Fragility fractures caused by osteoporosis are a major cause of morbidity and mortality in aging populations. Bone mineral density (BMD) is a useful surrogate marker for risk of fracture and is a highly heritable trait. The genetic variants underlying this genetic contribution are largely unknown.2, n = 319) and high  BMD at the lumbar spine. Significant findings were verified in two additional sample collections.We performed a large-scale association study investigating more than 25,000 single nucleotide polymorphisms (SNPs) located within 16,000 genes. Allele frequencies were estimated in contrasting DNA pools from white females selected for low (<0.87 g/cmPDE4D) gene region on chromosome 5q12. We subsequently tested the marker SNP, rs1498608, in a second sample of 138 white females with low (<0.91 g/cm2) and 138 females with high (>1.04 g/cm2) lumbar spine BMD. Odds ratios were 1.5 (P = 0.035) in the original sample and 2.1 (P = 0.018) in the replication sample. Association fine mapping with 80 SNPs located within 50 kilobases of the marker SNP identified a 20 kilobase region of association containing exon 6 of PDE4D. In a second, family-based replication sample with a preponderance of females with low BMD, rs1498608 showed an opposite relationship with BMD at different sites (p = 0.00044-0.09). We also replicated the previously reported association of the Ser37Ala polymorphism in BMP2, known to interact biologically with PDE4D, with BMD.Based on allele frequency differences between DNA pools and subsequent individual genotyping, one of the candidate loci indicated was the phosphodiesterase 4D (PDE4 gene family in human osteoporosis.This study indicates that variants in the gene encoding PDE4D account for some of the genetic contribution to bone mineral density variation in humans. The contrasting results from different samples indicate that the effect may be context-dependent. PDE4 inhibitors have been shown to increase bone mass in normal and osteopenic mice, but up until now there have been no reports implicating any member of the  COL1A1) gene has shown one of the most consistent associations to osteoporosis, even if the association is generally weak for BMD and varies between populations [BMP2, the gene encoding for bone morphogenetic protein 2, as responsible for the linkage [The postmenopausal loss of bone mass and subsequent increased risk of low-energy (fragility) fractures is an important public health problem, especially in countries with a high proportion of elderly individuals. More than 1 million fragility fractures, primarily in postmenopausal women, occur each year in the US. The annual direct medical costs exceed US$10 billion -5. Thereulations -11. Linkulations -19. So f linkage . NeverthPDE4D, the gene encoding cyclic AMP-dependent phosphodiesterase 4D, with lumbar spine BMD. PDE4D selective inhibitors have been shown to promote osteoblast differentiation in progenitor cells [Studies that rely on direct association approaches based on linkage disequilibrium within populations are expected to have greater statistical power and be more feasible to implement than traditional linkage studies to identify common variations that influence common, complex traits such as osteoporosis . Recentlor cells  and to ior cells  but the The population sample from which the discovery samples were chosen consisted of 5,436 female twins collected at the Twin Unit at St Thomas Hospital, London, England. They were selected without regard to health or trait. The volunteers had been recruited through advertisements and had undergone extensive investigation at the Twin Unit at St Thomas Hospital. Investigations included several questionnaires inquiring about present and past diseases, symptoms, family history, socio-economical factors, and medication. Subjects underwent an extensive clinical assessment including DEXA measurements of bone mineral density and anthropometric measurements ,24. All A twin sample from Royal North Shore Hospital, Sidney, Australia was collected similarly as the UK twin collection. 731 individuals including twin pairs and singletons with lumbar spine BMD assessments were available for genotyping  study that collected sib pairs concordant and discordant for bone mineral density . ProbandAll studies were approved by the appropriate research ethics committees. All participants gave their informed consent to participate in genetic studies before enrollment.Bone mineral density was estimated from the L1-L4 vertebrae, hip, and forearm using DEXA according to the user's manual for the Hologic QDR 4500W,  at all collection sites.A set of 25,494 SNP markers was selected from a collection of 125,799 experimentally validated polymorphic variations [For pooled DNA assays, 25 ng of case and control DNA pools was used for amplification at each site. All PCR and MassEXTEND™ reactions were conducted using standard conditions . RelativBMP2 were AGCTGGGCCGCAGGAAGTTCG (forward PCR primer), TCGTCAGAGGGCTGGGATGAG (reverse PCR primer) and TGAGGGGCGGCCCGACG (extension primer).Primers used to genotype rs1498608 were ATAACCTCGGGGTCCAGAAA (forward PCR primer), GAATCCCTGTTCATTCCTTG (reverse PCR primer) and CCCTAAAAACTGTTCCAGGTA (extension primer). The primers used to genotype the Ser37Ala polymorphism in -5, PCR/mass extension = 1.7 × 10-4, and chip measurement = 1.0 × 10-4.Tests of association between adjusted lumbar spine BMD group and each SNP using pooled DNA were carried out in a similar fashion as explained elsewhere . BrieflyTests of association using individual genotypes were carried out using a chi-square test of heterogeneity to compare allele frequencies, and Fisher's exact test to compare genotype frequencies (due to low frequency contingency table cells). Confidence intervals and P-values for odds ratios were derived using Fisher's exact test. When one or more cell counts were zero, non-infinite odds ratios were estimated by adding 0.5 to each cell . In the 2 = 0.82) [2 in the low BMD group and 1.11–1.60 g/cm2 in the high BMD group, corresponding to the upper and lower 22nd percentiles. Other characteristics of the samples are described in Table We carried out a genome-wide association study using 25,494 SNPs located within 10 kb of 15,995 LocusLink annotated genes. An overview of the investigative process is shown in Figure  = 0.82) . The selPDE4D on chromosome 5q12. Allele frequencies for the T allele based on genotyping were 0.91 in the low lumbar spine BMD pool and 0.88 in the high BMD pool . Complete genotype counts and summary statistics are reported in Table One of the associations was found with rs1498608, an A/T polymorphism within intron 5 of Genome-wide studies using tens of thousands of SNPs and liberal statistical selection criteria are expected to yield a high proportion of false positive associations. To distinguish the true genetic effects from among the false positives, the 78 selected SNPs were genotyped in a second twin sample from Australia.2, P = 0.049) or TT  genotypes, thus confirming the results observed in the unrelated tails of this sample. Similar GEE analyses carried out for femoral neck and hip BMD were not statistically significant.The Australian replication sample, a combination of female and male twin pairs and singletons, was analyzed in two ways. First, to create a design and carry out an analysis comparable to the discovery sample, unrelated individuals were selected from the lower and upper quartiles of the sex-specific adjusted lumbar spine BMD distribution Table . A simil2, P = 0.11) and the TT  genotypes. Using Z-scores at the femoral neck (P = 0.0007 and 0.0004), total hip (P = 0.003 and 0.007), and lumbar spine (P = 0.03 and 0.02) as dependent variables confirmed this pattern of association. In all cases the AT and TT genotypes had very similar point estimates.Being a sample of mostly affected sib pairs, this sample was unsuitable for formation of groups with contrasting BMD because of the preponderance of individuals with low BMD Table . TherefoPDE4D.In order to better define the extent of the region of association and possibly identify other SNPs more strongly associated with BMD, we performed DNA pool based association fine mapping in the UK sample using 80 publicly available intronic SNPs in the 100 kb region surrounding the incident SNP Figure . This an2 (P = 0.0029). There were no homozygous Ala individuals in this sample.As described in the discussion below, PDE4D inhibition is known to influence BMP2-induced alkaline phosphatase activity in osteoblast precursor cells. Recently, variation in the gene encoding BMP2 was found to be associated with osteoporosis in a study employing whole genome linkage and subsequent positional cloning . Since wPDE4D is associated with low bone mineral density at the lumbar spine in females. PDE4D encodes cyclic AMP-dependent phosphodiesterase 4D. Phosphodiesterases are a superfamily of enzymes involved in degradation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) [PDE4 genes in human osteoporosis. However, variation in PDE4D was recently reported to be associated with the risk of ischemic stroke [PDE4D variants may have effects on the risk for different common diseases. There are other examples of genes having such pleiotropic effects, the most notable being APOE in hyperlipidemia and Alzheimer's disease [PDE4D association with stroke was strongest for a region in the recently extended 5' end of the gene, which is close to 1,000 kb upstream of rs1498608 [PDE4D to the risk of osteoporosis as well as stroke, it is possible that different domains are involved in the different diseases.In an association study using SNPs in nearly 16,000 genes we obtained evidence that variation in the SNP rs1498608 located within e (cGMP) ,35. cAMPe (cGMP) -39, whice (cGMP) -45. The e (cGMP) , and dege (cGMP)  and to ae (cGMP) ,48. PDE4e (cGMP) . PDE4 ine (cGMP) . In spit disease ,51. It sBMP2 and measures of osteoporosis in an international family-based sample ascertained via low BMD probands. Although not statistically significant, this finding was supported by the results in the discovery sample of unrelated high and low spine BMD subjects. The allele frequency in the low BMD group was 2.2% and in the high group 1.4%, with an odds ratio of 1.6 (P = 0.28). The rare allele was less common in our low BMD group than the low spine BMD group (3%) in the Icelandic sample. However, our allele frequencies in the low and high BMD groups and the resulting OR corresponded well with the figures in the Danish sample  reported in the same paper [BMP2 and PDE4D in either sample. However, given the low minor allele frequencies of each SNP, there was very little power to test for interaction effects.Given the interaction between BMP2 and PDE4 for the inhibition on osteoblastic differentiation in vitro, it is interesting to note that variants in the gene encoding for BMP2 have also been found to increase risk of osteoporosis in humans . In the me paper . We founPDE4D and lumbar spine bone mineral density was detected, providing the first evidence that a variant of this gene could contribute to the risk of osteoporosis in humans. The effect was similar in size in premenopausal and postmenopausal women, indicating that the effect would be on the attainment of peak bone mass rather than the rate of decrease in BMD after menopause. The lack of a detectable interaction with female sex hormones is supported by having observed a similar genetic effect in the small sample of males in our study. The genetic contribution to peak bone mass is possibly bigger and definitely better documented than the as yet unproven genetic influence on postmenopausal bone loss [PDE4D could contribute to this effect, especially in light of the documented anabolic effect on bone by PDE4 inhibitors.The starting point of the present study was a large-scale association study of more than 25,000 SNPs located in 16,000 genes. After a stepwise selection process an association between an intronic SNP in one loss , and it An association with an intronic SNP provides little evidence for a change in amount or function of the protein that could explain the association. None of the 80 SNPs investigated as part of the association fine mapping were non-synonymous coding changes, which is consistent with the extraordinary lack of variation that others have reported for all PDE classes  and PDE4PDE4B in our assay set, rs1318475, was taken through to the second stage . These results suggest that further investigation into possible associations between variants in all PDE4 genes and bone mineral density may be justified.Given the functional similarity between different PDE4 enzymes, we went back and scrutinized our data for associations with SNPs in the other PDE4 genes that may have been overlooked during the first stage of the scan. The only SNP in PDE4D marker and bone mineral density was one result from over 25,000 hypothesis tests. A conservative Bonferroni adjustment to yield an experiment-wide type I error rate of 0.05 would demand a test-wise p-value on the order of 10-6. Given the modest sample size, only common variations with relatively large effects (OR > 2) would reach such significance levels. Instead, we chose to be more mindful of the role of type II error rates and apply a more liberal set of criteria in the initial phases of the study and verify true genetic effects by independent replication. The analysis of 78 selected markers in the Australian replication sample resulted in multiple associations of continuing interest, with rs1498608 displaying one of the strongest associations. A one-sided test of association comparing the results in the discovery and replication samples yields a p-value of 0.0074. This would not be considered significant on an experiment-wide level after Bonferroni adjustment, which would require a p-value on the order of 0.0006 or lower. The analysis in the international replication sample produced contradictory data in that the A allele, which in the first two samples was associated with increased lumbar spine bone mineral density, was associated with decreased BMD at all tested sites. The pattern of association evident from the first two samples, with AT and TT genotypes having very similar point estimates, was preserved in this sample, even in the face of the reverse direction of association. The highly statistically significant association between rs1498608 and femoral neck and hip BMD in this third sample and the consistency in the pattern of association would be unexpected from a spurious result. A possible explanation for the contradictory results could be the fact that the international sample consists mostly of individuals with low BMD since the probands all have a BMD Z-score < -1.5, and most of the siblings also have low BMD. It is possible that within such a selected sample the relationship between rs1498608 and BMD could be altered due to interactions with other genetic or environmental factors.The route by which these genetic associations were arrived at and the potential for spurious association must be considered. Recent published work has brought light to the need for proper validation to verify genetic findings for complex traits -56. In tPDE4D variants in the genetic contribution to BMD in humans merits further investigation.The well-documented anabolic effect on bone by PDE4 inhibitors lends indirect support for the association reported here, and it would seem that the possible role of The result of the present large scale association study together with data from previously published animal models suggest that genetic variation in the gene encoding PDE4D contributes to the variation in lumbar spine BMD in humans.The authors of this manuscript affiliated with Sequenom, Inc.  declare competing financial interests, which may include current or prior receipt of salary and/or stock ownership, as Sequenom, Inc. may be affected financially by the publication of this manuscript.RR drafted the manuscript and participated in study design and data analysis. SM was study project leader and participated in data analysis. SK supervised the operational aspects of the study, and was responsible for the development of the SNP marker assay set. CH and GM participated in the development of the SNP marker assay set. SW was project manager for the international multicenter study. PS was principal investigator for the Australian Twin collection. TS was principal investigator for the UK twin and international collections. MN participated in study design, performed the statistical analyses and helped draft the manuscript. AB had the overall scientific responsibility for the study.The pre-publication history for this paper can be accessed here:


Drosophila melanogaster genome were analyzed in vivo, confirming 15 active enhancer regions. A comparison with Drosophila pseudoobscura sequences revealed that conservation of binding-site clusters accurately discriminates functional regions from non-functional ones. 27 predicted gene-regulatory regions in the  Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters.The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns.We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: Measuring conservation of sequence features closely linked to function - such as binding-site clustering - makes better use of comparative sequence data than commonly used methods that examine only sequence identity. The transcription of protein-coding genes in distinct temporal and spatial patterns plays a central role in the differentiation and development of animal embryos. Decoding how the unique expression pattern of every transcript is encoded in DNA is essential to understanding how genome sequences specify organismal form and function.cis-acting sequences that control transcription, identifying which trans-acting factors act on each regulatory sequence, and determining how these interactions affect the timing and organization of transcription. The first step in this process is by no means straightforward. Regulatory regions are often large and complex. Functional cis-acting sequences are found 5' and 3' of transcripts and in introns, and can act over short or long distances. Most of the described animal regulatory sequences were identified by experimental dissection of a locus, and astonishingly few of these are well characterized.Understanding gene regulation requires discovering the cis-regulatory modules (CRMs) - tend to contain functional binding sites for several different transcription factors, often with multiple sites for each factor.Despite the paucity of good examples, as multiple regulatory sequences from different organisms were identified and characterized, some common features became apparent ,2. Most in silico methods to identify regulatory sequences on the basis of binding-site clustering have been developed and applied to animal genomes [in vivo regulatory activity [As the first animal genome sequences were completed -6, resea genomes -10. Someactivity -17, yet Drosophila development is perhaps the best system in which to apply and evaluate these methods. Development of the Drosophila embryo is arguably better understood than that of any other animal. Sophisticated genetic screens [in situ expression studies [The transcriptional regulatory network governing early  screens ,19 have  screens  and in s studies . TranscrDrosophila melanogaster genome with unusually high densities of predicted binding sites for the early-acting transcription factors Bicoid (BCD), Hunchback (HB), Krüppel (KR), Knirps (KNI) and Caudal (CAD). As nine of these regions overlapped previously known CRMs, we proposed the remaining 28 as predicted CRMs (pCRMs). We tested one of the previously untested pCRMs for enhancer activity in a standard reporter gene assay [giant (gt) in a posterior stripe. Here, we report the systematic testing of the remaining 27 untested pCRMs for enhancer activity, resulting in collections of both bona fide positive and false-positive predictions, allowing us to develop and evaluate methods to improve the accuracy of methods for identifying functional cis-regulatory sequences.In a previous study , we idenne assay ,23 and sD. pseudoobscura  was rec species ,26, and Drosophila suggest that similar methods will be valuable in insects as well [D. melanogaster and D. pseudoobscura showed that known regulatory regions are only slightly more conserved than the rest of the non-coding genome [Most methods used to identify regulatory sequences from interspecies sequence comparison are fairly simple. They identify 'conserved' non-coding sequences (CNSs), operationally defined as islands of non-coding sequence with relatively high conservation flanked by regions of low conservation, and assume that this conservation reflects regulatory function. Although crude, these methods have been remarkably effective in identifying mammalian regulatory sequences ,28, and  as well . However as well ,31. Simig genome , highligrunt (run), even-skipped (eve), hairy (h), knirps (kni) and hunchback (hb). To determine whether any of the remaining 28 pCRMs also function as enhancers, we generated P-element constructs containing the pCRM sequence with minimal flanking sequence on both sides fused to the eve basal promoter and a lacZ reporter gene . As the margins of the tested sequences do not precisely correspond to the margins of the clusters, we assigned a unique identifier (of the form CEXXXX) to each tested fragment .The 37 pCRMs are shown in Table We successfully generated multiple independent transgenic fly lines for 27 of the 28 pCRMs. We repeatedly failed to generate transgenes containing CE8007. This sequence contains five copies of an approximately 358 base-pair (bp) degenerate repeat. One additional pCRM (CE8002) also contains tandem repeats. While we were able to generate transgenes for CE8002 and assay its expression, these two tandem repeat-containing pCRMs (CE8007 and CE8002) were excluded from subsequent analyses.in situ RNA hybridization to the lacZ transcript in embryos at different stages in at least three independent transformant lines. Nine of the 27 transgenes showed mRNA expression during embryogenesis  of gt expression in the blastoderm as previously described [CE8001 is 5' of the gene for the gap transcription factor escribed .nub). The CRM recapitulates the endogenous blastoderm expression pattern of nub, first detected as a broad band extending from 50 to 75% egg length. Although nub expression continues in later embryonic stages, CE8011 expression is limited to the blastoderm stage.CE8011 is 5' of the gene for the POU-homeobox transcription factor nubbin (odd-skipped (odd) and drives expression of two of its seven stripes: stripe 3 at 55% and stripe 6 at 75% egg length. This CRM also has the ability to drive later, more complex, patterns of expression. During stages 6 and 7, expression is detected in the procephalic ectoderm anlage and in the primordium of the posterior midgut. By stage 13, expression is also detected in the anterior cells of the midgut which will give rise to the proventriulus, the first midgut constriction, the posterior midgut and microtubule primordial as well as cells in the hindgut, all similar to portions of the pattern of wildtype odd protein expression previously described [CE8010 is 5' of the pair-rule gene escribed .fushi-tarazu (ftz) and drives expression of two of its stripes: stripe 1 at 35% and stripe 5 at 65% egg length. Using a similar CRM reporter assay, this pattern of expression was also detected by [CE8024 is 3' of the pair-rule gene POU domain protein 2 (pdm2) and appears to completely recapitulate its stage-12 expression pattern, which is limited to a subset of the developing neuroblasts and ganglion mother cells of the developing central nervous system. A similar pattern of expression was previously described for the protein product of pdm2 [nub.CE8012 is in the third intron of  of pdm2 . It is wsqz) and recapitulates the wild-type expression pattern of sqz RNA in a subset of cells in the neuroectoderm at stage 12. The wild-type sqz expression pattern was previously described [CE8027 is 3' of the gene for the Zn-finger transcription factor squeeze (escribed .CG9650) that is maternally expressed and deposited in the embryo. This strong maternal expression potentially obscures a zygotic expression pattern. Two additional adjacent genes, CG32725 and CG1958, showed no expression in whole-mount in situ hybridization of embryos.The remaining three active pCRMs cannot be easily associated with a specific gene. CE8005 drives expression in the ventral region of the embryo. It is 3' of a gene encoding a ubiquitously expressed Zn-finger containing protein , which is expressed maternally, possibly obscuring an early zygotic expression pattern (a few in situ images show a hint of striping). sbb is also expressed later in development in the ventral nervous system. An additional potential target, Otefin (Ote), is also expressed maternally and relatively ubiquitously through germ-band extension. All other nearby genes displayed in Figure in situ hybridization or by microarray.CE8016 drives a seven-stripe expression pattern in the blastoderm. It is in the first intron of ftz stripe and two stripes at 39% and 87%. It is in the first intron of ome (CG32145), which is not expressed maternally and has no blastoderm expression, but is expressed late in salivary gland, trachea, hindgut and a subset of the epidermis. All other nearby genes displayed in Figure in situ hybridization or by microarray.CE8020 drives an atypical four-stripe pattern in the blastoderm - two stripes at 7% and 26% that are anterior to the first  ome CG3245, whichWith these results, and the nine previously known enhancers, at least 15 of the 37 highest density clusters of the five transcription factors used in our initial screen have early-embryonic enhancer activity. The remainder of this paper examines 35 of the original 37 clusters, with the two tandem repeat-containing clusters excluded. We divide these 35 into three categories - 15 positives (the nine overlapping previously known enhancers plus the six new enhancers identified here), three ambiguous (the three positives without a clear regulated gene), and 17 negatives (see Table reaper), so out of 16 pCRMs located within 20 kb of a gene with appropriate expression, 15 (94%) are active enhancers.All 15 positives are within 20 kb of the transcription start site  of transcripts expressed in spatiotemporal patterns consistent with regulation by the maternal and gap transcription factors used in our screen . Only one of the 17 negatives was located within 20 kb of a plausible target (PCE8021 is 7 kb upstream of p = 0.017). The positives have a slightly higher density of binding sites, but this difference was not significant. The binding site composition of the positives and negatives are similar . Although others have reported that some factors have characteristic spacings with respect to themselves and other factors [The positives are, on average, larger than the negatives , a difference that is significant by the Komogorov-Smirnov (KS) test  would have been successfully identified by such simple comparative methods, positive pCRMs do not collectively appear distinguishable from flanking sequence on the basis of conservation alone. Although positive pCRMs are almost all in highly conserved blocks, there is a surprisingly high amount of non-coding sequence conservation throughout these regions, and most negative pCRMs are also contained in highly conserved blocks. It remains to be seen whether this difference in the conservation landscape of Drosophila non-coding sequences compared to vertebrates reflects a significant difference in the functional organization of non-coding sequences, or simply indicates that there is too little divergence between D. melanogaster and D. pseudoobscura to detect useful differences in the rates of evolution (see Discussion).We first assessed whether positive pCRMs could be distinguished from their flanking sequences based on degree of conservation. In vertebrate comparative genomics, relatively simple methods (such as VISTA ) are comD. melanogaster and D. pseudoobscura. For each pCRM-containing region, we identified orthologous contigs from the D. pseudoobscura assembly and aligned them using the alignment program LAGAN [p = 0.002 by KS test), the distribution of conservation scores for positive and negative pCRMs overlap considerably, and thus conservation alone is not a useful way of distinguishing positive and negative pCRMs  regions in randomly chosen sections of the genome Figure . PositivD. melanogaster and D. pseudoobscura, and that most D. melanogaster enhancers should have functional orthologs in D. pseudoobscura. For those enhancers we seek to identify here - namely those where binding site clustering reflects their function - we expect clustering to be found in both D. melanogaster and D. pseudoobscura. Conversely, clusters that simply occur by chance in either genome but do not reflect the function of the sequence  should not be conserved. Thus, looking for conservation of binding-site clustering should provide a valuable way of distinguishing functional and non-functional binding-site clusters in the D. melanogaster genome.We expect that most genes will have similar expression patterns in D. melanogaster binding site that overlaps a predicted D. pseudoobscura binding site for the same factor in an alignment as an 'aligned' site. We require overlap and not perfect alignment to compensate for alignment ambiguity; the overwhelming majority (85%) of aligned sites are perfectly aligned. Although there is only a subtle difference in the binding-site density in the positive and negative pCRMs in D. melanogaster (22.7 sites/kb compared to 22.2), the density of aligned binding sites in positive pCRMs (13.8 sites/kb) is nearly twice that in negative pCRMs (6.8 sites/kb). This is a highly significant difference (p < 0.001 by KS test) and aligned site density better discriminates positive and negative pCRMs than sequence conservation , and soervation ,26.D. melanogaster to be 'preserved' if it can be matched to a corresponding site in the D. pseudoobscura pCRM . If we consider both aligned and preserved sites to be conserved, then roughly 80% of the sites in positive pCRMs are conserved compared with 40% in negative pCRMs.To characterize the extent of binding site conservation independent of positional conservation, we computed a second measure of binding-site conservation. We consider an unaligned binding site in D. pseudoobscura orthologs of active D. melanogaster CRMs we observe an increase in binding-site density that cannot be explained by the positional conservation of sites found in D. melanogaster or the random occurrence of sites in the genome. Several of the 15 positive CRMs have high densities of these preserved but unaligned sites, but two in particular, runt stripe 3 and hairy stripe 6, stand out from the rest. These two have almost as many preserved sites as strictly aligned sites.The density of preserved but not aligned sites in positive pCRMs (4.3/kb) is considerably higher than in negative pCRMs (2.2/kb) or random sequences (1.8/kb). Thus, in the Aligned plus preserved (conserved) site density Figure  almost pD. melanogaster and D. pseudoobscura successfully distinguishes positive and negative predictions made using the D. melanogaster sequence alone, we incorporated comparative sequence data into our enhancer-prediction algorithm CIS-ANALYST [As the conservation of binding sites and binding-site clusters between -ANALYST . Instead-ANALYST .Using 17 negative pCRMs and an expanded set of 25 positive pCRMs  as are five other known enhancers. One of our negative pCRMs, CE8021, is ranked number 12.As eCIS-ANALYST has markedly better specificity than CIS-ANALYST, we sought to identify BCD, HB, KR, KNI and CAD targets that were missed with the relatively stringent criteria used in our previous analysis. Rather than use a stringent cutoff (15 binding sites per 700 bp) as we did in , we perfDrosophila embryonic expression patterns [To focus our search for new enhancers on genes likely to be regulated by BCD, HB, KR, KNI and/or CAD, we searched FlyBase  and a dapatterns  and idencis-regulatory modules in Drosophila. Analysis of the in vivo activity of 36 high-density clusters of predicted BCD, HB, KR, KNI and CAD binding sites identified in our previous study [in vivo. An evolutionary analysis of these sequences - based on comparisons of the D. melanogaster and D. pseudoobscura genomes - shows that sequence conservation alone can not reliably discriminate cluster-containing regions that function in vivo from those that do not. However, a new method that combines binding-site clustering and comparative sequence analysis to search for binding-site clusters that are present in multiple species does reliably discriminate active and inactive clusters. Using this method, we make several hundred predictions of new CRMs, a large number of which are located near likely target genes.We performed a large and comprehensive evaluation of the efficacy of computational methods for the identification of functional us study  offers cThe success of relatively simple binding-site clustering methods here and in other work is remarkable given the crudeness of these methods. As our negative predictions demonstrate, the mere presence of a cluster of binding sites is not sufficient to make an active embryonically expressed CRM. Although these 17 sequences have binding-site densities and compositions indistinguishable from their functional cousins, they do not function as enhancers in a simple transgene assay.D. pseudoobscura orthologs of these negative pCRMs have binding-site clusters, and few are near genes with appropriate expression patterns. Thus it is unlikely that many function in their endogenous locations in vivo.It is possible that some of these negative pCRMs may be functional enhancers that respond to the factors used in our screen, perhaps requiring a different promoter or other flanking sequences not used in the transgene. While further experiments could address this possibility, we felt these were a low priority, as few of the cis-regulatory sequences.Both the general activity and, more important, the specific regulatory output of a CRM are a complex, and still poorly understood, function of the specific architecture of its sites. The emerging picture of the ordered multiprotein complexes that mediate enhancer activity suggests constraints on enhancer composition and architecture ,2,47 whoin vivo function related to their observed expression patterns. However, the poor conservation of these elements in D. pseudoobscura suggest that they do not have a regulatory function, and raises the possibility that some 'random' clusters of binding sites  have the necessary characteristics to be active enhancers in the proper genomic environment . That any such sequences exist suggests that the compositional and architectural constraints on binding sites in enhancers may be fairly weak.It is intriguing that three of the clusters we tested direct expression patterns that bear no obvious relationship to the expression of a neighboring gene despite our extensive efforts to identify such genes. We cannot yet exclude the possibility that these pCRMs have an Whatever the nature of these constraints, it is clear that binding-site density is not the sole defining characteristic of functional enhancers. However, it is a surprisingly effective distinguishing one, and the usefulness of this and related methods  suggestsftz and odd. ftz and odd are generally classified as 'secondary' pair-rule genes whose expression is governed by other pair-rule genes rather than by the maternal and gap transcription factors that govern the so-called 'primary' pair-rule genes    . Howeveftz enhancer is an evolutionary relic of the homeotic role played by ftz in primitive insects [D. melanogaster and D. pseudoobscura, it seems unlikely that this element is purely vestigial. In fact, Yu and Pick [ftz gene and show that stripes 1 and 5 appear before other ftz stripes and they postulate the existence of stripe-specific regulatory elements that may exist outside of the characterized zebra and upstream elements such as the one identified and characterized in this study. The conservation of binding sites in both the ftz and odd enhancers suggest that they play an important role in development, and further call into question the distinction between primary and secondary pair-rule genes.It has been suggested that the  insects , a view and Pick  examinednub  and pdm2 are expressed in the anterior and posterior midgut primordium and in neuroblasts. CE8011, found immediately upstream of nub, regulates its early expression, and not its later neuroblast expression. In contrast, CE8012, found in an intron of pdm2 regulates its expression only in neuroblasts and not earlier. While we did not detect a neuroblast enhancer for nub or a blastoderm enhancer for pdm2 in our single-species binding-site cluster search, a number of interesting pdm2 regions were discovered in our eCIS-ANALYST search (two are listed in Table Two of the new enhancers (CE8011 and CE8012) are adjacent to and apparently regulate two linked genes with very similar patterns of embryonic expression. Both Drosophila enhancers have been known to work at distances of up to 100 kb, but most are within 10 kb of their target gene. All of our true-positive predictions were within 10 kb of the known or predicted transcription start site of a gene with a pattern that was known, or plausibly could have been, regulated by the five regulators used in our screen . In contrast, only one of the negative predictions was this close to such a gene - an additional four were within 50 kb. As the comprehensive atlas of embryonic expression patterns is completed [The accuracy of our enhancer predictions would almost certainly be improved if we restricted our search space to genomic regions adjacent to genes known to be regulated by particular transcription factors. ompleted ,53 it wiDrosophila. Even assuming the availability of binding data for all of these factors, it will not be possible to search for targets of all combinations of these factors - there are too many possibilities. This is not just a practical problem - it is a fundamental statistical problem. While the false-positive rate for a single combination of factors is low, if we tried even all pairs of factors, it is likely that every region of the genome would have a high binding-site density for some collection of factors. Sequence data from other Drosophila species may allow us to determine which of these collections are conserved and therefore likely to be functional, but it is unlikely that all aspects of regulation can be inferred from comparative analyses and therefore it is essential that we continue to dissect the regulatory network by traditional means.Comprehensive methods for inferring regulatory interactions where they are not already known will be critical for the widespread application of binding-site clustering methods. In addition to allowing less stringent focused screens, they will also help overcome the combinatorial challenge raised by the existence of up to 700 sequence-specific transcription factors in Drosophila transcription factors. The initial success of methods that use in vitro binding data to predict regulatory targets has prompted the characterization of binding specificities for many additional factors. However, the heterogeneity of approaches used makes it difficult to combine these data in an optimal manner. In addition, most of the available transcription factor binding data consists of a few to several dozen high-affinity sites. While these data are very useful, they do not fully represent the binding capacity of a factor and thus do not permit the identification of intermediate or low-affinity sites which are known to be important in some regulatory systems [A greater current limitation in the widespread application of binding-site clustering methods is the absence of high-quality binding data for most  systems . We have systems  to charaD. melanogaster and D. pseudoobscura was surprising. A major motivation for the National Human Genome Research Institute (NHGRI) support of the D. pseudoobscura genome sequencing was the identification of conserved regions that would guide the annotation of functional sequences in D. melanogaster. D. pseudoobscura was chosen as the second member of this genus to be sequenced in part because it was felt that it had separated from D. melanogaster sufficiently long ago that non-functional sequences would exhibit substantial divergence. However, despite an evolutionary separation that is greater than human and mouse (an average synonymous substitution rate of 1.8-2.6 substitutions/site [The extent of non-coding sequence conservation between ons/site  comparedons/site ), and deons/site .One reason for the limited efficacy of these methods is that they do not recognize the specific patterns of conservation characteristic of different classes of functional sequences. For example, coding sequences can be easily recognized from the characteristic triplet pattern in evolutionary rates where the third (and often synonymous) position of codons tends to evolve at a greater rate than the first two positions ,57. Simiodd stripe enhancer) and PCE8015  that this sequence is actively responding to the presence of these binding sites. The poor conservation of binding sites in PCE8015 (no greater than is found in random regions of genome) suggests either that the BCD, HB, KR, KNI and CAD sites in this region are not functional or that the region is undergoing rapid functional diversification. Of course the absence of binding site conservation does not suggest that the sequence is non-functional, merely that these sequences are unlikely to have the particular function we are studying here.Contrast PCE8010  the probability of finding a site for the same factor in the orthologous region of D. pseudoobscura, even if the site is not in the same  position. Thus, in this set of positive pCRMs, there appears to be selection to maintain binding site composition, but not always the specific order and orientation of sites. This is consistent with models of enhancer plasticity that have been proposed and discussed elsewhere [In addition to this colinear conservation, we also observe that there is an overall enrichment for binding sites in positive pCRMs independent of the conservation of individual sites. Specifically, the presence of a binding site for a factor in a positive lsewhere ,59-61.D. melanogaster and D. pseudoobscura) most binding sites are conserved and found in the same place. Over longer evolutionary distances, individual binding sites are often poorly conserved even as the overall composition and function of a CRM is conserved.The relative importance of binding-site architecture and binding-site composition to maintaining the function of an enhancer over evolutionary time remains unclear. Over relatively short evolutionary distances . The PCR product was digested with AscI and NotI, and inserted in its native orientation into the AscI-NotI site of a modified CaSpeR-AUG-bgal transformation vector [eve basal promoter, starting at -42 bp and continuing through codon 22 fused in-frame with lacZ [w1118 embryos, as described previously [DNA fragments identified as candidate CRMs were amplified from either bacterial artifical chromosome (BAC) or n vector  containiith lacZ . The P-eeviously ,64. Traneviously  from eacin situ RNA hybridizations were performed as previously described [gt), RE14252 (odd), RE34782 (nub), RE49429 (pdm2), and RE47384 (sqz). Exon 1 of the ftz gene was amplified from genomic DNA using forward primer 5' GCGTTGCGTGCACATC and reverse primer 5' ATTCTTCAGCTTCTGCGTCTG. The PCR product was cloned into the TA vector (Invitrogen) and used to generate ftz RNA probe.Embryonic whole-mount escribed . RNA prolacZ RNA probes were labeled with digoxigenin-11-UTP (Roche). Hybridizations were performed as described above with the following modifications: (1) 2 μl of each probe were added to give a final concentration of 1:50; (2) sequential alkaline phosphatase staining was performed first with Sigma Fast red to detect endogenous transcripts, stopped by washing for 30 min in 0.1 M glycine-HCl pH 2.2, 0.1% Tween-20 at room temperature, and then continued as described to detect lacZ expression.RNA probes, using cDNAs or genomic DNA as templates, were labeled with fluorescein-12-UTP while The input to the genome assembly was the set of whole-genome shotgun reads from the Baylor Genome Sequencing Center retrieved from the National Center for Biotechnology Information (NCBI) Trace Archive, consisting of 2,607,525 total sequences. After trimming the sequences to remove vector and low-quality regions, the average read length was 607 bp. Approximately 75% of the reads were from short insert (approximately 2.5-3.0 kb) libraries, with another 25% from longer (6-7 kb) libraries. Another 46,040 reads came from the ends of 40-kb fosmids.j, which measures the probability that a contig is repetitive on the basis of its depth of coverage. The default setting is very conservative: if a contig has more than 50% likelihood of being repetitive, it is marked as such and is set aside during most of the assembly process. For large highly repetitive mammalian genomes this setting may be appropriate, but for D. pseudoobscura we found that setting it to 90% or higher produced considerably better contigs, while apparently causing few if any misassemblies.We ran the Celera Assembler several times, and found that by adjusting one parameter in particular we could produce considerably better assemblies. In particular, the assembler has an arrival rate statistic The overall assembly contained 10,089 scaffolds and 10,329 contigs, containing 165,864,212 bp. The estimated span of the scaffolds, using the gap sizes estimated from clone insert sizes, is 172,362,884. The largest scaffold was 3.05 million base-pairs (Mbp) and the scaffold N50 size was 418,046.  There are 308 scaffolds larger than 100,000 bp, whose total span is 129.5 Mbp. The N50 contig size, using 166 Mbp as the genome size (not counting gaps), was 43,555. Another measure of assembly quality is the number of large contigs: if we define 'large' as 10 kbp, then the assembly contains 3177 large contigs whose total length is 131,067,828 bp.  All of our contigs and scaffolds are freely available by anonymous ftp at [D. melanogaster and D. pseudoobscura in regions containing pCRMs were determined as follows. The D. melanogaster genomic sequence of the region of interest (with known repetitive elements masked) was extracted from a BioPerl genome database [D. pseudoobscura contigs/scaffolds were identified using WU-BLAST 2.0 [D. melanogaster sequence were treated as separate hits. After examining dot-plots of the hits, we noticed a large number of small, local inversions that were found in both our assembly and the assemblies released by the Baylor Human Genome Sequencing Center. We used BLASTZ [D. pseudoobscura sequence. Each D. pseudoobscura sequence was aligned to the D. melanogaster corresponding sequence using LAGAN 1.2 [eve and h loci contain three pCRMs each, and PCE8003 and PCE8004 are within 20 kb of each other). Twenty-eight regions had aligned D. pseudoobscura sequence that spanned all or most of the region. For three regions  we were not able to identify large regions of orthologous sequence; these were excluded from subsequent comparative analyses. Dot-plots of the alignments from all 30 regions are available at [The extent and pattern of conservation between database  containidatabase . PotentiLAST 2.0  using ded BLASTZ ) to autoAGAN 1.2  with deflable at .D. melanogaster bases aligned to the identical base in aligned regions (percent identity).The conservation of a specific genomic segment was scored as the fraction of D. pseudoobscura binding site for the same factor in the LAGAN alignment. Only overlap, and not strict alignment, was required to compensate for small errors in the alignment. A non-aligned binding site was considered 'preserved' if it could be matched to a D. pseudoobscura site for the same factor within the bounds of the pCRM, allowing each D. pseudoobscura site to be the match for only a single D. melanogaster site. The number of aligned plus preserved sites for each factor in a region is thus equal to the minimum number of sites for that factor in the two species.We used two definitions of binding-site conservation. A binding site was considered 'aligned' if it overlaps a predicted D. melanogaster genome (with annotated repetitive elements and transposable elements masked) and the D. pseudoobscura scaffolds described above. NUCmer was run with the command line parameters (-c 36 -g 10 --mum -d 0.3 -l 9). NUCmer generated a collection of short, highly conserved regions of homology ('anchors') spaced on average every 1 kb throughout the D. melanogaster genome. Anchors flanking either side of a D. melanogaster region of interest were used to pull out the corresponding D. pseudoobscura region, and additional flanking anchors were examined to ensure that the region was unambiguously orthologous. The region identified was re-aligned to the melanogaster region with LAGAN 1.2 using default settings.To develop an orthology map for genome-wide searches, we used NUCmer  to alignD. melanogaster genome, sampled uniformly from all non-coding sequence. For 3,300 of these, we could find orthologous regions in D. pseudoobscura, and these were used to calculate the properties of random non-coding sequence shown in Figure To characterize properties of non-coding sequences across the genome, we picked 4,000 1-kb segments of the D. melanogaster genome were determined as described in [Binding-site clusters in the ribed in , where tribed in  using thribed in . In the D. melanogaster cluster, we identified the corresponding D. pseudoobscura region using the homology anchors described above. A pairwise alignment was made using LAGAN 1.2 (default parameters), and the number of aligned and preserved binding sites were determined as described above. The 2-kb flanking either side of the pCRM was included in the alignment to avoid edge effects, and was subsequently removed when calculating pCRM properties.For each potential D. melanogaster sites followed an approximately logarithmic curve . From this observation, we classified a D. melanogaster binding site cluster as conserved if:We examined our functional (positive) and non-functional (negative) pCRMs and noticed that in the positives, the lower bound for the number of conserved sites as a function of NSm is the number of binding sites in the D. melanogaster pCRM and NSc is the number of conserved binding sites. Different values of the logarithmic base b give different behavior. The data shown in Additional data file 3 support values of b between 1.15 and 1.4. We defined a more intuitive parameter, CF (conservation factor), which can range from 0 to 1 where 0 is the least stringent threshold (b = 1.4) and 1 is the most stringent (b = 1.15)where b = 1.4 - (CF * (1.4 - 1.15))     (2)CF values of 0.25, 0.5, 0.55 and 0.75 and manually inspected the results with respect to false-negative and false-positive rates based on our 15 positive and 17 negative pCRMs . While we did not strictly optimize a single metric, we picked the values that gave a reasonable balance between false positives and false negatives, b = 0.25 for aligned sites alone, and b = 0.55 for aligned plus preserved sits.We performed genome searches with D. melanogaster. We merged overlapping clusters from runs 1-3, yielding 929 non-overlapping clusters as described in Results.eCIS-ANALYST genome searches were run with the following parameters: min_sites = 10, wind_size = 700 (run #1), and min_sites = 13, wind_size = 1,100 (run #2). All conserved clusters (with conservation defined as described in Equations 1 and 2 above) were combined. In order to capture weaker clusters, we performed an additional run (run number 3) using min_sites = 9, wind_size = 700. For this low stringency run, we used a non-standard conservation threshold different from the one described above, accepting all clusters with at least four aligned plus preserved sites, independent of the number of sites in Four metrics were then used to rank these 929 pCRMs: the number of aligned binding sites; the density of aligned binding sites; the number of aligned plus preserved binding sites; and the density of aligned plus preserved binding sites. All values were normalized according to background distribution of random non-coding sequences. The four normalized values were then summed to compute an overall score, which was then renormalized to arrive at a final z-score used to rank pCRMs in Tables The following additional data files are available with the online version of this article.D. melanogaster genome. The blue line plots the cumulative distribution. The colored asterisks show the average values for each class of pCRM. The panel below the histogram shows the values for each pCRM .Additional data file Additional data file D. melanogaster pCRMs to the number of aligned sites (top panel) and aligned plus preserved sites (bottom panel). Blue dots represent the 15 positive pCRMs from the text; green dots the ten known CRMs that were below the threshold used in [Additional data file  used in ; red dotD. melanogaster sequence relative to their D. pseudoobscura ortholog. The location of binding sites in D. melanogaster, binding sites in D. pseudoobscura and aligned binding sites along with the density of sites averaged over 700 bp are shown in the bottom three panels for each region.Additional data file Additional data file The binding site densities (column 1), aligned site densities (column 2), and aligned plus preserved site densities (column 3) for individual transcription factorsClick here for additional data fileExpression patterns of 65 genes adjacent to 122 pCRMs identified by eCIS-ANALYSTClick here for additional data fileD. melanogaster pCRMs to the number of aligned sites (top panel) and aligned plus preserved sites (bottom panel)Discrimination of positive and negative pCRMs. Comparisons of the number of predicted binding sites in Click here for additional data fileNew pCRMsClick here for additional data fileThe primers used to amplify pCRMs for transgenicsClick here for additional data fileAdditional information from Table 2Click here for additional data fileAll new pCRMs from genome-wide eCIS-ANALYST located within 20 kb of annotated transcriptClick here for additional data fileAll new pCRMs from genome-wide eCIS-ANALYST located more than 20 kb from annotated transcriptClick here for additional data fileGenes with anterior-posterior patterns and the source of the informationClick here for additional data fileAll new pCRMs from genome-wide eCIS-ANALYST located within 20 kb of gene with anterior-posterior patternClick here for additional data fileAll new pCRMs from genome-wide eCIS-ANALYST located between 20 kb and 50 kb from gene with anterior-posterior patternClick here for additional data file


Animal studies involving rabbits and a porcine model have demonstrated host tolerance of the implant. There have been no reports describing the histological changes in a human laryngectomy specimen with a Gore-Tex implant.Expanded polytetrafluroethelyne (e PTFE, Gore-TexThe histological findings in a laryngectomy specimen of a patient previously implanted with e PTFE for medialization of a paralyzed vocal fold following excision of a vagal neurofibroma were studied.Histopathology revealed a mild foreign-body giant cell granulomatous reaction with some associated fibrosis. The granulomatous response was limited to the periphery of the Gore-Tex and although it closely followed the profile of the material it did not encroach into or significantly break up the material. There was no significant neutrophilic or lymphocytic inflammation.Our findings are consistent with the animal models confirming that Gore-Tex implantation does not result in a significant granulomatous reaction in the human larynx over a 13-month period. Moreover, there is no evidence of resorption or infection. Further, the lack of lymphocytes in association with the granulomas indicates that there is no significant immunological hypersensitivity. Histologically, the slight permeation by connective tissue is similar to that seen in Gore-Tex vascular and cardiac implants. The degree of the slight giant cell response appears to be dependent on the profile of the material; a sharp edge incited more of a response than a flat surface. Unilateral vocal fold paralysis is symptomatic when it results in failure of the mobile vocal fold to approximate the paralyzed vocal fold during adduction. Despite the lack of movement, the paralyzed vocal fold will often contact the contra lateral mobile vocal fold permitting adequate glottic closure .Medialization laryngoplasty is a common procedure used to restore glottic competence. This procedure was popularized by Ishiki and initially used in patients with vocal fold paralysis. In recent years, the indications for this procedure have expanded to include most forms of glottic incompetence, including the use of bilateral medialization in mobile vocal folds for vocal fold bowing and atrophy .Although medialization is now widely accepted, the choice of implant material is still a subject of controversy. The ideal vocal fold injection material would be readily available, have excellent biointegration with no or minimal immunologic response, has an excellent biomechanical in vivo match to the injection site tissues, and is deliverable through a fine-gauge needle. Such a material does not presently exist.Teflon (Polytetrafluoroethylene) was the first modern material used for vocal fold injection. Long-term results, unfortunately, have shown an unacceptably high rate of granuloma formation associated with Teflon injection. This results in a gradual degradation of the injected material by foreign body giant cells and fibroblastic proliferation in the location of the implant . Autolog®) has been advocated as an implant material for medialization of the vocal fold [Recently, an expanded polytetrafluroethelyne implant  with cricothyroid approximation. Videofluroscopy and Speech assessment confirmed an improvement in aspiration and speech. Six months later, her symptoms recurred with subsequent aspiration pneumonia. A decision was made after lengthy counseling to proceed with a laryngectomy 13 months following the implantation of Gore-Tex. Figure The patient subsequently underwent right medialization using Gore-Tex implant  declare that they have no competing interests.All authors equally contributed towards the background research and writing of the paper.The pre-publication history for this paper can be accessed here:


Desmoid tumour is a benign, non metastasising neoplasm characterised by an elevated deposition of organic macromolecules in the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the degradation of ECM macromolecules. The MMPs and their natural inhibitors (TIMPs) have been implicated in tumour growth, invasion and metastasis. In this study we provide evidence that the in vitro cultured cell line from desmoid tumour accumulates more collagen fibres in the ECM than healthy fibroblasts.3H-thymidine incorporation, MMP expression by substrate gel zymography and TIMP expression by Western blot analysis.We investigated collagen accumulation by Desmoid fibroblasts showed a reduction in MMP activity and an increase of type I and III collagen and TIMPs compared to normal fibroblasts.The increase in collagen in desmoid fibroblasts was due to inhibited collagen degradation (reduction of MMP activity) rather than to increased collagen synthesis. Adding toremifene, an anti-estrogen triphenylethylene derivate, to desmoid fibroblasts reduced collagen accumulation by decreasing mRNA expression and increasing collagen degradation. Confluent cultures were obtained after 48 h of in vitro maintenance. The cells were cultured for 12 h in MEM. The medium was then discarded to avoid serum factor contamination. Toremifene was dissolved in ethanol and all the cultures were maintained in MEM containing ethanol or MEM containing toremifne in ethanol and treated as described below.Fibroblast cell lines were obtained from patients with Gardner's syndrome and were provided by NIGMS . The GMO 6965 cell line was obtained from phenotypically healthy fibroblasts, and the GMO 6888 cell line was obtained from desmoid fibroblasts. All cell lines were cultured in Eagle's minimal essential medium (MEM)  supplemented with 20% fetal bovine serum (FBS) , 2% non-essential amino acids (GIBCO), 2 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin in a humidified 5% CONormal (control) and desmoid fibroblasts were cultured for 24 h in MEM and ethanol or MEM containing 1 μM toremifene in ethanol . Then 50 μl of sterile 0.4% trypan blue solution  was added to each culture well; cultures were incubated at 37°C for 15 min. Viable cells (trypan blue negative) and dead cells (trypan blue positive) were counted by a Burker chamber.3H-proline  in the presence or absence of 1 μM toremifene. In a second set of experiments desmoid fibroblasts were cultured in MEM supplemented with L-ascorbic acid (50 μg/ml), β-aminopropionitrile fumarate (50 μg/ml) for 48 h with or without toremifene. 3H-proline was added for 48 h, for the last 24 and for the last 3 h. At the end of the labelling period collagen was extracted using the method of Webster and Harvey  dCTP (3000 Ci/mM) by random priming (Amersham RPN 1601). Hybridisation was performed at 65°C overnight using 106cpm/ml probe in the same buffer used for pre-hybridisation. After hybridisation, the nylon membrane was washed twice in 1 mM EDTA pH 8, 20 mM Na2HPO4 pH 7.2 and 5% SDS at 65°C (5 min each) and then washed twice with 1 mM EDTA pH 8, 20 mM Na2HPO4 pH 7.2 and 1% SDS at 65°C (5 min each). The filters were stripped and re-hybridised with a GAPDH probe to assess blot loading. For autoradiography the membranes were exposed to Kodak X-Omat film  at -80°C for 1 day. Autoradiographies were analysed by computerised scanning densitometry. Results are expressed as the ratio of procollagen α1(I)/control GAPDH densitometry signals.Total RNA was isolated from confluent cultures of normal and desmoid fibroblasts maintained for 48 h in MEM alone or supplemented with 1 μM toremifene using the method of Chomczynski and Sacchi . For NorA 670 bp Eco RI-Hind III cDNA fragment from human pro-collagen α1(I) and a 1.3 kb PstI cDNA fragment from rat glyceraldehyde-3-phosphate dehydrogenase were used as probes in hybridisation .2, 0.02% sodium azide, pH 7.4) and then dialysed overnight against the same buffer. The latent procollagenase was activated with trypsin (10 μg/ml) and then the trypsin was inactivated with soybean trypsin inhibitor (100 μg/ml). Acetic acid soluble type I collagen (25 μl of a solution 2 mg/ml) from bovine skin was incubated with the activated collagenase solution for 24 h. The products of the collagen digestion were separated by electrophoresis using 6% acrylamide gel containing SDS. The gels were stained with 0.25% Coomassie brilliant blue G-250 , destained appropriately  and fixed . The digestion products were quantified with a computerised scanner.Collagenase activity was determined using the method of Khorramizadeh et al., . Conflueg to remove cell debris, dialysed against bidistilled water for 24 h, lyophilised and used for zymography and Western blot analysis as described below.Confluent normal (GMO 6965) and desmoid (GMO 6888) fibroblasts were washed with 0.9% NaCl and cultured for 12 h in serum-free MEM. This medium was discarded to avoid contamination by serum factors and cells were cultured for the next 24 h in MEM and ethanol (control) or in MEM containing 1 μM toremifene in ethanol . Conditioned media (CM) were collected and centrifuged for 10 min at 350 2, 0.02% Brij-35 and 200 mM NaCl at 37°C for 24 h. Gels were stained with Coomassie brilliant blue G-250. Proteinase activity, observed as cleared (unstained) regions, was converted to dark regions to better observation of bands.CM were analysed for gelatinases and collagenases by zymography. Samples were separated under non reducing condition on 6% polyacrylamide gels containing 1 mg/ml of gelatin  or 1 mg/ml collagen (Sigma) . In one CM were analysed for type I and type III collagen, MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 by Western blotting using specific monoclonal antibodies. Aliquots of CM, containing 50 μg of proteins, were separated on SDS-10% polyacrylamide gels under reducing conditions and transferred on to a nitrocellulose membrane. The membrane was blocked with blocking solution  and incubated with the specific monoclonal antibody in antibody solution . Bound antibody was detected with a sheep anti-mouse peroxidase-conjugated antibody in antibody solution. Western analysis was performed using chemiluminescence reagents from Amersham Pharmacia Biotech.Protein concentrations were determined by the Lowry assay  of aliqut-test. Data are expressed as the means ± SD of four determinations. In other experiments, the results are reported as means ± SD of three separate experiments, each performed in quadruplicate. Statistical analysis was performed by Student's two-tailed t-test and by analysis of variance (ANOVA) followed by Sheffe F-test.In some experiments, statistical analysis was performed using Student's The amount of dead cells and viable cells in normal fibroblasts, desmoid fibroblasts and desmoid fibroblasts plus toremifene was evaluated after 24 h of in vitro maintenance in the presence of trypan blue Table . Granted3H-proline  mRNA level in normal and desmoid fibroblasts . Toremifene down regulated procollagen mRNA expression by 58% in desmoid cells.Northern blots were performed to analyse procollagen αMedia from normal and desmoid fibroblasts with or without toremifene were analysed by Western blotting to evaluate the presence of type I and III collagen using specific monoclonal antibodies Fig. . Densito1 and α2 chains of type I collagen into their corresponding 3/4 and 1/4 fragments than the collagenase in desmoid fibroblasts  gene expression, which showed mRNA levels were only lower in desmoid cells treated with toremifene. Normal and desmoid fibroblasts expressed different amounts of MMPs. Several studies suggest that MMPs are over-expressed in malignant tumour progression and facilitate both local tumour invasion and metastasis [Desmoid tumour is a benign non-invasive and non-metastasising neoplasm with an abnormal ECM macromolecule deposition which is stimulated by TGFβ1 ,23,24. T1 . There iroblasts , in the tastasis ,28. Difftastasis . They matastasis ,30. Nishtastasis  showed Mtastasis ,33. Thertastasis . In thistastasis . Moreovetastasis  which istastasis . Using Wtastasis ; hence, Upregulation in both inhibitors of MMPs may explain why the Desmoid tumour is characterised by an abundant deposition of ECM macromolecules and is neither malignant nor invasive. Toremifene addition to desmoid fibroblasts reduced the accumulation of collagen fibres but its mechanism of action remains unclear. Toremifene increased MMP-1 and MMP-2 activity by 8% and 25% respectively and decreased TIMP-1 by 18%. Despite these modest effects type I collagen degradation in 3/4 and 1/4 fragments increased almost 4-fold.1 was 6-fold higher in desmoid than in normal fibroblasts and that toremifene significantly reduced TGFβ1 activity and TGFβ1 membrane-receptors [1 because the growth factor enhances organic macromolecule accumulation in the ECM via a reduction in MMP-1 and MMP-2 [1 activity. Further studies on the regulation of MMP activities may clarify the role of toremifene on ECM degradation and provide important clues about pathogenesis of desmoid tumour.Our previous studies showed that TGFβeceptors . So the nd MMP-2  and an ind MMP-2 , so favoThe author(s) declare that they have no competing interests.CB carried out collagen synthesis, collagenase activity and drafted the manuscript.CL and GB participated in the design of the study and carried out Northern blot analysis.LM and GG carried out RT-PCR, zimography and oestrogen receptor assay.AB and LC carried out Western blot analysis and performed the statistical analysis.PL 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:


Current business models for drug development are inefficient and ineffective - drugs are not reaching all who need them. Hubbard and Love contend that it is time to explore some alternatives The AIDS crisis has brought to public notice what has always been generally true—that the existing business model for drug development leads to high prices and unequal access. There is now widespread dissatisfaction with drug prices in both the developed  and deveToday's high drug prices are a direct consequence of a business model that uses a single payment to cover both the cost of manufacture of a drug and the cost of the research and development (R&D) carried out by manufacturers to discover it. A 20-year patent-based marketing monopoly is then granted to the drug's developers to prevent their prices being undercut by ‘generic’ copies produced by manufacturers who do not have R&D costs to recover. Preventing such ‘free riding’ on R&D has become a global trade issue at the World Trade Organisation (WTO) . The impUnfortunately, monopoly-based business models have unpleasant side effects. Since the primary responsibility of any company is to maximise return on investment, it is unsurprising that there is pressure on pharmaceutical companies to set drug prices to whatever level gives the highest return, excluding those individuals who cannot afford to pay, rather than maximising the number of patients treated. There is also pressure to misuse the power given by patents, using them as anticompetitive weapons to block innovation and extend marketing monopolies. And there are growing fears that the huge growth in the use of patents is in itself starting to inhibit research . SomethiPropping up the present structure for financing R&D  is the wby any means, not just by the implementation of strict TRIPS intellectual property rules, as at present.Analysis of worldwide drug expenditure shows that spending varies, but is close to 1% of the gross domestic product (GDP) in most developed and developing countries . AssuminCountries that met the norm would then be free to decide whether they wanted to follow a strictly patent-based system as at present, with high drug prices for 20 years, or experiment with new models based on the creation of separate competitive markets for sales and R&D . CountriThe existing system , despiteOne obvious approach is direct funding of drug development. For example, the National Institutes of Health (NIH), the national agency in the United States, already spends $27 billion per year on research, a substantial amount of which is directed towards drug development, including clinical trials. The NIH already has a track record in developing important drugs for severe illnesses, such as cancer or AIDS, showing that this is a viable model. It is also widely recognised that much of the research carried out across the world by similar agencies underpins the existing commercial research that leads to new drugs.www.mmv.org), the Global Alliance for TB Drug Development , the International AIDS Vaccine Initiative (www.iavi.org), the Drugs for Neglected Diseases Initiative (www.dndi.org), and the Institute for One World Health .Governments could expand direct funding for drug development, either through the existing structures in academia or through funding R&D arms of existing companies to carry out specific drug R&D. Such directed drug development funding could be similar to existing nonprofit development projects, such as those currently resourced to address treatments for neglected diseases like malaria and tuberculosis (TB). Examples of such projects are the Medicines for Malaria Venture  could easily become bureaucratic and inefficient.As a possible alternative, we propose a competitive financing scheme that would work through R&D investment intermediators. These R&D funds would be licensed and regulated (like pension funds). Their role would be to manage R&D assets on behalf of consumers. Individuals (or employers) would be required to make minimum contributions into R&D funds, much as there are mandatory contributions to social security or health insurance or to pension funds. Government would set the required contribution, but the individual (or employer) would be free to choose the particular intermediator that received their contributions. Intermediators would compete to attract funds to invest in R&D on the basis of their prowess for drug development and upon their priorities. Different business models for financing R&D could be tested in such a market, with intermediators experimenting with prize systems, direct investments in profit or nonprofit entities, open collaborative public good models, or other approaches.We believe the economics of a change in the paradigm for funding R&D are highly favourable. Taken together, the two core steps of changing the trade framework and moving away from marketing monopolies can change the world in a positive way. We can raise global R&D levels as a matter of policy and ensure that resources flow into the areas of the greatest need, and we can do so knowing that the poor and the rich will have access to new inventions at marginal cost. Policy-makers will be weaned from their current unhealthy addiction to ever-higher levels of intellectual property rights as the only instrument to raise R&D levels, a path that has increasingly reached diminishing returns or become counterproductive. With new instruments to address the overall levels of R&D investment, policy-makers can more constructively address the well-known inefficiencies in the patent system without the fear that global R&D levels will suffer and explore alternative models . At the The most important is the World Trade Organisation (WTO) agreement on Trade-Related Aspects of Intellectual Property (TRIPS), which requires member countries to issue 20-year patents on all fields of technology. All but the least-developed countries must comply by 2005. Going much further than the TRIPS are a plethora of regional and bilateral ‘TRIPS-Plus’ trade agreements, pushed in particular by the United States, which require even higher levels of intellectual property protection, such as limitations on the use of compulsory licensing, a tool used by governments to override the strong exclusive rights of a patent in return for compensation to patent owners. In 2001 the WTO adopted the Doha Declaration on TRIPS and Public Health, which said that ‘the Agreement can and should be interpreted and implemented in a manner supportive of WTO Members’ right to protect public health and, in particular, to promote access to medicines for all'. In order to promote ‘access to medicines for all’, countries have to find new ways of financing R&D.


Anyone who's ever had a physical exam knows the monosynaptic stretch reflex arc. When the doctor taps your patellar tendon with a hammer, your quadriceps muscle briefly stretches and your knee responds with a quick kick. This involuntarily reflex is mediated by muscle spindles, specialized muscle structures containing both proprioceptive (sensory) and motor neurons. Proprioceptive neurons send information about how much the muscle is stretched to the spinal cord, and motor neurons emanating from the spinal cord tell the muscle to contract, which corrects the stretch.This reflex circuit is established in the developing embryo, when neurons migrate through and around developing tissues and send their axons to their signaling targets. Many neurons will successfully extend their axons into a target—which might be a muscle, another neuron, or some other tissue—but not all survive the process. Whether a neuron lives or dies depends on a family of growth factor proteins called neurotrophins. If a sensory neuron doesn't get enough neurotrophin-3 (NT-3), it will die.Proper development of the sensory/motor circuit also depends on NT-3, which is expressed in limb buds, muscle spindles, and the ventral spinal cord: muscle spindle development depends on sensory axons, and motor neuron connections depend on developing limb buds. It has not been clear, however, whether NT-3 simply ensures the survival of proprioceptive neurons or whether it also helps establish the proprioceptive reflex arc. In a new study, Reha Erzurumlu and colleagues demonstrate a clear role for NT-3 in axon guidance.Bax, which activates the cell death pathway for sensory neurons. This system removes NT-3 signaling without killing the sensory neurons, so the researchers can investigate any effects NT-3 may have on axon behavior.Attempts to investigate the axon guidance theory have been difficult since sensory neurons die without NT-3. To circumvent this problem, the authors developed a “double knockout” mouse model that deletes both the NT-3 gene and the apoptosis-promoting gene Erzurumlu and colleagues showed that sensory neurons, in the absence of NT-3 signaling, project to the right places but never reach their final destination. In normal development, sensory neuron axons travel into the ventral spinal cord and form synapses with motor neuron dendrites in the ventral horn to establish the reflex arc circuit. In the double knockout mice, sensory neurons manage to extend into the spinal cord, but then get lost; they can't find the ventral horn, so they never form a synapse with the motor neuron dendrites. The failure to establish connections between the sensory axons and motor neurons in mice lacking NT-3, the authors argue, indicates that NT-3 is required for proper axon targeting.Similarly, deprived of NT-3 signaling, sensory neuron axons fail to reach their ultimate targets in peripheral muscle. They project down toward the muscle but don't recognize the muscle and thus cannot enter or innervate it. Consequently, the muscles' sense organs, the muscle spindles, cannot differentiate. The authors argue that these findings, along with the results of tissue culture experiments, show that NT-3 acts as a short-distance cue for proprioceptive axons, which travel in the right direction but ultimately lose their way without NT-3.Altogether these results show that proprioceptive axons require NT-3 not just for survival, but to reach their proper endpoints in the peripheral and central nervous system. NT-3 also helps proprioceptive axons initiate muscle innervation and spindle differentiation. Researchers developing therapies to treat neurodegenerative injuries have increasingly focused their attentions on growth factors like NT-3. By identifying the molecules and mechanisms that establish connections between sensory and motor neurons during development, it may be possible to engage similar processes to attenuate neurodegeneration and even repair damaged nerves.


Thus, activation requires conformational changes across the interface that coordinate the positioning of catalytic residues in both GTPase sites. A distinct class of mutants exhibits half-site reactivity and thus allows us to further uncouple the activation of individual GTPases. Our dissection of the activation process suggests discrete conformational stages during formation of the active SRP•SRP receptor complex. Each stage provides a potential control point in the targeting reaction at which regulation by additional components can be exerted, thus ensuring the binding and release of cargo at the appropriate time.The signal recognition particle (SRP) mediates the cotranslational targeting of nascent proteins to the eukaryotic endoplasmic reticulum membrane or the bacterial plasma membrane. During this process, two GTPases, one in SRP and one in the SRP receptor , form a complex in which both proteins reciprocally activate the GTPase reaction of one another. Here, we explore by site-directed mutagenesis the role of 45 conserved surface residues in the Ffh-FtsY interaction. Mutations of a large number of residues at the interface impair complex formation, supporting the importance of an extensive interaction surface. Surprisingly, even after a stable complex is formed, single mutations in FtsY can block the activation of GTP hydrolysis in  A molecular dissection of the bacterial signal recognition particle-receptor complex reveals discrete stages that provide control points in the protein targeting reaction, ensuring appropriate timing of binding and release GTPases comprise a superfamily of proteins that provide molecular switches to regulate many cellular processes, including translation, signal transduction, cytoskeletal organization, vesicle transport, nuclear transport, and spindle assembly . In manyTwo homologous GTPases, one in the signal recognition particle (SRP) and one in the SRP receptor , mediate the cotranslational targeting of membrane and secretory proteins to the eukaryotic endoplasmic reticulum (ER) membrane or the bacterial plasma membrane. During the targeting reaction, SRP and SR switch between different functional states . SRP firHowever, the regulatory mechanism of the SRP family GTPases provides a notable exception to the “GTPase switch” paradigm. Unlike many other GTPases, no external GEFs or GAPs are known for the SRP and SR GTPases. Instead, Ffh and FtsY bind nucleotides weakly, and nucleotide dissociation and exchange are very fast ; thus, tRas GTPase fold. In addition, all SRP family GTPases contain a unique “N” domain, which together with the G domain forms a structural and functional unit called the NG domain. The crystal structures of the individual Ffh and FtsY NG domains show that both proteins have a wide-open GTP-binding pocket, and the apoforms of these proteins are stabilized by a network of side-chain interactions in the empty active site  How do these GTPases act as reciprocal activating proteins for one another, and (ii) how does the SRP family of GTPases switch between the “on” and “off” states, as the GTPases are predominantly in the GTP-bound state as they enter the targeting cycle and no stable, GDP-bound state exists under cellular conditions?Escherichia coli FtsY and showed that mutations that have deleterious effects on the Ffh-FtsY interaction define a large surface patch on FtsY that lies on its interaction surface with Ffh identified in the crystal structure  does not equal the dK (16 nM) of the Ffh•FtsY complex. Likewise, the mK of the reaction with mutant FtsY does not necessarily equal the dK of the mutant Ffh•FtsY complex, and thus cannot meaningfully distinguish between mutational effects on complex formation and GTPase activation.In contrast, for Class II and III mutants, the rate of GTPase reaction remains slow even at saturating concentrations of FtsY (data for representative mutants are shown in 0k). Addition of mutant FtsY [FtsY(mt)], which can form a complex with Ffh, will sequester the Ffh molecules into a less active Ffh•FtsY(mt) complex  complex.To circumvent these problems, we devised an assay to determine the ability of each FtsY mutant to inhibit the interaction of wild-type FtsY with Ffh. This assay allowed us to monitor selectively complex formation between Ffh and the FtsY mutants. The conditions of the assay were designed so that in the absence of any mutant FtsY as an inhibitor, a robust GTPase reaction mediated by Ffh and wild-type FtsY was observed A, k0. Ad2-fold weaker than the affinity of wild-type FtsY for Ffh  stood out as strong inhibitors. These mutants -specific Ffh mutant, Ffh D251(248)N. Asp251(248), located in the GTP-binding consensus motif, is conserved throughout the GTPase superfamily and forms a hydrogen bonding network with the N2 and N3 amino protons on the guanine ring . In the presence of XTP, Ffh D251(248)N stimulates the GTPase reaction of FtsY and, reciprocally, its XTPase reaction is stimulated by FtsY in the presence of GTP. We therefore used Ffh D251(248)N to monitor the individual hydrolysis events—XTP hydrolysis from Ffh D251(248)N and GTP hydrolysis from the mutant FtsY constructs—in the Ffh D251(248)N•FtsY complex.The mutants described above were identified by analyzing the sum of the two GTP hydrolysis reactions from both Ffh and FtsY. All of the Class II and III mutants must be defective in ine ring . The AspAs expected, all of the Class II and Class III mutants were defective in both hydrolysis reactions and, similarly, all but one Class I mutant and most of the neutral mutants showed no significant defect in either of the two reactions (unpublished data). Five half-site mutants, however, stood out from the pool of originally categorized Class I and neutral mutants see , green. 2-fold, whereas the reciprocal reaction, GTP hydrolysis by Ffh, is reduced only 2- to 4-fold N, thereby reversing the nucleotide specificity of the two binding partners. Upon complex formation, FtsY D449(248)N becomes XTP specific and reciprocally activates GTP hydrolysis in Ffh. Consistent with the results observed with Ffh D251(248)N, all Class IV mutations thus analyzed reduce the rate of XTP hydrolysis from mutant FtsYs by more than 10o 4-fold . Thus taThe mutational analyses described here define four distinct classes of mutants that map to the Ffh-FtsY interface. Each mutant class blocks the reaction in a different way and at a distinct stage, demonstrating that (i) multiple conformational rearrangements are required to form an activated Ffh•FtsY complex and (ii) some rearrangements can be blocked without preventing other rearrangements from taking place. The different classes of mutant interrupt the reaction in different ways, as represented by the states depicted in We previously showed that FtsY exhibits little discrimination between different nucleotides in its free, uncomplexed form, but gains substantial specificity for GTP only in the Ffh•FtsY complex . We therThe crystal structure of the Ffh•FtsY complex also shows that Ffh undergoes similar N-G domain rearrangements upon complex formation, although the effects of this rearrangement on nucleotide specificity are less apparent, as free Ffh already displays significant discrimination between nucleotides . Indeed, mutations at the N-G domain interface in either Ffh or FtsY impair complex formation, supporting the functional importance of this rearrangement in both binding partners . We propboth active sites, the defect in these mutants is not a consequence of simply removing a catalytic residue. Rather, this suggests that even after a stable, closed complex is formed, activation requires additional conformational changes (the “docking” process) that align active-site residues with respect to the bound nucleotides in both GTPase sites , suggesting that the mutant proteins have assumed the closed conformation in the complex. Because single mutations in FtsY can disrupt GTPase activation in The model in 135TFRAAA). As concluded from the structure, this loop can move relatively independently from the rest of the protein  comes from three of the Class II mutants, , which all map to the conserved IBD loop  to either Ala or Trp also results in a Class II phenotype. This residue in FtsY hydrogen bonds across the interface to the ribose 3′-OH of the nucleotide bound to Ffh. The ribose 3′-OH reciprocally donates a hydrogen bond back to the γ-phosphate of the twinned substrate in FtsY  to inhibit the interaction between Ffh and wild-type FtsY, as described in detail in the text see A. The da2+ as described in Fluorescence emission spectra were acquired as described in