In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein 1 (HS1) although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution.Cortactin homologues exist in sponges, worms, shrimps, insects, urochordates, fishes, amphibians, birds and mammalians, whereas HS1 exists in vertebrates only, suggesting that both genes have been derived from an ancestor cortactin gene by duplication. In agreement with this, comparative genome analysis revealed very similar exon-intron structures and sequence homologies, especially over the regions that encode the characteristic highly conserved F-actin-binding repeat domain. Cortactin splice variants affecting this F-actin-binding domain were identified not only in mammalians, but also in amphibians, fishes and birds. In mammalians, cortactin is ubiquitously expressed except in hematopoietic cells, whereas HS1 is mainly expressed in hematopoietic cells. In accordance with their distinct tissue specificity, the putative promoter region of cortactin is different from HS1.Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides inside into their origin and evolution. Our analysis shows that both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in cytoskeletal remodeling, which is of importance for the major role of HS1 in apoptosis and for cortactin in cell migration. EMS1 , CTTN, cttn, Amplaxin, see Genecard [EMS1 gene, which is located on chromosome 11q13 [Cortactin . The cein vitro ,23 and tin vitro . Cortactin vitro ). Tyrosiin vitro . Since cin vitro  and as aHS1 . Human Genecard ) was oriGenecard . HS1 oveGenecard , whereasGenecard . Only inGenecard ,30. SecoGenecard , T-cellsGenecard , mast ceGenecard  and erytGenecard , but at Genecard ,23. ThirGenecard , whereasGenecard ,37. FourGenecard  and WEH1Genecard . An HS1 Genecard . FinallyGenecard  and HS1-Genecard ) than thin vitro , although less efficient than cortactin [rd repeat of the F-actin binding domain in a systemic lupus erythematosus (SLE) patient resulting in increased apoptosis after B-cell receptor (BCR) stimulation [Cortactin and HS1 share also remarkable similarities. First, HS1 binds with its DDW-motif directly to Arp2/3 and is involved in Arp2/3 mediated actin polymerization ortactin . Second,ortactin , howevermulation . Fourth,mulation ,45 and tmulation , althougmulation . Finallymulation  and marcmulation , but alsmulation  and carcmulation .Although cortactin and HS1 share a high amino acid sequence and structural similarity, their functions differ considerable. In this paper, we compare their genomic organization in order to provide more insight into their evolution, which may form the basis towards understanding specific functions of both genes. We describe the genomic organization and the exon-intron boundaries for human cortactin. Both the genomic cDNA and deduced amino acid sequences of human cortactin were compared to cortactin and HS1 genes from other species. Genomic comparisons revealed the evolution and underscore the significance of the conserved F-actin binding repeat domain for HS1 and cortactin and the importance of alternative splicing for cortactin function.EMS1 cDNA [EMS1 cDNA sequence revealed homology with two human genomic clones (DDBJ/EMBL/GenBank Accession No. AP000487 and AP000405) . The splice donor and acceptor sequences, the sizes of the introns and exons of the human EMS1/ cortactin gene are provided in the supplementary materials [see We have previously described the isolation and sequencing of the MS1 cDNA ,49 . The seDrosophila melanogaster ) [Xenopus laevis ) [Other cortactin homologues have been reported in mouse , rat 1919, chickgaster ) , and frolaevis ) . We searDrosophila and mosquito) differ considerably with mammalian cortactin, despite the proteins sequences are very similar. Drosophila and mosquito carry 4 repeats in the actin-binding domain. In both species, repeat 1-to-3 and 4 are on separate exons with in mosquito the 4th repeat of the actin binding domain to be encoded by a single 111 bp large exon 2  are available and therefore we were able to reveal their genomic organization using BLASTn. The exon/intron-boundaries were determined and compared to human cortactin see . As sche2 Figure . Both, sBoth nucleotide and amino acid sequence comparisons with cortactin revealed the highest similarity with the hematopoietic lineage cell-specific protein 1 (HS1). So far, HS1 homologues have been reported in human , mouse , rat andX. laevis HS1), the cDNA (and corresponding protein) sequences were deduced from the genomic DNA with BLASTn or were predicted by Ensemble program. In these lower species, two cortactin related proteins exist. To distinguish between cortactin and HS1 variants, only the most conserved N-terminal part of cortactin and HS1 protein variants, including repeat 3 (corresponding to amino acid 1–190 of human cortactin) was used in BLASTp analysis. In each species, one protein variant turned out to be more homologous to human cortactin, and was called cortactin, whereas the other protein variant appeared to be more related to HS1 and was called HS1. This analysis unveiled HS1 proteins with more than 3 repeats in chicken and pufferfish Tetraodon nigroviridis (containing 4 1/2 repeats), pufferfish Takifugi rubripes and Xenopus laevis HS1 (5 1/2 repeats) and zebrafish HS1 (6 1/2 repeats)  genomic sequences in chicken, pufferfish, zebrafish and frog Table  that werMoreover, alignments of the exon/intron boundaries of these HS1 genes to the mammalian HS1 genes see  revealedDrosophila and mosquito cortactin shares putative transcription factors found both in mammalian cortactin and HS1. Thus at least in mammalians, the nature of the promoters seemed to determine the broad distribution of cortactin expression in various tissues except most hematopoietic cells and the limited expression of HS1 to hematopoetic cells.Cortactin is widely expressed in most cell types suggesting to be important for vital functions, while HS1 expression is restricted to hematopoietic cells suggesting to be tailored later in evolution to serve a specific function in these cells. In concordance with their tissue-specific expression pattern, we suppose that their expression might be differently regulated. Therefore, we compared the upstream promoter regions of several cortactin and HS1 genes Figure . The mamth repeat and SV2 lacking the 5th and 6th repeat resulting in a different F-actin binding properties and decreased cell migration [Xenopus laevis ), the SV1-cortactin variant exists despite the splice donor of intron 11 begins with a GT [We recently reported the identification of two alternative splice variants of human cortactin; SV1-cortactin lacking the 6igration . As showigration  except figration . As has igration . Howeverith a GT . Thus, cith a GT . The relith a GT  suggesterd repeat (exon 7) has been found in an SLE patient [rd repeat and its NLS links HS1 to a role in apoptosis, while such a role has not been described for cortactin lacking a NLS. Since the cytoskeleton architecture in hematopoietic lineage cells is very different from that in adherent cells, it is likely that HS1 plays an important role in the construction of tissue-type specific actin networks. Other types of actin cytoskeleton factors, such as the Arp2/3 complex activators of the WASP family have been reported to have distinct tissue specific expression profiles as well. Thus, the apparent role of HS1 in apoptosis is likely due to its actin remodeling related function. Additionally, our genomic comparisons revealed that the 3rd repeat of HS1 corresponds with the 5th repeat of cortactin, and therefore it might be of interest to investigate whether cortactin SV2 variant (lacking the 5th and 6th repeat) might be involved in apoptosis.Alternative splicing also occurs in human HS1. Recently a splice variant lacking the 3 patient , resulti patient . With reth repeat of cortactin has been suggested to be required for F-actin-binding [th repeat. Nonetheless, HS1 does bind to F-actin and activate the Arp2/3 complex, although at a lower efficiency than cortactin [2 binding site in each of its 3 repeats, whereas cortactin has only one in the 4th repeat. PIP2 reduces F-actin cross-linking by cortactin, probably due to competition for the same binding site. Due to its higher affinity for PIP2 [2. This might be of importance in platelets and megakaryocytes where both, cortactin and HS1 are expressed. Taken together, the composition of the repeat domain is also involved in diverting the functions of both genes.The 4-binding . Genomicortactin . This suortactin ,18. In afor PIP2 , HS1 resDrosophila cortactin gene were viable and fertile, except impaired border cell migration during oogenesis [An elegant way to study the function of a protein is to perform loss-of-function experiments. So far, cortactin knock-out models have not yet been generated successfully, because deletion of one allele of cortactin leads to premature differentiation of embryonic stem cells . Howeveogenesis . Down-reogenesis  and in Eogenesis . Mice laogenesis , howeverDrosophila (Dm)), urochordate ) and sponge (Sd) cortactin. In this last cluster all the species with only one gene (with the highest similarity with cortactin) are present. This suggests that with the appearance of the vertebrates, an ancestral gene became duplicated to create two genes, which later evolved into cortactin and HS1. This hypothesis is supported by the fact that many genes duplicated at this stage in the evolution, the overall amino-acid sequence in both genes is very similar and the introns are located at the same amino acid position. Furthermore, gene duplication often correlates with a tissue specific expression pattern of the duplicated genes, which is true for mammalian cortactin and HS1.To examine the genesis of the cortactin family, we studied the relationship between the cortactin and HS1 homologues by generating a phylogenetic tree based on a multi-sequence alignment with the ClustalW 1,83 program see . We compFigure Takifugu rubripes contains two cortactin-related genomic sequences both including 5 1/2 repeats. Most likely, an ancestor vertebrate cortactin gene underwent gene duplication. From this moment on during evolution, two cortactin/HS1-releated genes are present in all higher species. One gene evolved to mammalian HS1 with a specific function in apoptosis in hematopoietic cells. For its function, exon 8 and 9 (encoding repeat 3 and 4) were not useful and lost during evolution. However, the HS1 protein in pufferfish Takifugu rubripes and frog Xenopus laevis contains 5 1/2 repeats, while chicken and pufferfish Tetraodon nigroviridis HS1 carries 4 1/2 repeats. It might be of interest to investigate the function of these HS1 proteins and their functional differences to mammalian HS1. The other gene evolved to a ubiquitously expressed mammalian cortactin protein with a vital function in the organization of the cytoskeleton and cell migration. The 6th repeat of cortactin most likely originated from a duplication event of the 5th repeat, since the 6th repeat is most similar to the 5th repeat in all species with 6 1/2 repeats. We recently demonstrated that 6 1/2 repeats are necessary for optimal F-actin cross-linking activity and cell migration, while the splice variant lacking both the 5th and 6th repeats (SV2) was less efficient [th repeat (dotted arrows), since both zebrafish cortactin and HS1 contain 6 1/2 repeats.The genome of pufferfish fficient . Thus, tWe report the genomic organization of cortactin and HS1 genes of several species. These genes display a conserved genomic organization as the coding regions have almost identical exon/intron structure. Comparison of 5' sequences allows possible regulatory elements that stress their specific tissue distribution. Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides insight into the evolution of the conserved actin-binding repeat domain, which forms the basis towards understanding specific functions of both genes. Most likely, both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in cytoskeletal remodeling, which is of importance for the major role of HS1 in apoptosis and for cortactin in cell migration.To determine the genomic structure of the human cortactin gene, an algorithm was applied based on the consensus sequence of exon-intron junctions ('gt ... ag' rule of intronic sequence) as well as on the codon usage within ORF. Nucleotide sequence comparisons with human cortactin sequences  using BLASTn  revealedTo confirm the predicted genomic structure, we determined the intron/exon boundaries using a cloning procedure as described . GenomicBecause no overlapping genomic sequences immediately 5' of the first exon were present in the database, we performed sequence analysis of a 2.7-kb HincII-HincII fragment representing the first exon and its 5'-flanking sequences from cosmid COS-7.12 cloned into pUC18 (p5'EMS_3135). In addition, we sequenced a 5-kb PCR product using a 5'-primer in the vector (within the TET gene) and 3'-primer (p3135p601: 5'-ccgggtcggccctggattcc-3') within exon 1, subcloned in pUC18 (p5'EMS_4911). Nucleotide sequences of both products were compared with the genomic clones representing the cortactin gene present in the NCBI database (Accession number AP000487 (GI 8118774 and GI 6277297) and AP000405 (GI 8118742)) and used to define the 7.4 kb 5'-flanking region. The PROSCAN program  from BIMThe (deduced) protein and genomic sequences of all cortactin and HS1 genes were retrieved from various WEB-sites and their available sequence data are summarized in Table To determine the exon/intron boundaries of all cortactin and HS1 genes, available genomic sequences were subjected to sequence alignments of each species-specific cDNA sequence using the BLAST program of NCBI. Using the same algorithms, as described for human cortactin, the exon/intron-boundaries could be predicted. The complete genomic sequences of the 5' flanking region of cortactin of human, chimpanzee, mouse, rat, fruit fly, and mosquito were determined using the various accession numbers of genomic DNA in Table Sequence alignments were carried out using the BLAST program of NCBI. The multiple sequence alignments of various cortactin proteins were constructed using Basic GeneBee ClustalW 1.83 . The genaa, amino acid(s); bp, base pair(s); BCR, B-cell receptor; EST, expressed sequence tag; HS1, hematopoietic lineage cell-specific protein 1; NLS, nuclear localization signal; RT-PCR, reverse transcriptase polymerase chain reaction; SH3, Src homology; UTR, untranslated region.AGSHvR designed the study on comparative genome analysis, performed database searches, sequence alignments and gene structure prediction and drafted the manuscript. ESS designed, conducted and analyzed the cloning and sequencing of the promoter of human cortactin. VvBvS conducted and analyzed the PCR and sequencing experiments of the exon-intron boundaries of human cortactin and its splice variants. PMK read the manuscript and provided comments. ES helped with writing the paper, provided overall technical guidance and coordination. All authors read and approved the final manuscript.Splice donor and acceptor sequences of cortactin in different species.Click here for fileSplice donor and acceptor sequences of HS1 in different species.Click here for fileMultiple amino acid sequence alignment of cortactin and HS1 homologues.Click here for file


If you could peer inside someone else's head, you'd see a scrunched-up gelatinous mass of tissue, weighing roughly a kilogram, homogeneous to the naked eye—in other words, a brain. The seeming uniformity of the overlying cerebral cortex, which has so outstripped other parts of the brain over the course of evolution that it makes up more than 80% of the brain, is belied by centuries of painstaking neuroscience. Some of the most compelling early evidence that parts of the cortex are specialized in their duties came from gun-shot wounds during the first world war. For instance, bullets lodged in the back of the brain disrupted sight in discrete portions of the visual scene, prompting insights into the localization and function of visual cortex.The study of the front of the brain has a similar history of injury leading to insight. Phineas Gage, a railroad worker, had a 3.5-foot-long tamping iron blow straight through his frontal lobes and turned from a responsible, mild-mannered geek into an unruly exhibitionist overnight. Parts of the prefrontal cortex that he damaged have since been much studied for their involvement in motivation and emotional control.More recent work has implicated other parts of the prefrontal cortex in working memory. Working memory is famously illustrated by your ability to temporarily remember a seven-digit telephone number, roughly the amount of information that you can store on-line in working memory for the duration of a task like phoning for a pizza.Monkeys can be trained to remember information much like you remember a phone number, and then use the memory for gaining a reward . They can learn to remember the specific location of a briefly flashed target on a screen and then, when cued, make an eye movement to look directly at that location. Previous research has shown that neurons in the prefrontal cortex maintain high rates of activity while monkeys remember the target location, and gradually the idea that the prefrontal cortex specializes in maintaining these transient memories has risen to dominance over other ideas about its functions.PLoS Biology, Mikhail Lebedev and his colleagues challenge this prevailing view with evidence that most prefrontal cortex neurons may not be so closely tied to working memory after all. As in previous research, they also trained monkeys to make an eye movement to a remembered target, but instead of only seeing one target, the monkeys saw two potential target locations during the course of the task. The monkeys had to pay attention to one of the potential targets, but this was not necessarily the one they would have responded to and was not the one they had to remember. To perform the task successfully, the animals had to engage their working memory, but most of the neurons the researchers recorded increased their activity selectively to the target that was the focus of attention.In this issue of Despite decades of research, the degree to which one region of the brain can be thought of as dedicated exclusively to a particular function is still much debated. These results do not refute the idea that the prefrontal cortex plays an important role in working memory. However, the authors suggest that this area may be more important in focusing the attention needed to remember that phone number, rather than actually holding that number in your mind.


Close to a quarter of the world's mammals are at high risk of extinction. Save for the periodic “great extinctions,” mammalian extinction has been a relatively rare event in geological terms, with one species disappearing from the fossil record every 1,000 years or so. Over the past 400 years, species have been disappearing 50 times faster than this “background” rate, with one vanishing every sixteen years. Human population growth and all its consequences—habitat destruction, propagation of invasive species, poaching—are largely to blame. Top predators often suffer heavily from encounters with humans, especially when those predators are perceived as economic threats. Thirty-four Mexican gray wolves have been reintroduced in Arizona since 1998, and five have been shot, reportedly by ranchers.Species in the most densely populated areas are expected to face the greatest risk, yet some survive while others perish, suggesting biological factors play a role in their fate. If, for example, the same external force drastically reduces populations of species with different biological profiles, then a species with a relatively short gestation period may stand a better chance of recovering than a long-gestating species.Effective conservation strategies depend on understanding which factors are likely to increase extinction risk, but it's unclear how important intrinsic biological traits are relative to external pressures from humans and whether biology's influence on survival depends on the intensity of the threat. Ecologists often use human population density as a proxy for anthropogenic threats such as habitat destruction and hunting. To tease out the relative importance of all these factors, Marcel Cardillo et al. analyzed the impact of various biological traits and human population density on extinction risk in the mammal order Carnivora, which includes the red panda, lion, and members of the photogenic weasel-like viverrid family. By identifying the most salient factors in predicting extinction, the authors have created a model to identify those species at greatest risk.The biology of a species combined with human population density, the researchers found, is a stronger predictor of risk than exposure to humans alone; those biological traits that increase risk vary depending on a species' exposure to human populations. Carnivores with low exposure to humans, for example, are likely to be at greater risk if their population density is low and they have small ranges, possibly because this makes them more vulnerable to loss of habitat. Species living near densely populated human areas must often contend with hunting and other direct threats on top of habitat loss and are more at risk if they also have long gestation periods—they can't repopulate fast enough to offset the additional pressures. Based on projected human population growth, this model predicts the addition of a number of species—mostly from Africa, where population growth rates largely exceed the global average—to the endangered list by the year 2030. Most of these species—including African viverrids such as the common genet, which not only lives in areas where human populations are rapidly expanding but is also biologically predisposed to decline—are currently considered a low conservation priority.While it's possible that the direct effects of human population density are past—that is, species most sensitive to human incursions are already gone—human population density likely modulates biology. That might explain why gestation length didn't predict risk for species living in sparsely populated areas—all else being equal, their numbers remained relatively stable. A species with a small population forebodes a high extinction risk regardless of human density, though species with long gestation periods, again, face greater danger in the company of humans.before a species reaches the brink of extinction. “There is no room for complacency about the security of species,” the authors warn, “simply because they are not currently considered threatened.”Altogether, these results suggest that as human population pressures increase, the importance of species-specific biology in predicting extinction risk also increases, with biology affecting which species are most vulnerable to external threats. With most conservation efforts focused on damage control, these findings make the case for interceding


Evolution of new complex biological behaviour tends to arise by novel combinations of existing building blocks. The functional and evolutionary building blocks of the proteome are protein domains, the function of a protein being dependent on its constituent domains. We clustered completely-sequenced proteomes of prokaryotes on the basis of their protein domain content, as defined by Pfam (release 16.0). This revealed that, although there was a correlation between phylogeny and domain content, other factors also have an influence. This observation motivated an investigation of the relationship between an organism's lifestyle and the complement of domains and domain architectures found within its proteome.Agrobacterium species and in plants. We identified the 0.5% of Pfam protein domain families that were most significantly over-represented in the plant-associated Proteobacteria with respect to the background frequencies in the whole set of available proteobacterial proteomes. These included guanylate cyclase, domains implicated in aromatic catabolism, cellulase and several domains of unknown function.We took a census of all protein domains and domain combinations (architectures) encoded in the completely-sequenced proteobacterial genomes. Nine protein domain families were identified that are found in phylogenetically disparate plant-associated bacteria but are absent from non-plant-associated bacteria. Most of these are known to play a role in the plant-associated lifestyle, but they also included domain of unknown function DUF1427, which is found in plant symbionts and pathogens of the alpha-, beta- and gamma-Proteobacteria, but not known in any other organism. Further, several domains were identified as being restricted to phytobacteria and Eukaryotes. One example is the RolB/RolC glucosidase family, which is found only in We identified 459 unique domain architectures found in phylogenetically diverse plant pathogens and symbionts that were absent from non-pathogenic and non-symbiotic relatives. The vast majority of these were restricted to a single species or several closely related species and so their distributions could be better explained by phylogeny than by lifestyle. However, several architectures were found in two or more very distantly related phytobacteria but absent from non-plant-associated bacteria. Many of the proteins with these unique architectures are predicted to be secreted.Pseudomonas syringae pathovar tomato, those genes encoding genes with novel domain architectures tended to have atypical GC contents and were adjacent to insertion sequence elements and phage-like sequences, suggesting acquisition by horizontal transfer.In in vivo and in vitro is time-consuming and expensive, this computational approach may be useful for reducing experimental search space. Furthermore we discuss the biological significance of novel proteins highlighted by this study in the context of plant-associated lifestyles.By identifying domains and architectures unique to plant pathogens and symbionts, we highlighted candidate proteins for involvement in plant-associated bacterial lifestyles. Given that characterisation of novel gene products  Pseudomonas aeruginosa, which has a relatively large genome that encodes enormous regulatory and metabolic flexibility, allowing it to colonise diverse niches. At the other extreme are highly specialised intracellular symbionts , whose small genomes have undergone reductive evolution and which lack many common metabolic and regulatory features. With the availability of complete genome sequences for many model plant-associated bacteria, we are particularly interested in how genome analyses can be used to gain insights into the mechanisms and evolution of associations between bacteria and plants.The Proteobacteria comprise a phylum of Gram-negative bacteria that includes an extraordinary diversity of lifestyles, ecology and metabolism. At one end of a spectrum are free-living organisms such as Agrobacterium tumefaciens [Bradyrhizobium japonicum [Mesorhizobium loti [Sinorhizobium meliloti [Caulobacter crescentus [Rickettsia species [Ralstonia solanacearum [Neisseria [Bordetella [Nitrosomonas europaea [Xylella fastidiosa [Xanthomonas campestris [Xanthomonas axonopodis [Pseudomonas syringae pathovar tomato [P. aeruginosa [There are complete annotated genome sequences available for several phylogenetically diverse proteobacterial plant pathogens and symbionts, along with many of their non-pathogenic and non-symbiotic relatives. For example, among the alpha-Proteobacteria, complete genome sequences are available for the phytopathogen efaciens -3, the naponicum , Mesorhiium loti  and Sinomeliloti ,7, the nescentus , and the species -11. Ralsnacearum  is the seisseria ,14 and Brdetella  and the europaea  whose gestidiosa ,18, Xantmpestris , Xanthomonopodis  and Pseur tomato  as well ruginosa , which iEach of these three divisions of the Proteobacteria contains a wide variety of different lifestyles, so it is logical to assume that bacteria-plant interactions have evolved independently in multiple separate Proteobacterial lineages. Ultimately the differences between these lifestyles are determined by the organisms' genes acting through their expressed proteins and RNAs. Given the abundance of complete genome sequence data now available, a high priority is to understand which features of an organism's proteome determine its lifestyle, and the evolutionary processes underlying environmental adaptation and evolution of novel traits. Two main sources have been proposed for the evolution and acquisition of novel traits by bacteria: (i) duplication, mutation and recombination of existing genes within a single lineage, and (ii) lateral gene transfer between lineages. A combination of both bioinformatic and experimental studies are needed to determine the relative importance of these two processes in the evolution of plant-associated lifestyles in bacteria.Evolution of new complex biological behaviours tends to arise (but not exclusively) by novel combinations of existing building blocks. The functional and evolutionary building blocks or units of the proteome are protein domains. Protein domains can be classified into families; examples of widely used classification schemes are those of Pfam  and SMARTo gain an overview of the similarities and differences between their protein domain content, we classified representative prokaryotes into hierarchical clusters based on their complement of protein domain families described. For each proteome we generated a 7,677 binary state element vector where each element represented the presence or absence of one of the 7,677 Pfam protein domain families. Pairwise distances were calculated for each pair of proteomes based on the level of similarity between the pair of vectors, and tree was built by neighbour-joining (see Methods for more details). One hundred trees were built, each time leaving out 10 % of the vector elements, selected at random. The tree shown in Figure Treponema denticola  is restricted to Bradyrhizobium japonicum and Sinorhizobium meliloti. Although it is possible that these species-specific domain families are involved in pathogenesis or symbiosis it is equally likely that they have some unrelated function. However, several domains are potentially interesting from the point of view of plant-microbe interactions either because they are found in phylogenetically disparate species of phytobacteria or because they are also found in eukaryotes. Table X. campestris and NolX from the alpha-proteobacterium Rhizobium fredii and Rhizobium species NGR234. In these rhizobia, NolX  has been shown to play a role in nodulation specificity and is exclusively expressed during the early stages of interactions with plants [X. campestris HrpF [R. solanacearum  appears to be restricted to h plants ,26. NolXh plants , and a sris HrpF . The imp species .R. solanacearum and the gamma-Proteobacteria P. aeruginosa and X. campestris (Table Nicotiana (see Table Agrobacterium-like proteins in Rhizobium (Agrobacterium) vitis reflects another key feature of the biology of these plant-associated bacteria, the fact that many of the genes involved directly in Agrobacterium and Rhizobium- plant interactions are encoded on large plasmids that facilitate lateral gene transfer of complex and novel traits between bacteria. Rhizobium (Agrobacterium) vitis is not a symbiont, but rather causes a tumorigenic disease of grapevine through the action of a number of A. tumefaciens-like genes [A further protein family limited to plant-associated bacteria is characterised by the ice nucleation repeat (Pfam:PF00818)and is found in proteins that may have a role in frost damage to host plants. It remains to be seen whether the remaining two domain families (DUF811 and DUF1427) are involved in the plant-associated lifestyle. DUF1427 (Pfam:PF07235) is restricted to several plant-associated alpha-Proteobacteria, the beta-proteobacterium ke genes .Erwinia carotovora, Pseudomonas syringae pathovar tomato, Ralstonia solanacearum, Sinorhizobium meliloti, Bradyrhizobium japonicum, Mesorhizobium loti, Agrobacterium tumefaciens (Washington strain and Dupont strain), Xanthomonas campestris pathovar campestris, Xanthomonas axonopodis pathovar citri, Xylella fastidiosa and Xylella fastidiosa (strain Temecula1). We then calculated a P value for the probability of observing at least this number of occurrences given the background frequency in the Proteobacteria and assuming a binomial distribution. The smaller the P value, the less likely that the observed frequency occurred by chance. In other words, the smaller the P value, the more over-represented is the domain family. The most over-represented domains are listed in Table Bacterial physiology and behaviour is determined not only by the presence or absence of particular proteins but also by numbers of representatives of protein families. For example, gene duplication events may lead to a lineage-specific expansion that results in novel orthologues that can take on novel functions different from that of the parent gene. Therefore we investigated whether any protein domain families were over-represented in the plant-associated proteobacteria with respect to the background distribution of domains in all Proteobacteria for which complete sequences were available. For each of the 7,677 Pfam domain families, we counted the numbers of proteins in which that domain family occurs in the complete proteomes of B. japonicum (32 proteins) and S. meliloti (24 proteins). No other fully-sequenced proteobacterium encodes more than three, although the spirochaete Leptospira interrogans encodes 17 proteins matching PF00211). Cyclic-diGMP, the product of guanylate cyclase, is a secondary messenger that plays a role in cell-cell and cell-surface contact in several bacteria by regulating cellular adhesion genes [C. crescentus has been shown to become sequestered to the cell pole following phosphorylation [The domain with the statistically most significant over-representation in the plant-associated bacteria was the guanylate cyclase domain (Pfam:PF00211). This domain was particularly abundant in on genes . Such inrylation . This isAgrobacterium tumefaciens, 11 proteins in B. japonicum and 9 proteins in M. loti containing this domain. The related alpha-Proteobacteria C. crescentus, B. melitensis, B. suis and Rhodopseudomonas palustris have 3, 2, 2 and 0 luciferase (PF00296) proteins respectively. Other species containing large numbers of luciferase-like proteins include Mycobacterium bovis (13 proteins) and M. tuberculosis (14 proteins).Another domain with statistically significant over-representation in the plant-associated bacteria was the bacterial luciferase-like monooxygenase domain (Pfam:PF00296). This domain was particularly abundant in the plant-associated alpha-Proteobacteria with 15 proteins in B. japonicum and 14 and 13 in M. loti and S. meliloti respectively. Other prokaryotes encode between 0 and 5 DUF636 proteins, whilst Arabidopsis thaliana and Homo sapiens each encode one.Several domains of unknown function are amongst those most over-represented in the phytobacteria. For example, DUF636 is unusually abundant in the rhizobia with 16 representative proteins in The functionality of the proteome depends not only on the repertoire of protein domains but also on the interactions and cellular context of those domains. One important aspect of this context is the range of combinations of domains within a protein; that is the domain architecture of proteins.R. solanacearum, P. aeruginosa, E. carotovora (subspecies atroseptica), P. syringae (pathovar tomato), B. japonicum, S. meliloti, M. loti, A. tumefaciens, X. fastidiosa, X. campestris, X. axonopodis. 459 of the 3,774 domain architectures encoded in genomes of plant-associated bacteria were absent in all other bacteria for which complete genome sequences were available. These 459 architectures are listed in the supplementary data. However, many of these architectures were restricted to a single species or several closely related species and so were of limited interest for this study.We used the Pfam database to ascertain the domain architecture of every protein sequence from each bacterial species for which a complete annotated genome sequence was available. 3,774 distinct protein domain architectures were found in A. tumefaciens, M. loti, S. meliloti, X. fastidiosa and X. fastidiosa.The metallo-beta-lactamase domain (Pfam:PF00753) is common and widespread, being found in over 2000 different proteins from a wide range of organisms. However, only in these proteins from plant-associated bacteria is the metallo-beta-lactamase domain fused to DUF442. This suggests that the catalytic domain may have been recruited to some new function connected to a plant-associated lifestyle in these bacteria.We were particularly interested to discover whether any domain architectures are related to plant-associated lifestyle rather than simply resulting from phylogeny. The 15 protein architectures illustrated in Table Pseudomonas genome is the PAS domain (Pfam PF00989) [P. aeruginosa PAO1 and 30 ORFs in P. syringae pathovar tomato. The average number of PAS-containing ORFs in complete proteobacterial genomes is about 10. Although PAS domains are only found in a limited subset of bacterial regulators, they are at the forefront of molecular innovation with 9 of the novel architectures identified in P. aeruginosa, and 5 of those in P. syringae pathovar tomato containing PAS domains (see supplementary data for more details). Xanthomonas genomes also encode a large number of PAS-containing polypeptides, (18 and 21 in X. axonopodis and X. campestris respectively). However, each X. fastidiosa encodes only one: PhoR, a regulator generally associated with responses to phosphate limitation. Ten novel PAS architectures are present in each Xanthomonas genome, of which 7 are common and 3 are unique to each strain  , which iP. syringae and Xanthomonas was a phytochrome domain (Pfam:PF00360)  as well as plant associated bacteria  and soil bacteria (e.g. P. putida) [Bradyrhizobium species ORS278 uses phytochrome to regulate the photosynthesis gene cluster and a similar induction was seen with Rhodopseudomonas pallustris but not with several other photosynthetic bacteria [Agrobacterium may play a role in detecting depth in soil strata as a means of optimising interactions with roots [Rhodobacter sphaeroides  Figure . This do putida) ,39. An ubacteria . It is nPseudomonas genomes allowed us to study in more detail the distribution, genomic context and properties of Pseudomonas gene products highlighted by this analysis. Closer examination of the genomic context of the P. syringae genes encoding proteins with unusual domain architectures showed that most were flanked on either or both sides by genes that have few or no orthologues in other Pseudomonas strains, suggesting that these novel genes have been recruited simultaneously with other genes, possibly of related function, or that they have recombined into the genome at hotspots for recombination and insertion of alien DNA.The availability of multiple finished and unfinished pvsA, PSPTO4084, PSPTO2413 and cfa6. Low GC3 content genes include hrpZ, PSPTO3210, glf, PSPTO4696, hopPtoS, PSPTO2259, PSPTO0400, avrF and PSPTO1070. The GC content of flanking genes frequently reflected that of the novel gene, most strikingly for glf, PSPTO2441, PSPTO4696, hopPtoS, PSPTO4699, PSPTO1070 & PSPTO2632, which were each associated with low GC regions containing few ORFs with orthologues in other Pseudomonas genomes.To further address the hypothesis that at least some of these architectures have been acquired by horizontal gene transfer we examined the GC content and third position GC content of each of these genes, in comparison to the total genome . Sixteen of the genes deviated from the average GC3 content by more than 0.05. High GC3 content genes include One other feature frequently associated with horizontally transferred genes is the presence of IS elements, tRNAs, plasmid and phage genes in flanking regions. PSPTO3229, PSPTO4569, PSPTO2312, PSPTO2829, PSPTO2310, Glf, PSPTO2441, PSPTO4696 and PSPTO2326 are all located in close proximity to IS elements and phage-like sequences, or in defined regions of the genome flanked by IS elements and phage-like sequences . Some proteins will inevitability be included or excluded because of the limitations of current domain prediction technology. However, in addition to identifying protein candidates for further investigation, this type of analysis can be used to challenge and improve current models for domain prediction and expose errors and limitations of genome sequence data and protein prediction. For example, consider a case in which a protein is identified as having the "unique" architecture B~C~D. Additional examination of the protein may reveal that the protein has a similar sequence to proteins with the architecture A~B~C~D. The absence of the A domain may indicate a genuine alteration in structure and potentially in function, or a frameshift in the genome sequence data, or a functional "A" domain that fails to meet current predictive criteria. Each of these hypotheses can be tested by further research and experimentation, both Although our approaches to identifying candidate genes and proteins of significance to lifestyle have led to several potential leads and interesting hypotheses, there are some caveats. Firstly, evolution does not proceed exclusively through loss and gain of domains and domain shuffling; for example, protein innovation can also occur through mutation and divergence within domain families. Also, it is becoming increasingly apparent that an organism's physiology, behaviour and ecology depend as much on higher order 'systems level' phenomena as on the inventory of molecular components.e.g. compared with SMART) and its data is of high quality. Furthermore, its data is distributed in a form that is ideally suited for constructing database queries such as those in this study. Another advantage is that in Pfam no two domains ever overlap in their coverage of a protein sequence, which significantly simplifies the analysis. However, it should be noted that Pfam is not absolutely infallible and some of its threshold values are rather stringent, leading to failure to identify some 'outlying' members of a domain family.We chose to base our surveys of protein domains on the Pfam because this mature database is relatively comprehensive in its coverage .Click here for file"domains.tab.gz" Species distribution of each of the 3,774 Pfam domains. This tab-delimited file has been compressed using gzip.Click here for file


Hepatocellular carcinoma is a common malignancy for which chronic hepatitis B infection has been defined as the most common etiologic factor. The most frequent metastatic sites are the lung, bone, lymphatics, and brain, respectively. Metastases to the chest wall have been reported only rarely.We report a patient with hepatocellular carcinoma who presented with an isolated metastatic mass on the left anterolateral chest wall in the axillary region.Metastasis of HCC should be included in the differential diagnosis of rapidly growing lesions in unusual localizations, particularly in patients with chronic liver disease even if a primary tumor can not be radiologically identified. Chronic hepatitis B has been described as the most frequent cause [Hepatocellular carcinoma (HCC) is the most frequent primary malignant tumor of the liver . It is unt cause -4. Hematnt cause ,4. Rarelnt cause -7.This report presents an unusual metastasis of HCC which presented as a mass on the left chest wall in the axillary region.3; Plt: 126 000/mm3; PT: 15.6 sec; INR:1.3; total protein: 6.5 g/dL (N: 6.4–8.5 g/dL); albumin: 2.2 g/dL (N: 3.4–4.8 g/dL); AST: 124 iu/L (N: 15–40); ALT: 52 iu/L (N: 10–40); ALP: 173 iu/L (N: 37–147); GGT: 213 iu/L (N:0–40); total bilirubin: 3.8 mg/dL (N: 0.1–1.2), and direct bilirubin: 1.57 mg/dL (N: 0–0.3). Hepatitis B virus surface antigen, IgG antibody to the core antigen, anti-HBe and HBV DNA with polymerase chain reaction were positive. HBe antigen, anti-Delta and serological markers of hepatitis C were negative.A 71-year-old male was admitted to our clinic with complaints of left shoulder pain, swelling in the left anterolateral chest wall, jaundice, weight loss, dyspnea and weakness. At three months prior to admission, he had noticed a less movable mass approximately 2 cm in diameter and then the tumor had grown rapidly, becoming increasingly painful during exertion. There was no history of expectoration, fever, or night sweats. The patient's physical examination revealed yellow discoloration of the sclerae, pitting edema up to the knees and a 5 cm × 6 cm fixed mass in the left axillary region. There was mild splenomegaly but no hepatomegaly, liver masses or ascites. Laboratory findings were as follows: sedimentation rate: 90 mm/hr; Hb: 11.5 mg/dL; WBC: 3900/mmAbdominal ultrasonography showed ascites, splenomegaly and diffusely nodular and heterogeneous echogenic patterns in the liver. There was no history of chronic liver disease. Upper gastrointestinal endoscopy was normal except for esophageal varices. Computerized tomography of the thorax revealed a mass on the left anterolateral chest wall Figure . CytologAlthough extrahepatic metastasis of HCC was reported in 18% of untreated patients in a retrospective study, metastatic lesions were found at a higher incidence in an autopsy study of deaths related to primary liver cancer . The mosAlthough bone metastases are seen in 10% of HCC patients , it infrth rib [The establishment of the diagnosis of metastatic HCC can occasionally be problematic, particularly when the primary tumor has not been identified. To the best of our knowledge there are only two reports in the literature describing cases of solitary metastasis to the chest wall from an unknown primary HCC. One patient had an HCC metastasis involving the sternum, and the other patient had a metastasis on the right 4th rib ,6.In the present patient, the etiology of HCC was due to chronic hepatitis B, and the HCC was diffuse in nature. Yuki et al. reported an association between HBs antigen positivity and diffuse-type HCC. Intrahepatic, hematogenous and lymphogenous metastases have been frequently observed in diffuse-type HCC . NeverthAFP, when elevated, usually correlates with tumor size. AFP doubling time is also closely related to tumor doubling time. A rapidly growing axillary mass, diffuse tumoral infiltration of the liver and a very high level of AFP in our patient are consistent with these observations. However, nearly one-fourth of patients with HCC may have normal AFP values .The need for liver biopsy might be questionable in the our patient. The presence of chronic liver disease with a very high AFP level was highly suggestive for HCC. However, there are several reports in the literature describing ectopic HCC without a liver origin. These patients can be treated with surgical resection and have a good prognosis ,20. FurtIn conclusion, metastasis of HCC should be included in the differential diagnosis of rapidly growing lesions in unusual localizations, particularly in patients with chronic liver disease even if a primary tumor cannot be radiologically identified.The author(s) declare that they have no competing interests.SC, involved in the patient active management and preparation of the figures. OY, carried out the literature search. SK, revised it for scientific content. KC, performed the cytological examination. MB, involved in the patient active management. AD, edited the manuscript and coordinated the submission. All authors contributed to the preparation of the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


Alignment and comparison of related genome sequences is a powerful method to identify regions likely to contain functional elements. Such analyses are data intensive, requiring the inclusion of genomic multiple sequence alignments, sequence annotations, and scores describing regional attributes of columns in the alignment. Visualization and browsing of results can be difficult, and there are currently limited software options for performing this task.The Application for Browsing Constraints (ABC) is interactive Java software for intuitive and efficient exploration of multiple sequence alignments and data typically associated with alignments. It is used to move quickly from a summary view of the entire alignment via arbitrary levels of resolution to individual alignment columns. It allows for the simultaneous display of quantitative data,  and annotation data . It can be used to facilitate basic comparative sequence tasks, such as export of data in plain-text formats, visualization of phylogenetic trees, and generation of alignment summary graphics..The ABC is a lightweight, stand-alone, and flexible graphical user interface for browsing genomic multiple sequence alignments of specific loci, up to hundreds of kilobases or a few megabases in length. It is coded in Java for cross-platform use and the program and source code are freely available under the General Public License. Documentation and a sample data set are also available  Functional elements in a genome accumulate inter-specific substitutions more slowly than neutral DNA throughout evolution . TherefoSimultaneous visualization of complex data such as these is of utmost importance both for experimentalists and for computational biologists. Several options currently exist for such visualization, but there are a variety of characteristics that distinguish the ABC from them. VISTA, for example, generates a static image that is not interactive . Other p. The file formats used by the ABC are quite similar, with only minor modifications, to other standard formats, such as fasta-formatted sequence files and standard parenthesis notation for phylogenetic tree descriptions. A sample data set is available, and is the source of the screenshot depicted in Figure ST7 gene in the human genome, near the CFTR gene. Repeats were identified in the human sequence using RepeatMasker [The ABC requires Java 1.4 or later and has been successfully tested on Windows, Linux, and OS X. There is no specific upper limit in the size of potential data sets, but system memory usage can be high on large alignments. The ABC can efficiently handle a 2 Mb alignment of 29 sequences on a machine with a 1 GHz processor and 1 Gb of RAM. Details about file formats and instructions for use are available in the documentation that is available along with the source code atMasker  and geneatMasker . The aliatMasker , and hasBy default, the ABC displays graphical summaries of the quantitative information associated with the alignment. Scores are summarized regionally in consecutive non-overlapping windows. The size of these windows depends on the resolution, defined as the number of alignment columns summarized per pixel. The ABC has three distinct display modes, chosen automatically depending on the density of the information. At very low resolution, a histogram is displayed that plots the number of data points in each window that are at or below a specified value; note that regions containing many low scores will stand out as peaks in the histogram, as demonstrated by the clear association of peaks and the location of exons . This flexibility should be beneficial to researchers whose primary interest is comparative sequence analysis, but should also be valuable to those who use comparative analyses in support of other types of projects, such as experimental characterization of constrained elements. We also note that this flexibility distinguishes the ABC from other browsers that require built-in or specific types of score data and/or the use of a particular alignment program. The software is written in Java for cross-platform support, and the source code is freely available under the General Public License (GPL).Project name: Application for Browsing Constraints (ABC)Project home page: Operating system: Platform independentProgramming language: JavaOther requirements: Java 1.4 or laterLicense: GPLABC: Application for Browsing ConstraintsGUI: Graphical User InterfaceGPL: General Public LicenseSNP: Single Nucleotide PolymorphismUCSC: University of California, Santa CruzRGB: Red-Green-BlueGMC and AS conceived of the project, organized the feel and design of the browser, generated the underlying data, and wrote the manuscript. SAGS wrote the Java code and provided comments on the manuscript. All authors read and approved the final manuscript.


Beginning two years ago, the US Dept of Health and Human Services began "special reviews" of all current research grants that involved harm reduction, sex and drugs, and continues its ban on funding of needle exchange. With Bush's second term, the campaign was extended to all US funded international programs that dealt with these issues and populations. And, most recently, the US has again undertaken to dominate the discourse within international organizations charged with drug control and AIDS policies – especially those of the UN. But the international harm reduction and human rights community is fighting back in several important ways, including "An Open Letter to the delegates of the Forty-eighth session of the Commission on Narcotic Drugs (CND) of the UN" prepared by a group of 334 well respected public health experts and human rights advocates, protesting U.S. pressure on the U.N. to withdraw its support from harm reduction. This editorial includes the letter and signatures as well as French, Spanish, and Russian versions of the letter as additional files. Now, by God's grace, the shining sun is up, and them that fear not light will surely praise it."Arthur Miller, The Crucible, Act III"I do not believe that the meaning of our Eighth Amendment, any more than the meaning of other provisions of our Constitution, should be determined by the subjective views of five members of this court and like-minded foreigners"Justice Antonin Scalia, in his dissent from the US Supreme Court majority decision barring capital punishment for crimes committed by minors.It is indeed "a sharp time" in the US for those of us who agree with "like-minded foreigners". This is especially so regarding matters of drug and AIDS policies based on harm reduction (HR) and public health – decriminalization of drug users, the need for safer injections, low thresholds for access to care, sex education and social supports that work to reduce risk. Today more people are imprisoned for drug use in the US than are incarcerated in the European Union for all crimes.US conservatives have, in the same lethal moralistic tradition of our Salem witch-hunts of the 1600's and the McCarthy era, effectively obstructed and undermined our HR efforts at home for two decades. But now the Bush administration, emboldened by its re-election and in full warrior mode, has undertaken a newly invigorated global jihad against harm reduction. Americans who support HR are now to be made to feel "foreign", their moral compass, patriotism, and loyalty to "American core – values" placed in doubt.Parallels to the death penalty are not casual: the failed punitive drug policies of the war on drugs are also official "death sentences", with many more lives lost to them each year than to all the judicially sanctioned executions of all the countries on earth. And, because preventable death is more then an analogy when it comes to public health, we can learn a lot from this Supreme Court case. Justice Anthony M. Kennedy, writing for the courts majority, recognized the "evolving standards of decency" that should shape our judgment of what constitutes a violation of our Constitution's Eighth Amendment and its prohibition against cruel and unusual punishments: "it is fair to say that the United States now stands alone in a world that has turned its face against the juvenile death penalty."But Justice Scalia, significant as the most likely nominee for Chief Justice with the ailing incumbents imminent departure, saw it differently, the NY Times reporting that he reserved "his strongest dissent for (the majority's) reference to international developments that have left the United States alone in supporting juvenile executions". For while the majority opinion said the court was not bound by foreign developments, "it is proper that we acknowledge the overwhelming weight of international opinion" for its "respected and significant confirmation for our own conclusions", Scalia objected that this position implied that "the views of our own citizens are essentially irrelevant," and had wrongly given "center stage" to the "so-called international community." Assumedly this would be that same " international community" that our Declaration of Independence refers to in its famous opening paragraph where, attempting to justify our nations throwing off British rule, we are told that " a decent respect to the opinions of mankind requires that " we explain our course of action. But as the worlds "sole super- power" (tell China that) it appears that we no longer have to show a decent respect for any other nations opinions about human rights, nor it would seem for the relentlessly insistent biology of HIV.The US harm maximization drug policies, which violate both human rights and the realities of infectious diseases, are immoral and dangerously misguided – sustained by demagogic politicians and mad moralists now in near absolute power in our country. As surely as capital punishment, these policies mete out death sentences on a massive scale to our most vulnerable citizens. These failed policies account for the continued annual incidence of 40,000 new HIV infections in the US.Beginning two years ago, the US Dept of Health and Human Services  began "special reviews" of all current research grants that involved sex and drugs. Washington re-asserted the drive for mandated "abstinence only" and "faith based" programs, and continues the Federal ban on funding of needle exchange. Don't even bother applying for work on gay sex. With Bush's second term, the campaign was extended to all US funded international programs that dealt with these issues and populations. And, most recently, the US has again undertaken to dominate the discourse within international organizations charged with drug control and AIDS policies – especially those of the UN.Now the US seeks to impose them as extra judicial capital punishment on the rest of the world. But the international harm reduction and human rights community is not going quietly – it is fighting back in several important ways and we can cite many successes that have already saved hundreds of thousands of lives. Henceforth we will chronicle this struggle in this journal.As a start we are publishing An Open Letter to the delegates of the Forty-eighth session of the Commission on Narcotic Drugs (CND) of the UN prepared by a group of well respected public health experts and human rights advocates, protesting U.S. pressure on the U.N. to withdraw its support from harm reduction see . The letcohenj@hrw.orgFor more information about this letter contact Jonathan Cohen at Human Rights Watch – We at HRJ welcome your views, which can be submitted online (as Comments) at harmreductionjournal.com• E Drucker PhD is Director, Division of Public Health and Policy Research, Professor of Epidemiology and Social Medicine and Professor of Psychiatry, and Family Medicine at Montefiore Medical Center/Albert Einstein College of Medicine in New York City. Dr. Drucker was a founder of the International Harm Reduction Association and Founding Chairman of Doctors of the World / USA. He is a currently a senior Soros Justice Fellow.An Open Letter to the delegates of the Forty-eighth session of the Commission on Narcotic Drugs (CND).In a year when the United Nations Office on Drugs and Crime (UNODC) is chair of the governing body of the UN's Joint Programme on HIV/AIDS (UNAIDS), we write to express concern about U.S. efforts to force a UNODC retreat from support of syringe exchange and other measures proven to contain the spread of HIV among drug users. Injection drug use accounts for the majority of HIV infections in dozens of countries in Asia and the former Soviet Union, including Russia, China, all of Central Asia, and much of Southeast Asia. In most countries outside Africa, the largest number of new infections now occurs among injection drug users. As UNODC director Antonio Maria Costa noted at the July 2004 International AIDS Conference, effective responses to injection driven AIDS epidemics require expanded HIV prevention, including syringe exchange, rather than policies that accelerate HIV infections through widespread and indiscriminate imprisonment.Unfortunately, recent events suggest that UNODC – under pressure from the United States – is being asked to withdraw support from proven HIV prevention strategies at precisely the moment when increased commitment to measures such as syringe exchange and opiate substitution treatment is needed. It is particularly alarming that the silencing of UNODC is occurring in a year when the agency is chair of UNAIDS' Committee of Co-sponsoring Organizations and in a year when HIV prevention is a focus of thematic debate at the 48th meeting of the CND. Among the events that have particularly heightened our concern are:* Mr. Costa, who last year expressed support for positive changes in the Russian criminal code, expansion of syringe exchange in countries facing injection driven epidemics and other measures to reduce drug-related harm, has apparently been rebuked by the U.S. State Department. Following a meeting with Robert Charles, U.S. Assistant Secretary for International Narcotics and Law Enforcement Affairs, Mr. Costa pledged to review all UNODC electronic and printed documents for references to "harm reduction" and to be "even more vigilant in the future."* In Southeast Asia, UNODC has suspended a program that sought reduce drug users' vulnerability to HIV prevention through approaches that emphasized public health and drug users' human rights, rather than punishment.* Even syringe exchange, affirmed as an effective and essential part of HIV prevention by UNAIDS, WHO, and UN member nations, has become politically unpalatable. A November e-mail from a senior UNODC staff member asked junior staff to "to ensure that references to harm reduction and needle/syringe exchange are avoided in UNODC documents, publications and statements."We recognize that UNODC is dependent on contributions from donor nations, and that the U.S. is the single largest donor to UN drug control. At the same time, the lives of hundreds of thousands depend on sound, scientific approaches to HIV prevention. Numerous studies, including U.S. government studies, have found that strategies such as syringe exchange and methadone maintenance demonstrably diminish HIV transmission and other health risks. The fact that U.S. delegates declare the evidence in support of syringe exchange "unconvincing," as they did in last year's CND session, should not be allowed to determine the course of the UN drug control and HIV prevention efforts, which are inextricably and essentially linked. Nor should UNODC – a co-sponsor of UNAIDS, and an agency with an essential role to play in the course of the HIV epidemic – be asked to refrain from public statements about needle exchange simply because they do not fall within the realm of what the U.S. deems acceptable.Strategies that attempt solely to achieve abstinence from drug use do not constitute an acceptable alternative to programs, such as syringe exchange, that help active drug users protect themselves from HIV/AIDS. Experience has shown that "zero tolerance" drug control efforts can have the effect of driving injection drug users underground and away from drug treatment and other health services. This is particularly true where, as in many countries, counter-narcotics efforts lead to false arrest, beatings and extortion by police, prolonged detention without trial, forced drug treatment, disproportionate incarceration in cruel conditions and, in some cases, extrajudicial execution. Programs such as syringe exchange and opiate substitution, by contrast, both prevent HIV infection and can provide a bridge to other health services. Restricting these programs is a blatant infringement of drug users' human right to health.As you gather this year to debate HIV/AIDS prevention and drug abuse, we respectfully urge you to support syringe exchange, opiate substitution treatment and other harm reduction approaches demonstrated to reduce HIV risk; to affirm the human rights of drug users to health and health services; and to reject efforts to overrule science and tie the hands of those working on the front lines. No less than the future of the HIV epidemic is at stake.cc: Joint United Nations Programme on HIV/AIDSWorld Health OrganizationOffice of the High Commissioner for Human RightsInternational Narcotics Control BoardOrganizations and individuals who have signed this letter as of March 1, 2005 are listed in For the French version of this Open Letter please see Open Letter to the delegates of the Forty-eighth session of the Commission and other additional information.Click here for fileFrench version of the Open LetterClick here for fileSpanish version of the Open LetterClick here for fileRussian version of the Open LetterClick here for file


Excessive sequential stereotypy of behavioral patterns  in Tourette's syndrome and obsessive compulsive disorder (OCD) is thought to involve dysfunction in nigrostriatal dopamine systems. In sequential super-stereotypy, patients become trapped in overly rigid sequential patterns of action, language, or thought. Some instinctive behavioral patterns of animals, such as the syntactic grooming chain pattern of rodents, have sufficiently complex and stereotyped serial structure to detect potential production of overly-rigid sequential patterns. A syntactic grooming chain is a fixed action pattern that serially links up to 25 grooming movements into 4 predictable phases that follow 1 syntactic rule. New mutant mouse models allow gene-based manipulation of brain function relevant to sequential patterns, but no current animal model of spontaneous OCD-like behaviors has so far been reported to exhibit sequential super-stereotypy in the sense of a whole complex serial pattern that becomes stronger and excessively rigid. Here we used a hyper-dopaminergic mutant mouse to examine whether an OCD-like behavioral sequence in animals shows sequential super-stereotypy. Knockdown mutation of the dopamine transporter gene (DAT) causes extracellular dopamine levels in the neostriatum of these adult mutant mice to rise to 170% of wild-type control levels.We found that the serial pattern of this instinctive behavioral sequence becomes strengthened as an entire entity in hyper-dopaminergic mutants, and more resistant to interruption. Hyper-dopaminergic mutant mice have stronger and more rigid syntactic grooming chain patterns than wild-type control mice. Mutants showed sequential super-stereotypy in the sense of having more stereotyped and predictable syntactic grooming sequences, and were also more likely to resist disruption of the pattern en route, by returning after a disruption to complete the pattern from the appropriate point in the sequence. By contrast, wild-type mice exhibited weaker forms of the fixed action pattern, and often failed to complete the full sequence.Sequential super-stereotypy occurs in the complex fixed action patterns of hyper-dopaminergic mutant mice. Elucidation of the basis for sequential super-stereotypy of instinctive behavior in DAT knockdown mutant mice may offer insights into neural mechanisms of overly-rigid sequences of action or thought in human patients with disorders such as Tourette's or OCD. Overly rigid sequential patterns of movement and thought characterize several human brain disorders involving dysfunction in basal ganglia systems . For example, pathological repetitions of spoken words in Tourette's syndrome, and the tormenting habits and thoughts of obsessive-compulsive disorder (OCD), involve overly rigid sequential patterns of action, language or thought -9, whichNormal sequential patterns of action, language and thought also have been suggested to depend on proper basal ganglia function ,15. For Almost all behavior is sequential, so what do we mean by 'syntactic sequence'? In the simplest terms, a syntactic sequence is one that follows normative rules that determine the temporal progression of its elements and impart a lawful predictability to the sequence as a whole ,20,21. HSpermophilus beecheyi), syntactic chains have been even further ritualized into a stereotyped display, and adapted for territorial communicative use [Neuroethological studies of natural behavior in animals have shown that neostriatum, substantia nigra, and their connecting dopamine projections are critical to sequential stereotypy for complex serial patterns of instinctive behavior -35. In ptive use . As is ttive use ,40. Thustive use ,24,29-31The firing of some basal ganglia neurons in neostriatum and in substantia nigra codes the serial pattern of syntactic grooming chains as an entire sequence in rats ,28. In adisrupt behavioral sequences indicate a potential sequencing function for the targeted structures. However, factors besides sequencing loss may contribute to disrupted serial patterns after lesions. An alternative and stronger proof for dopamine mediation of action syntax would be to demonstrate enhanced stereotypy of behavioral sequences, by boosting nigrostriatal dopamine neurotransmission. Enhanced sequential stereotypy would be reflected if the complex serial pattern as a whole entity became more sequentially rigid or persistent. Indeed, in rats, pharmacological boosting by dopamine D1 agonists administered systemically or into brain ventricles produces sequential super-stereotypy of syntactic grooming chains [Brain lesions that g chains -45. In ag chains ,44. Suchg chains -50.In human pathologies such as Tourette's or OCD, complex sequential super-stereotypy often occurs spontaneously in human patients. If sequential super-stereotypy of complex instinctive behavior sequences is to serve as a model of human disorders involving sequential super-stereotypy, it ought to be able to occur spontaneously in some individual animals too. In addition, it should possess features of compulsive behavioral sequences.perseverative tendencies and more rigid sequences of entire serial patterns. To date, prior genetically-modified mouse models of spontaneous compulsive behavior have successfully captured the perseverative feature, but it is not yet clear whether these animal models also share the exaggerated serial pattern feature of compulsive behavior. For example, the Hoxb8lox mutant model has been reported to exhibit OCD-like increased persistence of self-directed grooming and body-licking, and even mutual grooming of other mice [D1CT mutant mouse, caused by transgenic potentiation of D1-associated brain circuits, shows OCD-like persistence of grooming, as well as persistence of other behaviors such as digging, climbing, and tics [Compulsive behavior may have several features, including both her mice ,51,52. Sand tics ,53-57. HDAT-KD mutant mice with genetic knockdown of the dopamine transporter (DAT) [DAT-KD mutant mice have 10% normal DAT expression in dopamine neurons [DAT-KD mutant mice show other behavioral evidence for high levels of dopamine activation. They tend to be hyperactive, to walk in perseverative straight paths, and to over-pursue certain incentive stimuli [Here we show that this serial pattern feature of sequential super-stereotypy indeed appears spontaneously without drugs in er (DAT) . DAT-KD  neurons , which i neurons . DAT-KD  stimuli -60. The DAT-KD mice had virtually all the typical features of wild-type chains and of syntactic chains previously reported for outbred mice and D1 receptor knockout mice [Hyper-dopaminergic mutant mice and wild-type control mice each generated syntactic chains of grooming as described above Figures , 2 &3. Sout mice ,41  and consists of 1–4 unilateral or highly asymmetrical strokes made by one forepaw. The unilateral stroke is typically of small or medium amplitude ascending to about the level of the eye. In mice, the other paw not participating in the Phase II stroke often makes a smaller Phase-I ellipse-type stroke simultaneously ,41. ThusPhase III is highly visually distinctive, and consists of 1–5 large bilateral strokes with both paws. Both paws move very symmetrically almost as mirror images of the other, typically ascending together high up the side of the face, and passing forward synchronously over the ears. Phase III strokes are extremely stereotyped, usually all of the same height, and with both paws traveling back down to the nose between successive Phase III strokes . The entPhase IV concludes the prototypical chain, and consists of a postural turn to the side and caudally, and lowering of the head to bring the tongue towards the flank or side of the body, followed immediately by a 2–5 s bout of body licking directed to the flank.started during a grooming bout, DAT-KD mutant mice initiated marginally more syntactic chains overall than wild-type mice  = 3.592, p = 0.061; Figure  = 17.315, p < 0.001; Figure In terms of the number of syntactic chains The nature of the context-dependence of the difference was further clarified by a closer look at the time course of exactly when syntactic chains were begun by mice during a grooming bout. The overwhelming majority of syntactic chains tended to be initiated early in a grooming bout by all mice Figure . MutantsIn mice and rats, once a syntactic chain pattern begins with Phase I, each remaining action can be predicted with roughly 80–90% accuracy. The entire syntactic chain occurs with a frequency over ten thousand times greater than could be expected by chance (based upon the relative probabilities of the component actions). However, several types or degrees of chain completion are possible.A prototypical or perfect syntactic chain requires Phases I, II, III and IV in order, with no deviations, additions or omissions. Perfect chains were occasionally performed by both mutant and wildtype mice. After performing Phase I, II and III strokes over the face, a mouse performs Phase IV by transitioning to body grooming. For this transition to body licking, the mouse must bend down and backward to bring its mouth toward a side flank, and then begin a vigorous bout of body licking that continues for 1–4 s.imperfect syntactic chains were observed in both mutant and wildtype mice. Imperfect sequences proceed from Phase I to IV with some minor deviation from the prototypical pattern along the way. In this study, we recognized three forms of imperfect completion. All involved a minor imperfection, which was either an insertion, reversal or replacement of a component action within the syntactic chain. Imperfect completion forms were: (i) Reversal of Phases II-III, where Phase II unilateral strokes were emitted after Phase III bilateral strokes (instead of before them), but the chain was otherwise syntactically correct; (ii) Insertion of an unexpected movement component in between phases, usually a quick paw lick or several paw licks inserted between Phases III and IV; (iii) Skip or omission of one phase en route to completion, where a chain lacked either any Phase II unilateral face stroke, or any Phase III face strokes (never both), but was otherwise syntactically correct (e.g. an observed order of I-III-IV). Finally, a fourth type of syntactic chain was observed that failed to be completed in the prototypical sense, but where the mice substituted paw licking in the terminal place of Phase IV . We called this Terminal substitution: the final Phase IV component (body licking) was completely replaced with a different type of licking movement (paw licking), and the chain was otherwise syntactically correct (e.g. I-II-III-paw lick bout).In addition, several types of Terminal substitution never attains a prototypical Phase IV, and so is not really a form of syntactic completion by criteria used in earlier studies. However, the terminal substitution of paw licking might be viewed as an attempt to complete syntactically with a transition from paw strokes to licking, compared to other forms of incompletion such as either simply stopping or immediately launching into a sequentially flexible series of grooming strokes. Thus for the purpose of analysis, we examined the consequences of allowing terminal substitution to count provisionally as a form of "weak" completion.At the completion of Phase IV (strong or weak), over 93% of syntactic chains led to continued grooming of body or face in sequentially flexible and much less predictable patterns compared to syntactic chains. After 7% of chains, the end of Phase IV terminated the entire grooming bout, and the mouse rested quietly afterwards or began to explore the chamber.DAT-KD mutant mice not only started more syntactic chain patterns, they were also more likely than wild-type mice to complete the syntactic chain patterns they started – in both laboratory and home environments  = 12.33, p < 0.001; Figure  Figures  &3. Sequ = 5.96, p < 0.001). Hyper-dopaminergic mutant mice nearly always completed their syntactic chains with the strong form of Phase IV (body licking), whereas wild-type mice completed roughly half their chains with only the weaker form of Phase IV (paw licking). Mutant mice completed a higher percentage of insertion, reversal and omission types of syntactic chains than wild-type mice  = 129.01, p < 0.001; each subtype; Figures The higher syntactic rigidity of mutants was visible qualitatively and verified quantitatively Figures  &3, and In other words, mutant mice were better than wild-type mice at resisting disruption of the pattern by minor flaws that occurred along the way, and mutants more often returned to the full-blown pattern after any distraction. For example, insertion chains included 1 or 2 extraneous movements, such as a nonsyntactic paw lick action inserted between Phases III and IV. After an insertion, mutants were nearly 50% more likely than wild-type mice to reach a strong form of Phase IV completion . Similarly, a reversal error reversed the serial order of Phases II and III, or followed a Phase II stroke with a late Phase I ellipse stroke, and after a reversal mutant mice were nearly 50% more likely than wild-type mice to go on to complete a strong form of Phase IV. Finally, in an omission chain, a mouse would omit either Phase II or Phase III (never both), and after an omission mutants were again roughly 50% more likely than wild-type mice to successfully return to the full pattern and reach a strong form of terminal Phase IV completion .In contrast, wild-type mice had a greater proportion of terminal substitution chains that never achieved a full syntactic transition to body grooming. Wild-type mice instead substituted a weaker paw-lick form of Phase IV as terminal component. In terminal substitution, a mouse completely omitted the normal Phase IV shift to body licking, and instead simply continued to lick its paws, never changing posture or moving its head caudally out of the normal facial grooming position . Wild-type mice had nearly twice the proportion of terminal substitutions as mutant mice = 11.47, p < 0.001).If terminal substitution is regarded as failure to complete the pattern, then wild-type mice simply failed to complete over half the syntactic chains they began. More leniently, wild-type mice could approach an 80% – 90% rate of syntactic completion – if we took the unprecedented step of allowing Phase IV terminal substitution to count as weak completion Figure . AllowinDAT-KD mutant mice had more rigid sequential patterns than wild-type controls in several ways. Mutant mice were more likely than wild-type mice to proceed syntactically through Phases I, II and/or III to reach the syntactic final Phase IV (body licking). Even after encountering minor imperfections along the way, mutants persevered in the sequential pattern. Wild-type mice introduced the same imperfections in their syntactic pattern, but did not return to the full pattern or complete Phase IV as strongly, ending their chains without ever reaching the full-blown transition to body grooming that normally terminates a syntactic chain pattern.In summary, Finally, syntactic completion was highest in home environment grooming for all mice (even though more syntactic chains were begun in laboratory) 14.31, p < 0.001). This difference suggests that stress may promote the initiation of stereotyped sequences, but impede their lawful completion, and is consistent with reports that stress disrupts completion of syntactic chain sequences . HoweverIn order to reject motor confounds that might have provided an alternative explanation of some results, we assessed whether wild-type mice were simply less able to perform body licking movements than mutant mice. If wild-type mice had motor deficits that impaired their ability to perform body-lick posture/movements, then wild-types might have had weakened syntactic chains simply because of their motor incapacity to perform Phase IV movements, rather than because mutants had stronger sequencing tendencies. Therefore we analyzed whether wild-type mice spent a lower proportion of their total grooming behavior time making body licking movements compared to mutant mice. However, wild-type mice did not have significantly lower total cumulative duration scores for body licking overall than mutant mice  = 0.56, n.s.), indicating there was no motor impairment of Phase IV movements. That suggests the difference in tendency to complete syntactic chains represents a true difference in sequence rigidity or pattern strength, and not in simple motor capacity. = 1.773, p < 0.001; Figure DAT-KD mutant mice spent 10%–50% more time than wild-type control mice in grooming behavior overall  = 3.949, p < 0.05), and the mutant propensity to groom more was most visible in the home environment (p < 0.1).All mice groomed twice as much in their home cages than in the laboratory environment, suggesting that the relatively novel laboratory environment might have acted to suppress spontaneous grooming behavior  = 4.083, p = 0.008), while bout numbers did not differ.Closer analysis of grooming compared the relative contributions to increased grooming time of greater bout numbers versus longer bout durations Figure . The inc = 3.403, p < 0.07), a separate analysis of grooming specifically in the home cage showed that mutants at home had longer cumulative durations of body licking , consistent with prolongation of the later components of cephalocaudal grooming bouts in that home environment [DAT-KD mutant grooming in the home cage therefore may overlap with perseverative body grooming tendencies reported for other genetic animal models of compulsive behavior, such as Hoxb8lox and D1CT mutant mice [Although mutants emitted only marginally more body licking than wild-type mice in an analysis that combined data from both home and laboratory environments . In the laboratory environment, mutants began more grooming bouts than wild-type mice  = 3.478, p = 0.026), but their duration of bouts did not differ. Thus, different features of grooming bouts (length versus number) were enhanced in mutant mice depending on their environmental context of the moment. However, as described above, in both home and laboratory the hyper-dopaminergic mutants were always more likely than wild-type mice to perform more rigid and strongly stereotyped syntactic chain sequences.Conversely, in the laboratory environment, Our results reveal that hyper-dopaminergic mutant mice show excessively strong and rigid manifestations of a complex fixed action pattern compared to wild-type mice. Their sequential super-stereotypy was produced by DAT knockdown mutation, which reduces DAT to 10% of wild-type levels and causes extracellular dopamine elevation to 170% in neostriatum . Mutant DAT-KD mutant mice were more likely to begin a syntactic chain pattern than wild-type mice, especially during the early minutes of a grooming bout , and especially in the novel laboratory environment . Further, once the complex sequence began, DAT-KD mutant mice went on to execute chains that were more stereotyped and rigid, both qualitatively and quantitatively. Qualitatively, mutant mice almost always achieved the strongest form of the terminal phase (Phase IV), successfully making a transition from head grooming to body grooming. By comparison, wild-type mice ended far more of their chains with a weaker terminal substitution for Phase IV, which left them stuck in head grooming without ever making a transition to body grooming. Quantitatively, DAT-KD mutant mice returned more often to the prototypical pattern after minor mistakes, whereas wild-type mice failed to reach full Phase IV after such mistakes. Mutant mice returned more often to the full pattern after extraneous component insertion, phase omission, or serial reversal of phases. The mutants' elevated pattern strength for this complex sequence was evident in both home and laboratory environmental contexts.The stronger pattern was evident in several ways. First, sequential super-stereotypy, representing the exaggerated serial rigidity feature of compulsive behavior. Here sequential super-stereotypy is manifest in a complex behavioral sequence that is instinctive and naturally stereotyped to begin with, but becomes even more stereotyped or excessively rigid as a consequence of the DAT mutation.If the less-stereotyped sequential patterns of wild-type mice are viewed as the norm , then the mutant tendency to complete stronger syntactic patterns must be viewed as It may be important that the mutant pattern strength is revealed not as an elimination of errors, but rather primarily as a resistance to disruption by errors. In other words, mutants did not have more frequent perfect chains than wild-types: both generated similar moderate rates of minor errors . Instead, the mutants' stronger syntactic pattern was like a tightened elastic band, pulling them back after such errors to finish the prototypical pattern. Stronger return to the pattern could only be possible if DAT knockdown strengthened the entire pattern as a global whole, facilitating the mutants' ability to maintain a neural representation of the pattern during an error and to resume the remaining pattern after the error. That suggests that neural mechanisms of pattern coordination were better able to persist in rule maintenance in the face of disruption, and to successfully compete to re-establish control of the behavioral stream after the disruption.Thus, stronger patterns were not simply the results of strengthened Markov sequential transitions among individual pairs of actions, producing a stimulus-response (S-R) reflex chain. If sequence composition was simply a probabilistic construction based only on the frequency of transitions between individual pairs of actions, then stronger perfect completion might have been expected in mutants, but not stronger return after an error. Errors would still terminate or weaken the pattern. Instead, we observed the opposite result: mutants kept errors but recovered better after them, and took the full pattern up again where it had left off.D1CT mice, and hyperdopaminergic DAT-KD mice [Hoxb8lox and D1CT mutant mice, also show any exaggerated serial rigidity features in their fixed action patterns similar to those found here [These results are the first demonstration to our knowledge of sequential super-stereotypy of a complex behavioral pattern, occurring spontaneously without drugs. In previous studies, dopamine D1 agonists were needed to cause sequential super-stereotypy of syntactic grooming chains, whereas D2 agonists in contrast reduced initiation and completion of syntactic grooming chains (even though D2 agonists can cause simple repetitive movement stereotypies) -45,64. F-KD mice ,55,57,64und here -55,57,65weakening of the syntactic chain pattern by other genetic manipulations. For example, the ability to complete syntactic grooming chains is impaired in several types of mutant mouse, caused by either a knockout of D1 dopamine receptors [DAT-KD findings provide an opposite demonstration to complement that D1 knockout study: DAT knockdown strengthens the same pattern presumably by elevating extra-cellular dopamine. Both results might therefore reflect essentially linear effects on the sequential stereotypy of this complex behavior pattern, mirroring up or down changes in basal ganglia dopamine neurotransmission.We should note that although our study is the first to produce spontaneous sequential super-stereotypy, several previous studies reported eceptors , or by aeceptors ,66. In teceptors . Our DATSpermophilus beecheyi [We acknowledge that there is one other known form of genetically-related sequential super-stereotypy for syntactic grooming chains. However, that sequential super-stereotypy is not caused by a single targeted gene mutation but rather is a naturally evolved adaptation of the fixed action pattern in a species of ground squirrel, beecheyi , which ibeecheyi .Spermophilus beecheyi syntactic chains are ritualized, more sequentially rigid and predictable than normal self-grooming chains, and occur as a single grooming chain with no other grooming before or after [Display forms of or after . Phase Ior after .Spermophilus beecheyi ground squirrels appears to have exapted the pre-existing pattern of a syntactic grooming chain, which likely evolved in ancestral rodents over 60 million years ago, and co-opted it into a sequentially super-stereotyped form for specific communicative use [Thus the evolution of tive use . It may tive use . The gentive use ,65,66.DAT-KD mutants. Similarly, neurons in the substantia nigra pars reticulata appear especially to code initiations of the complex behavioral sequence, and so modulated input to them might be more relevant to the elevated mutant tendency to begin the syntactic pattern [Altered neurochemical signaling within basal ganglia neural circuits may be the mechanism by which DAT knockdown produces sequential super-stereotypy of grooming syntax. Electrophysiological studies have shown that neurons in neostriatum and in substantia nigra pars reticulata code the sequential pattern of syntactic grooming chains and other natural sequences of behavior ,27,28. F pattern ,28.DAT-KD mutant mice might also have elevated extra-cellular dopamine concentrations in other target structures, including prefrontal cortex and amygdala. Such systems might also contribute to OCD and Tourette's syndromes in humans and to some aspects of compulsive-like behavior in mutant mice. Elaborated applications of dopamine-related circuits for sequencing may thus extend from instinctive animal behavior to abstract human cognition and behavior, including syntactic sequencing of action plans, linguistic syntax, and the serial order of streams of thought [Nigrostriatal mechanisms for sequencing instinctive action may have been co-opted in subsequent mammalian and human evolution into use in sequencing learned and cognitive psychological elements -69. In t thought ,73.A clinical implication of the embeddedness of basal ganglia in sequencing function may be a vulnerability to sequential dysfunction in some human disorders involving nigrostriatal systems ,75. BothFinally, while highly speculative, it is at least conceivable that an evolutionary specialization of dopamine-related neural mechanisms for self-grooming sequences, suggested by our current results, might also influence the theme or content, as well as the syntactic stereotypy, of some human super-stereotypies involving washing or purifying compulsions .DAT-KD mutant mice show sequential super-stereotypy in a complex instinctive fixed action pattern.Pathologically-intense rituals of cleanliness, security behavior, or concerns with contamination, all share a focus that might relate to grooming of oneself . ConceivDAT-KD mutant mice  and wild-type control mice  were generated at the University of Chicago by breeding heterozygous mutants on a 129 Sv/J genetic background as described earlier [Slc6a3 locus. DAT knockdown was achieved by insertion of the tetracycline regulatable system into the 5' untranslated region in the second exon of the DAT gene (Slc6a3). Such an insertion reduced the DAT promoter strength without affecting its expression pattern. It also allows regulation of DAT expression by dietary tetracycline, although that feature was not used in this study. DAT knockdown reduces adult DAT expression to 10% of wild-type levels and raises extracellular dopamine levels in neostriatum to 170% (wild-type control = 100%) [ earlier . Such a  = 100%) . Once hoIt was important to determine whether any sequential stereotypy difference between mutant and wild-type mice in grooming behavior was a stable difference in action syntax strength, and not merely an artifact of testing conditions. Grooming behavior of rodents is sensitive to environmental contexts, both in quantity and in fine structure, and stressors in particular can either suppress or increase grooming behavior depending on type . All micImmediately prior to testing, mice were transported in their home cage on a cart down a 30 m hallway to a laboratory testing room with standard white fluorescent lighting, and placed individually in a test chamber . The laboratory test chamber consisted of a transparent cylinder  suspended over a tilted mirror. A camera lens focused on this mirror gave a close-up view of the mouse's face, forepaws, and upper body. For behavioral testing, each mouse was placed individually in a test chamber and videotaped for 30 minutes. Each mouse received 3 habituation days in the laboratory test procedure before grooming behavior data were collected over the next 2 consecutive days in 30 min sessions.Testing in the home environment took place during the dark phase under dim red light conditions. Mice were housed singly in transparent rectangular cages (12 cm high × 19 cm long × 10 cm wide). Videotaping of grooming sequences took place from the side of the transparent home cage, for 30 min each day on 2 consecutive days, with the camera focused closely on the mouse.th actual speed; scorer blind to genotype) for grooming amount (cumulative durations), grooming bout number and bout length, and occurrence of syntactic chains. Syntactic grooming chains were identified and classified in frame-by-frame analysis as either Perfect, Imperfect but completed by full Phase IV , Terminal substitution of paw lick for Phase IV body licking, or Incomplete  [Videotaped grooming behavior was scored in slow motion (frame-by-frame to 1/10strokes) ,41, [43,strokes) . We alsostrokes) . BehavioKCB conceived and supervised the study and drafted the manuscript; JWA co-conceived the study and participated in interpretation and writing; KRH carried out behavioral testing, videoanalysis, and statistics; XZ developed and generated the mutant mice, and participated in writing the manuscript.Movie: Sequential super-stereotypy of an instinctive fixed action pattern in hyper-dopaminergic mutant mice. Windows Media Player movie file (.avi): DAT Knockdown grooming fixed action pattern.aviExamples of syntactic grooming chains performed by three hyperdopaminergic mutant mice are shown in the accompanying movie file. Choreograph diagrams of component movements' form and sequence are displayed for each syntactic chain, and strokes are illuminated sequentially in synchrony with their corresponding movements. Note that the first two syntactic chains contain insertion or reversal errors . However, the syntactic chains are not disrupted by these errors, and the mutants continue on with the sequential pattern to successfully complete Phase IV (body licking). Mutant mouse 3 also shows the ventral view that permits the viewer to see both forepaws simultaneously, which was used to score all syntactic chains in the laboratory.Click here for file