that unambiguously correspond to the same binding sites. Conservation scores for weight matrices were calculated as described for k-mers in Materials and methods, except that we used the weight-matrix score thresholds that maximize the significance of the overlap between the two sets of ORFs containing matches to the weight matrices in each species. This involves progressively lowering the score threshold by small increments, and for each threshold, calculating the overlap and its hypergeometric p-value. We then choose the score threshold corresponding to the most significant p-value, and use the negative natural logarithm of this p-value as the conservation score. As shown in Table k-mers. These results suggest that k-mers provide results that are almost as good as those obtained using weight matrices, when utilizing the network-level conservation criterion. One reason why, in many cases, k-mers have a higher conservation score than weight matrices may have to do with the more narrow selection of k-mers for binding sites with similar or identical affinities. In fact, we recently showed that PWM scores, widely seen as proxies for binding affinity, are statistically conserved in a comparison between S. cerevisiae and S. bayanus [k-mers representing each transcription factor binding site may be defining affinity classes that are more strongly conserved than a looser definition of a binding site represented by a weight matrix. Recent work in bacteria has established the importance of binding affinity, especially with respect to coordinating the temporal order of events [While powerful, our approach has potential limitations. Our current approach allows matches to a given patterns . To eval bayanus . In the f events .k-mer. This suggest that our k-mer based approach is limited in its ability to discover highly degenerate binding sites.However, Table Drosophila and mosquito genomes (which diverged approximately 400 million years ago), we only found a handful of k-mers (interestingly including GATA-factor and Myc/Max binding sites) to have conservation scores above those obtained from randomized data.As shown by our inter-group analysis, many regulatory elements have remained functional across evolution, but few have remained upstream of the same genes. The network-level conservation principle thus appears less applicable to species that diverged very long ago. For example, when we compared the k-mers could be used to seed weight matrices whose individual weights could be optimized for network-level conservation, using stochastic optimization procedures . Introns and downstream noncoding regions could also be explored using our approach, as these regions are known to harbor functional regulatory elements in metazoan genomes. While our approach can deal with genomes presenting arbitrary levels of divergence and rearrangements, it would be interesting to investigate how global alignments or suboptimal and non-overlapping local alignments [There are also several directions in which our approach could be extended. From a methodological standpoint, the approach could be extended to take into account local over-representation of identical or nearly identical copies of the same binding sites, a well known feature in the promoter regions of higher eukaryotic species . To discignments  could bek (k-mer, with k between 7 and 9) as a candidate regulatory element. For each k-mer, we found the set of ORFs whose upstream regions contain at least one exact match to the k-mer, anywhere in the upstream region, in the first genome. We did the same for the second genome, obtaining another set of ORFs. Then, we calculated the overlap between the two sets and assessed its statistical significance  for all yeast species considered in this paper; worm (C. elegans and C. briggsae), Drosophila (D. melanogaster), human (H. sapiens) and mouse (M. musculus) sequence data were downloaded from Ensembl [D. pseudoobscura genome sequences (contigs) were downloaded from [Sequence data were downloaded from the  Ensembl . The D. ded from . The upset al. [D. melanogaster and D. pseudoobscura, or between distant species such as S. cerevisiae and C. elegans), we determine orthologs using the reciprocal best BLAST hits approach.Orthology information provided by Ensembl or by Kellis et al.  was usedk, we first generated the set of all possible k-mers and removed half of them on the basis of reverse complementarity. We also removed k-mers with very low complexity and which are over-abundant in the intergenic regions of the genomes we analyzed (that is, those that contain k - 1 or more As or Ts), as these sequences are unlikely to be regulatory elements. Every remaining k-mer (that is, 8,170 for k = 7) is then considered as a candidate regulatory element. For each k-mer, we found the set of ORFs in the first species that have at least one exact occurrence of the k-mer in their upstream regions. We then found the set of ORFs in the second species that have at least one occurrence of the same k-mer in their upstream region. Importantly, the matches can be anywhere in the upstream regions: they do not have to be at the same positions in two orthologous upstream regions  and can be on any strand. Since both functional and non-functional elements are expected to be conserved between two closely related species, the two sets are expected to overlap. However, under the network-level conservation principle, the extent of the overlap - and therefore its statistical significance - will be even greater for k-mers that represent functional transcription factor binding sites. The significance of the overlap can be measured using the hypergeometric distribution. The probability of two sets of size s1 and s2, drawn from a set of N elements, to have i or more elements in common is given by :Given a value of k-mers can be ranked by their hypergeometric p-values. It is important to note that due to basal conservation (that is, conservation arising from common ancestry), the hypergeometric p-values will generally be very small for most k-mers. Therefore, we only use these p-values as relative measures of network-level conservation and focus on k-mers with the greatest conservation. For simplicity, we define the 'conservation score' to be the negative logarithm (base e) of the hypergeometric p-value obtained for a given k-mer. Therefore, the more extensive the overlap between the two sets, the higher the conservation score. Also, for the same k-mer, we call 'conserved set' the set of ORFs corresponding to the overlap between the two sets of orthologous ORFs containing at least one exact match to the k-mer in their upstream regions. Conserved sets are used throughout this study to get insights into the function of the most conserved k-mers, using functional annotation [k-mers are constrained in terms of position or orientation.In this way, all notation ,82, chronotation , known mk-mers with a user-specified gap (termed gapped k-mers), which is a straightforward extension of the approach described above. The conservation score returned by FastCompare is independent of the size of the patterns ; therefore k-mers with different sizes, and gapped k-mers (for example, CGTNNNNNNTGA) can be compared.The current FastCompare implementation handles m highest-scoring 7-mers, with m chosen according to independent biological data . We then replace each of the retained 7-mers by an 8-mer (if there is one) with higher conservation score for which the considered 7-mer is a substring. We also include within the final list the 8-mers which do not have any substrings within the m 7-mers. We then repeat the same process for the retained 8-mers, replacing each of them by its higher scoring 9-mer superstring if there is one, and add the 9-mers that do not have any substring within the 8-mers. This strategy thus allows the optimal length for candidate regulatory elements to be determined.We use the following strategy when applying FastCompare to pairs of genomes. First, we calculate conservation scores for all 7-mers, 8-mers and 9-mers. We then retain only the k, the worst-case time complexity is O(kn + 4k(p + k)), where n is the total amount of upstream sequences and p is the total number of orthologous pairs. Note that the first term is generally much larger than the second one; therefore the complexity of our approach can be seen as linear in the combined sizes of the genomes to be compared (when k is restricted to 7, 8 and 9). The calculation of hypergeometric p-values involves factorials of large integers, so we use specialized C routines, as described in [FastCompare is implemented in the C language and uses efficient data structures (hash tables and prefix trees ). For a ribed in . FastComk-mers found at the previous stage in the following ways.As described in Results, we applied FastCompare to 1 kb (yeast) or 2 kb upstream regions . While these lengths are reasonable, they are somewhat arbitrary, and it is known that some regulatory elements are constrained to be within specific distances (often shorter than 1 kb) from the start of transcription, reflecting mechanistic constraints for transcription factor-transcription factor or transcription factor-RNA polymerase interactions . Moreovek-mer, we calculated the median distance to ATG  for the set of all (non-overlapping) occurrences of this k-mer within the upstream regions of its conserved set (see previous section for a definition of the conserved set of a given k-mer). To statistically assess whether the median distance to ATG for a given k-mer is unusually small or large, we built the distribution P(d) of median distances to ATG, for the entire set of 8,170 7-mers. We first created a histogram by binning the median distances to ATG for all 7-mers into 20-bp bins, and then smoothed the histogram . Then, using numerical integration, we sought the distance thresholds d0.025 and d0.975 such that P(d <d0.025) = 0.025 and P(d <d0.975) = 0.975. We then considered the median distance to ATG for a given k-mer as unusually short or long when it is less than d0.025 or greater than d0.975, respectively.First, for each high-scoring k-mer, we also sought the sequence window which maximizes the conservation score by progressively shortening all upstream regions  by 100 bp increments from the 5' end. Then, we did the same from the 3' end using the optimal 5' end found in the previous step. Evaluating every possible window whose length is a multiple of 100 bp almost always yields identical results. We then calculated the conserved sets for these windows, and output the orientation (strand) for each k-mer occurrence within its conserved set .For each k-mer, we used the binomial distribution to assess whether the proportion of occurrences of this k-mer (within its conserved sets) on one strand is significantly smaller (or larger) than 0.5. Binomial p-values less than 0.05 (after Bonferroni correction) are considered significant.Finally, using the results of the previous step, for each k-mers. To focus on heterotypic interactions, we only examined k-mers that differ by more than l nucleotides, after optimal ungapped alignment. We tested several values of l and found that l = 4 was most appropriate when using 7-, 8- and 9-mers. Then, we proceeded as described above, except that instead of seeking two sets of ORFs (one for each species) whose upstream regions contain a single k-mer, we sought the two sets of ORFs that contain the two k-mers simultaneously. Once these two sets were available, we evaluated the extent of their overlap as described above, and rank interaction pairs according to their conservation score.It is now known that the regulatory code governing the expression of genes is combinatorial ,85,86. TWe used randomized data to show that high conservation scores (obtained as described above) are unlikely to be obtained by chance, and independent biological information to assess the ability of FastCompare to predict functional regulatory elements by giving them a high conservation score. We also estimated the over-representation of predicted regulatory elements in upstream regions compared to coding regions.Our goal was to generate new pairs of upstream regions that are conserved at the same level of divergence as the actual sequence data. We align each pair of orthologous sequences using the Needleman-Wunsch algorithm , and calThe proportions of 7-mers supported by each type of independent data, as presented in Figures S. cerevisiae), worm (C. elegans), fly (D. melanogaster) and human (H. sapiens) functional categories and corresponding ORF annotations were downloaded from the MIPS [p-values for functional enrichment were not corrected for multiple testing, but only p-values smaller than 10-4 are reported, providing a slightly less stringent thresholds than Bonferroni corrections.Yeast (the MIPS  and GO [the MIPS  websitesk-mer as a known motif if it meets the following two criteria. The first is significant overlap (p < 10-4) between the conserved set for the given k-mer and the set of ORFs whose upstream regions contain at least one match to the known motif . The sempareACE  to calcuS. cerevisiae [p-value < 0.001  7-mers and removing consensus patterns that matched more than 50 7-mers.The 309 weight matrices and corresponding consensus patterns for known transcription factor binding sites were downloaded from ,91. k-meS. cerevisiae, 555 conditions for C. elegans, 156 conditions for D. melanogaster, and 1,384 conditions for H. sapiens. We use these expression data in the following way.Expression data for all species considered were downloaded from diverse sources ,93. Overk-mer in the upstream regions of genes that are themselves over- or underexpressed in certain microarray conditions. Over- or underexpressed genes are operationally defined as having a log ratio of intensity above average plus two standard deviations, or below average minus two standard deviations, respectively . To evaluate the over-representation of a given k-mer in a given microarray condition, we defined as O1 the set of overexpressed genes in this condition, and as O2 the set of ORFs whose upstream regions contain at least one occurrence of the considered k-mer, genome-wide. Then, we evaluated the significance of the overlap between O1 and O2 using the hypergeometric distribution, as described above. Overlaps whose hypergeometric p-value is smaller than 0.05 (after Bonferroni correction) were considered significant. We proceeded separately with the set of underexpressed genes in the same way. The total number of microarray conditions (overexpressed plus underexpressed) for which a k-mer was found to be significantly over-represented is reported. Note that we do not use the conserved set for the considered k-mer here, as we do not want to restrict our analysis to orthologous genes. Indeed, except for yeast, microarrays often contain only a fraction of all genes within the considered organism. In these cases, the overlap between conserved sets and over- or underexpressed genes can be very small, reducing statistical power. Using all genes, therefore, increases our power to detect significant associations, while retaining a uniform approach for all species considered.We evaluated the over-representation of each k-mer in their upstream regions over the number of genes that have the k-mer in their coding regions (using only exons), and we correct this ratio using the average length of the upstream and coding regions.As shown in  for the The FastCompare implementation, all the sequences, and results are available on our website .


Both paclitaxel (P) and carboplatin (C) have significant activity in non-small cell lung cancer (NSCLC). The weekly administration of P is active, dose intense, and has a favorable toxicity profile. We retrospectively reviewed the data of 51 consecutive patients receiving C and day 1 and 8 P chemotherapy (CT) regimen in advanced stage NSCLC to evaluate the efficacy and toxicity.2 intravenously (IV) over 1 hour on day 1 and 8, followed by C AUC 5 IV over 1 hour, repeated in every three weeks. PC was given for maximum of 6 cycles.Patients treated in our institutions having pathologically proven NSCLC, no CNS metastases, adequate organ function and performance status (PS) ECOG 0–2 were given P 112.5 mg/mMedian age was 58 (age range 39–77) and 41 patients (80%) were male. PS was 0/1/2 in 29/17/5 patients and stage was IIIA/IIIB/IV in 3/14/34 patients respectively. The median number of cycles administered was 3 (1–6). Seven patients (14%) did not complete the first 3 cycles either due to death, progression, grade 3 hypersensitivity reactions to P or lost to follow up. Best evaluable response was partial response (PR) in 45% and stable disease (SD) in 18%. Twelve patients (24%) received local RT. Thirteen patients (25%) received 2nd line CT at progression. At a median follow-up of 7 months (range, 1–20), 25 (49%) patients died and 35 patients (69%) progressed. Median overall survival (OS) was 11 ± 2 months , 1-year OS ratio was 44%. Median time to progression (TTP) was 6 ± 1 months , 1-year progression free survival (PFS) ratio was 20%. We observed following grade 3 toxicities: asthenia (10%), neuropathy (4%), anorexia (4%), anemia (4%), hypersensitivity to P (2%), nausea/vomiting (2%), diarrhea (2%) and neutropenia (2%). Two patients (4%) died of febrile neutropenia. Doses of CT were reduced or delayed in 12 patients (24%).P on day 1 and 8 and C every three weeks is practical and fairly well tolerated outpatient regimen. This regimen seems to be comparably active to regimens given once in every three weeks. Lung cancer is the leading cause of cancer related deaths all around the world. About 80% of all lung cancers are non-small cell lung cancer (NSCLC) and more than 50% of these patients present with locally advanced or metastatic disease.Meta-analysis of several randomized trials have demonstrated a modest survival advantage for treatment with cisplatin-based regimens in patients with advanced stages of NSCLC ,2. FurthBeing the first of the taxane antimicrotubule agents, paclitaxel (P) demonstrated overall response rates of 21–24% and 1-year survival rates of 37–42% in the phase II trials where it was used as a single agent . AntiangP and C used in combined chemotherapy regimens have significant activity in NSCLC. PC given every three weeks is considered to be one of the standard regimens being used worldwide . The wee3, hemoglobin ≥ 9 g/dl, and platelet count ≥ 100000/mm3), and adequate liver functions  and kidney functions (creatinine ≤ 1.5 mg/dl). No prior chemotherapy or radiotherapy  was allowed. Patients presenting with known central nervous system (CNS) disease and uncontrolled cardiac arrhythmia were excluded from this study and they were treated with other chemotherapy regimens (single agent or combination of platinum and vinorelbine, docetaxel or gemcitabine).All patients with stage III or IV NSCLC treated at Medical Oncology Units of Marmara University Hospital, Dr. Lutfi Kirdar Research and Training Hospital, SSK Sureyyapasa Chest and Cardiovascular Diseases Hospital and Gulhane Military Medical Academy Hospital within July 2002 and August 2003 were considered for this protocol. Eligible patients were required to have pathologically proven NSCLC, stage III or IV disease at presentation or progressed after surgery, performance status (PS) ECOG 0–2, objective measurable disease, adequate bone marrow functions (white blood cell count ≥ 3500/mm2/day) on days 1 and 8, followed by C (AUC 5/6) on day 1, repeated in every three weeks. Both drugs were diluted in 250 ml of normal saline and given intravenously (IV) over 1 hour. No growth factors were administered. Anti-allergic premedication included IV diphenhydramine 50 mg, IV ranitidine 50 mg, and IV dexamethasone 16 mg 1 hour prior to P administration.Patients were treated with P  was done on days 1 and 8 of each cycle, liver and kidney function tests on every 2 cycles. Cranial computed tomography scans (CT), magnetic resonance imaging (MRI) and bone scans were performed as clinically indicated. Side effects of the treatment were graded according to the National Cancer Institute Common Toxicity Criteria (CTC), version 2.0 . Colony rd cycle and standard World Health Organization (WHO) criteria were used to determine response [Response was evaluated with CT of chest and/or abdomen on every 3response . IndepenPatients were irradiated with CT based treatment planning and multiple fields arrangements with custom blocking to all fields and involved hilar and mediastinal lymph nodes up to 40–41.4 Gy. Boost was given to the primary tumor. Total dose of 60–61.2 Gy was administered in 1.8–2 Gy daily fractions for 5 days a week and completed in 6 weeks.Overall survival (OS) and time to progression (TTP) were assessed from the date of diagnosis to the date of death (any cause) and the date of objective disease progression (death was considered a progression event in patients who died before disease progression), respectively. Survival rates were calculated by using the Kaplan-Meier method . The preThe data of 51 patients receiving PC treatment were collected retrospectively between July 2002 and November 2003. Median follow-up time was 7 months (range, 1–20). Median age was 58 years (range 39–77) and 45% of patients were 60 year-old or above. Eighty percent were male. PS was 0 in 57% of patients and 67% had presented with stage IV disease. Most frequent metastatic sites were the other lung (17), adrenal (10), liver (7) and bone (7). Eighty-two percent of the patients had smoking history, median of which was 40 pack-years (range, 0–135). Patients' baseline characteristics are presented in Table The median number of cycles administered was 3 (range, 1–6). Seven patients (14%) did not complete the first 3 cycles either due to death (2), progression (3), grade 3 hypersensitivity reaction to P (1) or lost to follow up (1).Best evaluable response was PR in 45% and SD in 18%. Only 22 (43%) patients continued the treatment after the 3rd course. At the end of treatment of these 22 patients 10 (46%) had PR and 6 (27%) had SD, but the other 6 patients (27%) had PD. No complete remission was seen. Twelve patients (24%) received local RT and 4 of these patients were given low dose gemcitabine (75 mg/m2/week × 5–6 weeks) as radiosensitizing agent. Of these 12 patients 3 presented with stage IIIA and all had PR to PC therapy. But of the 5 patients with IIIB disease who were irradiated only one patient had PR, 3 had SD and another one had PD after the 3 cycles of CT. Four patients with stage IV were offered RT for palliation. Thirteen patients (25%) received 2nd line CT at progression and of those only one patient had PR and another SD to this treatment. For 2nd line CT gemcitabine ± cisplatin or C was used in 10 patients (78%) and the rest received other agents like vinorelbine, C or docetaxel. Details of this data can be seen in Table At a median follow-up of 7 months 25 (49%) patients died and 35 patients (69%) progressed. Median OS time was 11 ± 2 months , 1-year OS ratio was 44% , neuropathy (42%) and anorexia 35%). We observed the following grade 3 toxicities: asthenia (10%), neuropathy (4%), anorexia (4%), anemia (4%), hypersensitivity to P (2%), nausea/vomiting (2%), diarrhea (2%) and neutropenia (2%). Two patients (4%) died of febrile neutropenia due to a three day delay in referral to hospital after the onset of fever > 38°, although they were warned about the side effects of the therapy. Doses of CT was reduced or delayed in 12 patients (24%)  and C (AUC = 2) given weekly for 6 out of 8 weeks for a total of two cycles. Greater percentage of the patients on arm I received intended CT (30% of P and 55% of C) compared with the other arms (28–29% of P and 21–22% of C). Patients on arm I received more than half of the planned C dose. The main reasons for discontinuation of therapy were progression of disease (31%) and adverse events (15%). Median time to progression and median survival time were significantly higher for arm I than arm II for patients with stage IIIB disease. Performance status of the patients was also statistically related to the survival times. Patients with PS-0/1 had longer median PFS with treatment arm I than arm II and patients with PS-2 had higher median OS with arm I than arm II. Although arm I was the most easily tolerable schedule between the three arms, grade 3 or 4 neutropenia was observed in 22% of the patients included. In this trial treatment arm I had a response rate of 32%, median TTP of 6.9 months, median OS time of 11.3 months and 1-year survival rate of 47%. In our study, response rate was 45%, median TTP was 6 months, median OS time was 11 months and 1-year survival rate was 44%. The majority of our patients comprised of stage IIIB and IV disease, similar to the patient group in Belani's study resulting in similar response rates and survival data [Weekly dose of P in combination with cisplatin or C had been administered in NSCLC patients by Belani et al. ,22. Theyval data ,16.2) given in every three weeks and divided into two consecutive weeks. C dose was calculated according to Calvert formulation with an AUC of 5. This is a lower dose than the dose of C being used in other phase III trials in the literature. In our study only 4 patients (8%) had dose reduction of 10% and 16% of patients had treatment delays of 1 week because of side effects. According to this data, 76% of patients have received the total planned doses of the drugs on scheduled date. Two patients (4%) died of febrile neutropenia due to a three day delay in referral to hospital after the onset of fever > 38°, although they were warned about the side effects of the therapy. It is worth mentioning that none of our patients received any colony stimulating factors.We used the standard dose of P , only 4% of our patients experiencing grade 3 sensory neuropathy. Grade 3 or 4 neuropathy has been reported to be 10–20% in schedules given every three weeks . Belani 2/week × 3 weeks out of four weeks cycles) in maintenance arm or observed until disease progression has occurred. They reported that the maintenance arm was compared to the observation arm and had a median PFS of 38 weeks vs. 29 weeks, median OS of 75 weeks vs. 60 weeks, respectively [Besides the reduced toxicity, weekly administration of P also increases the drugs' anti-angiogenic and apoptotic effects. The metronomic schedule of P has been studied widely during the last few years. P had been shown to inhibit endothelial cell proliferation, motility, invasiveness, and cord formation both in vitro and in vivo Matrigel assays in a dose dependent manner . Belani ectively . AlthougAlthough our study is a retrospective analysis, it is one of the few manuscripts on this PC scheduling in NSCLC in the literature.Paclitaxel on day 1 and 8 and carboplatin every three weeks is a practical and fairly well tolerated outpatient regimen. This regimen seems to be comparably active to regimens given every three weeks. This schedule needs to be further evaluated by well planned randomized phase III trials where it could be compared to the standard regimens in patients with advanced stage NSCLC.The author(s) declare that they have no competing interests.PFY designed the study, followed the patients, collected the data, performed the statistical analysis and drafted the manuscript. NST followed the patients and helped with the manuscript. MG followed the patients and helped with statistical analysis. NFH, OT, AO, TS, MA followed the patients. RA confirmed the diagnosis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Understanding how the cell functions—or breaks down—implies an understanding of the assembly lines, transportation systems, and powerhouses that keep it running. Global approaches are needed to identify the numerous proteins essential to each cellular machine. But which techniques are best? Lars Steinmetz and colleagues applied and evaluated a variety of methods to define mitochondrial proteins, and report that sets of complementary approaches are needed to characterize a cellular subsystem.Yeast mitochondria make an ideal subject for study. About two-thirds of the estimated 700 mitochondrial proteins have been identified to date, leaving fertile ground for new finds. The researchers previously compiled a list of 477 proteins with compelling evidence for mitochondrial involvement. This list provides a well-defined reference set against which to test protein-finding methods. Moreover, the well-studied and accessible yeast genome is well-suited for exploration and genetic manipulation. Since mitochondria are very similar among all eurkaryotes (organisms whose cells have nuclei), the results will prove relevant across species.Steinmetz and colleagues triangulated results from multiple techniques to identify new candidate mitochondrial proteins. They compared the reference protein list to their new data from protein, mRNA, and gene knockout studies, and to 19 published datasets from other researchers, to evaluate the success of different techniques at finding known mitochondrial proteins. Then they combined evidence across studies to identify a set of proteins that likely characterizes most of the mitochondrial machinery.The researchers first identified proteins from yeast mitochondria using a technique called liquid chromatography mass spectrometry, which separates the proteins by water insolubility , then identifies each by the mass and molecular charge of its constituents. By comparing this approach with others, the authors show that this proteomic technique alone is by no means comprehensive, nor error-free. Mass spectrometry is biased toward finding more abundant proteins, and the purified mitochondria can contain contaminants from elsewhere in the cell. To address these issues, the authors compared their protein data with a protein study from another group, the reference protein set, and a recent subcellular localization study. Potential mitochondrial proteins identified by more than one protein approach were more likely to localize to mitochondria in the localization study than were proteins identified by only one approach. This finding suggests that, compared to either method alone, a combination of protein and localization measures can more robustly identify proteins residing in mitochondria.But since mitochondria, as the cell's power plants, are integrated into other cellular machinery, the authors argue, methods targeting proteins that are physically located to mitochondria should be complemented with functional approaches. Proteins with mitochondrial roles, regardless of concentration or location in the cell, are better identified by approaches that associate mRNA expression or gene deletion—which removes proteins or renders them inoperable—with changes in mitochondrial function.By comparing results from multiple methods against the reference protein list, the researchers evaluated the likelihood that each protein was mitochondrial. They compiled a list of 691 top candidates. This multi-technique analysis easily outperformed any single study in terms of its ability to identify proteins in the reference set, and of the proportion of known versus unconfirmed proteins located. As mitochondria are well-conserved across species, the results provide a candidate gene list for finding human counterparts that might be associated with mitochondrial disorders.Future studies can use this analysis to evaluate which research methods are likely to be most informative in other cell systems. This paper demonstrates the power of combining techniques with differing strengths in order to zero in on proteins that might elude any single approach, resulting in a more complete parts list for specific cellular machinery.


Raised activity of the renin-angiotensin system (RAS) may both amplify inflammatory and free radical responses and decrease tissue metabolic efficiency and thus enhance cerebral injury in the preterm infant. The angiotensin-converting enzyme (ACE) DD genotype is associated with raised ACE and RAS activity as well as potentially adverse stimuli such as inflammation. The DD genotype has been associated with neurological impairments in the elderly, and thus may be also associated with poorer motor or cognitive development amongst children born preterm prematurely.The association of DD genotype with developmental progress amongst 176 Caucasian children born at less than 33 weeks gestation  was examined at 2 and 5 1/2 years of age. Measured neuro-cognitive outcomes were cranial ultrasound abnormalities, cerebral palsy, disability, Griffiths Developmental Quotient [DQ] at 2 yrs, and General Cognitive Ability [British Ability Scales-11] and motor performance [ABC Movement], both performed at 5 1/2 yrs. All outcomes were correlated with ACE genotype.The DD genotype was not associated with lower developmental quotients even after accounting for important social variables.These data do not support either a role for ACE in the development of cognitive or motor function in surviving infants born preterm or inhibition of ACE as a neuroprotective therapy. Delight over recent survival gains for the very premature infant has been tempered by the frequent presence of cerebral injury and developmental impairment. One quarter of those born before 26 weeks postmenstrual age (at least 11 weeks premature) show evidence of severe cerebral injury including cognitive dysfunction by 30 months of age .Three factors seem to play important roles in the aetiology of preterm cerebral injury. Firstly, exposure to inflammatory stimuli is associated with white matter injury and cerebral palsy in the preterm . SecondlCandidate systems that might influence motor or cognitive outcome after premature birth are likely to be those which affect these responses. The human renin-angiotensin systems may be such a system. Angiotensin converting enzyme (ACE), a key component of the circulating (or endocrine) renin-angiotensin system (RAS), cleaves angiotensin I to yield the potent vasoconstrictor angiotensin II. In addition, ACE degrades vasodilator kinins. In these ways, endocrine RAS plays an important role in circulatory homeostasis. However, local RAS also exist in diverse human tissues including lung, myocardium, vasculature, lymphocyte and brain tissue. These are powerful regulators of mitochondrial respiration and whole-cell metabolism  and exerA common variant of the human ACE gene provides a tool to determine if ACE activity does influence developmental progress after preterm birth. The presence  rather than the absence  of a 284-base-pair fragment in the human ACE gene is associated with lower ACE activity in organs including both circulating inflammatory cells  and the The study was approved by the ethical committees of Southmead Hospital and United Bristol Health Care Trust. Parental consent was obtained for participation in neurodevelopmental follow-up   . BrieflyThe Griffiths Mental Development Scales, used to assess motor and cognitive performance, was performed at 2 years corrected age . The GriA psychologist performed the Griffiths Scales of Mental Development and a second psychologist performed the British Ability Scales (second edition) (BAS). The ABC Movement tests were performed by a trained research nurse. All assessments were blind to the child's neonatal course and subsequent progress.DNA was extracted from the Guthrie card blood spots (newborn metabolic screening cards). ACE genotype was determined using 3-primer PCR amplification . Primer An estimate of sample size suggested that 144 patients would be needed for this study. The assumptions made for this calculation were that DD genotype infants had a mean DQ of 92.5 (1/2 SD below the norm) compared to a mean DQ of 100 in the ID+II group, assumed typical genotype distributions, and a significance of 0.05 with 80% power.Data were stored in SPPS v9.0 for Windows. Lymphocyte  and tissp = 0.047) (table p = 0.65).Guthrie cards were located for 230 of 308 children. After exclusion of non-Caucasians and, at random, 1 child of any identical twin pairs (based on genotypes and gender) 176 babies with ACE genotype formed the study population  with follow-up data at 2 years. 122 of these also had follow-up at 5 1/2 years. The ACE genotype distribution was 49 [27.8%] DD, 73 [41.5%] ID, 54 [30.7%] II, demonstrated Hardy-Weinberg equilibrium, and was similar to that observed in the newborn term population from the same region of the UK (203 [24.1%] DD, 433 [51.5%] ID, 205 [24.4%) II). Baseline characteristics were independent of genotype, except that fewer individuals of DD genotype were from twin births . Thus the effect of any one polymorphism, with a relatively minor effect, may be swamped in the newborn infant by other protective mechanisms.The lack of any association between ACE genotype and scores of developmental progress was also surprising because we have demonstrated an association between DD genotype and markers of poor cardio-respiratory instability in the perinatal period in this patient group . This asWe cannot support an association of ACE genotype with cognitive or motor development in survivors born preterm or, thus, the use of RAS inhibition as a neuroprotective agent in the preterm. Given the current lack of understanding of the mechanisms leading to cerebral injury and subsequent impairment – particularly of higher function – in such patients, further genetic association studies of other candidate genes are warranted.ACE, angiotensin-1 converting enzyme; DQ developmental quotient, BAS, British ability scales (second edition); GCA, general cognitive ability; RAS, renin angiotensin system; PCR, polymerase chain rection.The author(s) declare that they have no competing interests.DH, HM, AW, NM conceived the study and its design and wrote the manuscript. DH, DD and SD performed data collection, DNA extraction and PCR and participated in analysis of the data with SH and HM. NM reviewed all cranial imaging. All authors participated in the writing of the manuscript and approved the final manuscript.


Nidation of floating tumour cells initiates peritoneal carcinosis and limits prognosis of gastro-intestinal tumours. Adhesion of tumour cells to extracellular matrix components is a pivotal step in developing peritoneal dissemination of intraabdominal malignancies. Since phospholipids efficaciously prevented peritoneal adhesion formation in numerous animal studies we investigated their capacity to reduce adhesions of gastric cancer cells to extracellular matrix components (ECM).Human gastric cancer cells (NUGC-4, Japanese Cancer Research Resources Bank, Tokyo, Japan) were used in this study. Microtiter plates were coated with collagen IV (coll), laminin (ln) and fibronectin (fn). Non-specific protein binding of the coated wells was blocked by adding 1% (w/v) BSA (4°C, 12 h) and rinsing the wells with Hepes buffer. 50.000 tumour cells in 100 μl medium were seeded into each well. Beside the controls, phospholipids were added in concentrations of 0.05, 0.1, 0.5, 0.75 and 1.0/100 μl medium. After an incubation interval of 30 min, attached cells were fixed and stained with 0.1% (w/v) crystal violet. The dye was resuspended with 50 μl of 0.2% (v/v) Triton X-100 per well and colour yields were then measured by an ELISA reader at 590 nm. Optical density (OD) showed a linear relationship to the amount of cells and was corrected for dying of BSA/polystyrene without cells.The attachment of gastric cancer cells to collagen IV, laminin, and fibronectin could be significantly reduced up to 53% by phospholipid concentrations of 0.5 mg/100 μl and higher.These results, within the scope of additional experimental studies on mice and rats which showed a significant reduction of peritoneal carcinosis, demonstrated the capacity of phospholipids in controlling abdominal nidation of tumour cells to ECM components. Lipid emulsions may be a beneficial adjunct in surgery of gastrointestinal malignancies. In the treatment of gastro-intestinal cancer the detection of free, isolated tumour cells in the peritoneal cavity serve as a prognostic marker for postoperative survival -4. Sincein vitro study was focused on the influence of phospholipids on adhesion of gastric cancer cells to extracellular matrix components with broad reactivity to several integrins. Collagen IV (coll IV), and laminin (ln) are main components of the basement membrane and fibronectin (fn) plays an important role in wound healing [Phospholipids, polar phosphoric acid di-esters, are natural constituents of the abdominal fluid. The substance is able to form a lubricant layer on the peritoneal surface . Additio healing ,18.2, Falcon, Becton Dickinson-Gambil, Heidelberg, Germany) in RPMI 1640 medium (GIBCO, Karlsruhe, Germany), supplemented with 10% foetal bovine serum (GIBCO), penicillin and streptomycin (GIBCO). Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were passaged after treatment with 0.125% trypsin for 6 min. The cells were pelleted after centrifugation for 10 min at 200 g, suspended in 20 ml PBS, and pelleted. The cell pellet was resuspended in 30 ml complete medium and seeded with a splitting ratio of 1:3. Only cells from three passages were used for the experiments.The human gastric cancer cell line NUGC-4 was purchased from the Japanese Cancer Research Resources Bank (Tokyo, Japan). The cells were maintained in monolayers in tissue culture flasks (75 cm2 in air (ln), respectively. Nonspecific protein binding of the coated wells was blocked by adding 1% (w/v) BSA (4°C, 12 h) and rinsing the wells with Hepes buffer.Flat-bottom polystyrene microtiter plates (Becton Dickinson, Heidelberg, Germany) were coated for adhesion experiments. The purified ECM components were dissolved in PBS with the following concentrations: coll IV – 2,5 μg/ml (Biomol, Hamburg, Germany), fn – 10 μg/ml (Boehringer, Mannheim, Germany), ln – 50 μg/ml (Boehringer, Mannheim, Germany). We found these concentrations to be optimal in foregoing dilution series. They were added to the wells and incubated at 4°C for 24 hours (coll IV, fn), or at 37°C for 45 min in a humidified atmosphere of 5% CO3 and 5 × 104 cells per well, as determined by a dilution series.For adhesion experiments gastric cancer cells were detached with collagenase I (15 min, 37°C, Worthington, Freehold, USA), washed once with RPMI 1640, centrifuged (200 g for 10 min), resuspended in RPMI 1640, and preincubated for 30 min in a humidified atmosphere of 5% CO2 in air (37°C). Fifty thousand tumour cells in 100 μl medium were seeded into each well. Evaluation of adherent cells was performed using crystal violet staining according to the method described by Aumeilley et al., and Tietze et al. ,20. AfteControl dying of BSA/polystyrene without cells led to Optical Density (OD) values of 0.01–0.07. These values were subtracted from those obtained in the experiments.After complete preparation of the tumour cell suspension, the PL solution was added in the following concentrations: 0.05, 0.1, 0.5, 0.75, and 1 mg per 100 μl medium. The concentrations used were correlated to our in vivo experiments. The phospholipid solution consists of phosphatidylcholine 70% by weight, phosphatidylethanolamine 15% by weight, neutral lipids 8% by weight, sphingomyelin <3% by weight and lysophosphatidylcholine <3% by weight.All experiments were performed three times in quadruplicate. The data are expressed as means +/- standard error of the mean (SEM). Student's t-test for unpaired data was used for statistical analysis. Differences were regarded as significant for p values < 0.05.The analysis of tumour cell adhesion to BSA 1% resulted in a mean extinction of 0.27 (SEM 0.01) at 590 nm. Coating with ln and fn led to a nearly twofold increase of tumour cell adhesion with mean values of 0.59 (0.03, ln) and 0.63 (0.03, fn). The cancer cells showed a most pronounced adhesion to coll IV with a mean extinction of 0.97 (0.02).The tumour cell adhesion to ln registered after addition of PL was significantly reduced. The effect was concentration dependent compared to the controls. Even the minimum amount of PL 0.05 mg/100 μl led to a reduced extinction of 0.4 (0.01). Treatment with 0.1 or 0.5 mg/100 μl PL revealed extinction values of 0.32 (0.02) and 0.28 (0.02), respectively. The maximum effect could be demonstrated with 0.75 mg/100 μl PL with an extinction of 0.24 (0.02). The relative reduction of tumour cell adhesion compared to the control amounts to 59%. Treatment with 1 mg/100 μl PL showed no further decrease of tumour cell adhesion to ln. The mean extinction was 0.26 (0.01)  and 0.59 (0.01). However, a significant reduction of tumour cell adhesion could be observed after treatment with 0.5 mg/100 μl PL, 0.42 (0.02); as well as with 0.75 mg/100 μl PL (0.39 (0.02)) and 1 mg/100 μl PL (0.38 (0.02)). We found a similar situation compared to ln with equal effects of 0.75 mg/100 μl and 1 mg/100 μl PL indicating that the maximum influence on adhesion is reached. The relative reduction of tumour cell adhesion compared to the control values amounts to 40%  after administration of 0.05 mg/100 μl PL to a maximum effect after treatment with 1 mg/100 μl PL with a value of 0.44 (0.02) table . In compCell adhesion to the extracellular matrix plays a fundamental role in peritoneal carcinosis. The adhesion is mediated by transmembrane Integrins. Several proteins including fibronectin in the interstitial matrix, laminin and collagen IV in the basement membrane were identified as important ligands ,22. Manyin vitro as well as a reduction of tumour weight after intraperitoneal tumour injection [In our experiments the reduced rate of cell attachment in the presence of phospholipids was independent from the extracellular matrix. A similar effect on intraperitoneal tumour growth was described by Jacobi et al. who could demonstrate that taurolidine/heparin and povidone iodine lead to a significant reduction of tumour cell growth njection . Predominjection . Dextrannjection ,32. Howenjection ,34. PhosThe hypothesis is that phospholipids form a lubricant layer on the peritoneum by binding with its negatively charged cholin branch chain to the positively charged peritoneal surface ,14,35. Pin vitro experiments with tumour cells as soluble agents added to ECM immobilized onto plastic surfaces cannot appropriately mimic the situation in situ. Recently we found that phospholipids significantly reduce the attachment area and the tumour volume of peritoneal carcinosis caused by the colonic cancer cell line DHD/K12/TRb in rats. These results were supported by a prolonged survival rate of the treated animals as compared to the control group. Additionally, we found a similar effect of phospholipids on the adhesion of the human rectal cancer cell line HRT-18 on the same ECM-components in vitro [The in situ tumour cell – ECM interaction is influenced by adhesive and non-adhesive ECM components and can be understood as a three dimensional network . Therefoin vitro . Consistin vitro ,6,38,39.We performed this study to ascertain the results of the foregoing animal experiments and to demonstrate the influence of phospholipids to three different ECM components, even though matrices of collagen IV, laminin and fibronectin alone may not be predictive of peritoneal membrane nidation.These results, within the scope of additional experimental studies on mice and rats which showed a significant reduction of peritoneal carcinosis, demonstrated the capacity of phospholipids in controlling abdominal nidation of tumor cells to ECM components. Lipid emulsions may be a beneficial adjunct in surgery of gastrointestinal malignancies.The work was financially supported by Fresenius Kabi, Bad Homburg, Germany.The results are part of an international patent application.The pre-publication history for this paper can be accessed here:


Pregnancy in patients with lipoprotein lipase deficiency is associated with high risk of maternal pancreatitis and fetal death. A very low fat diet  is the primary treatment modality for the prevention of acute pancreatitis, a rare but potentially serious complication of severe hypertriglyceridemia. Since pregnancy can exacerbate hypertriglyceridemia in the genetic absence of lipoprotein lipase, a further reduction of dietary fat intake to < 1–2% of total caloric intake may be required during the pregnancy, along with the administration of a fibrate. It is uncertain if essential fatty acid deficiency will develop in the mother and fetus with this extremely low fat diet, or whether fibrates will cross the placenta and concentrate in the fetus.A 23 year-old gravida 1 woman with primary lipoprotein lipase deficiency was seen at 7 weeks of gestation in the Lipid Clinic for management of severe hypertriglyceridemia that had worsened with pregnancy. While on her habitual fat intake of 10% of total calories, her pregnancy resulted in an exacerbation of the hypertriglyceridemia, which prompted further restriction of fat intake to < 2% of total calories, as well as administration of gemfibrozil at a lower than average dose. The level of gemfibrozil, as the active metabolite, in the venous and arterial fetal cord blood was within the expected therapeutic range for adults. The clinical signs and a biomarker of essential fatty acid deficiency, namely the ratio of 20:3 [n-9] to 20:4 [n-6] fatty acids, were closely monitored throughout her pregnancy. Despite her extremely low fat diet, the levels of essential fatty acids measured in the mother and in the fetal blood immediately postpartum were normal. Normal essential fatty acid levels may have been achieved by the topical application of sunflower oil.An extremely low fat diet in combination with topical sunflower oil and gemfibrozil administration was safely implemented in pregnancy associated with the severe hypertriglyceridemia of lipoprotein lipase deficiency. Primary lipoprotein lipase (LPL) deficiency is a rare autosomal recessive disorder characterized by severe hypertriglyceridemia, due to the accumulation in plasma of chylomicrons and very low density lipoproteins (VLDL) that result from the absence of LPL activity . The estThe management of severe hypertriglyceridemia in a pregnant patient with LPL deficiency is directed toward preventing pancreatitis in the mother and delivery of a healthy infant. Lowering of plasma TG in the prevention of pancreatitis is managed primarily by dietary fat restriction, but additional TG lowering may be required and has been reported with use of fibrates, such as gemfibrozil ,9,10. TwThe proband presented to her pediatrician at age 3 month with failure to thrive. She was evaluated at the Children's Hospital Medical Center in Seattle, where an elevated TG level of 14,000 mg/dl (158 mmol/L) suggested hyperchylomicronemia with LPL deficiency. Plasma post-heparin LPL activity was absent, consistent with defective catabolism of TG rich particles. Further study at the University of Washington of her post-heparin plasma at age 19 revealed absent LPL activity due to a defective LPL protein, while her hepatic lipase activity was normal . She wasth week of gestation. A 5 lb 3 oz baby girl with a 5-minute Apgar score of 9 was delivered vaginally. A short time after the delivery, the baby was briefly intubated for about 48 hours due to respiratory distress but did well subsequently. The patient's plasma TG rapidly decreased to 2015 mg/dl (22.8 mmol/L) within the first postpartum 24 hours, accompanied by improved abdominal symptoms. Resumption of low fat solid food brought back the symptoms of pancreatitis and she was placed back on the IV fluids followed by a more gradual incremental introduction of oral intake. Along with 1,200 mg/day of gemfibrozil, she had complete resolution of abdominal symptoms by postpartum day-8 and eventual resolution two weeks after discharge of peri-pancreatic fluid accumulation demonstrated by CT imaging studies. Now, 11 years later, the proband and her daughter are both healthy and doing well. The proband's TG levels are back to baseline and stable on 10–20% fat diet. Her daughter has had normal TG and cholesterol levels on regular diet.At the age of 23, the proband presented at week 7 of gestation for management of anticipated worsening of hypertriglyceridemia in pregnancy. She had been in excellent physical condition and had continued her routine 10–20% fat diet during the first trimester , also developed at week 27. A concomitant regnancy .Because of concern for unfavorable fetal neurological development due to EFA deficiency, EFA profile was monitored in the mother at each visit starting at gestational week 23. The initial analyses were performed at the Clinical Nutrition Research Unit (CNRU), Harborview Medical Center campus of the University of Washington. After separation from cells, the fatty acids from the phospholipid fraction of the plasma were extracted and subsequently measured by capillary gas chromatography. In addition to the total amount and % of each FA, the ratio of eicosatrienoic acid (ETA, 20:3(n-9)) to arachidonic acid (AA, 20:4(n-6)) was calculated  by high performance liquid chromatography (HPLC) and revealed similar concentrations of the drug and its active metabolites in both umbilical vein and artery, which were within the normal reference range for adults Figure .Children with primary LPL deficiency can be effectively managed on fat-restricted diets and grow normally into adulthood. However, they can present with extreme elevation of TG levels with serious acute pancreatitis. This LPL-deficient subject developed severe hypertriglyceridemia in early pregnancy, with eruptive xanthomas and pancreatitis. With the diligent efforts from the patient, her family, and a team of specialists in lipid metabolism, dietetics, high-risk obstetrics and gastroenterology, a successful outcome was achieved. Outcome goals were clearly set at the onset of her pregnancy care, including nutritional management of the expected rise in triglyceride levels associated with the estrogen surge of pregnancy to prevent acute pancreatitis, and avoidance of clinical EFA deficiency in both the mother and the fetus.Pregnancy-induced hypertriglyceridemia is estimated to be the cause in 4–6% of all pancreatitis cases during pregnancy, while most cases result from cholelithiasis . SuccessPregnancy is a well known situation in which the physiologic estrogen surge profoundly alters the TG-rich lipoprotein metabolism, resulting in a gradual rise in TG levels over the course of non-complicated pregnancy, peaking at the level of 200–300 mg/dl (2.26 – 3.39 mmol/L) at term . During Arachidonic acid [AA, 20:4(n-6)], an important precursor of the prostaglandin compounds, cannot be synthesized de novo from FFA in mammals and must be derived from another EFA in the diet, namely linoleic acid [LA, 18:2(n-6)]. In the case of life long low oral fat intake, as in our patient, clinical EFA deficiency might occur with depletion of n-3 and n-6 FA stored in adipose tissue. Therefore, her source of EFA would be entirely from recent dietary intake and deficiency might occur sooner than in individuals with normal LPL and abundant EFA storage . EicosatUse of TG lowering drugs, such as gemfibrozil (a fibrate), can be used to directly lower the triglyceride level in the prevention of acute pancreatitis. Pregnancy induces hepatic production of TG-rich VLDL and may respond to fibrates through inhibition of hepatic production of VLDL. Gemfibrozil, which is an FDA category C drug, has not been observed to be associated with adverse drug effects in reports of pregnancy-related severe hypertriglyceridemia ,10,36,37In conclusion, a successful pregnancy outcome was achieved in our LPL deficient patient, confirming previous reports ,43 that The author(s) declare that they have no competing interests.ECT was a senior fellow in Metabolism, Endocrinology and Nutrition and drafted the manuscript. JAB was the dietitian. Both MSV and GJA contributed to the measurement of fatty acids. GJA was also a consulting scientist in fatty acid metabolism. AC and JDB were the faculty associated with the case at the UW GCRC. JDB conceived of the research, supervised the fellow, and coordinated the manuscript revisions.The pre-publication history for this paper can be accessed here: