A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 μM, while it contains as many as 50–80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment), that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are simultaneously analyzed in an array format, the likelihood of each interaction occurring in any given physiological settings can be evaluated. WISE can be easily extended to a variety of protein interaction domains, including those binding to modified peptides, thereby offering a powerful proteomic tool to help completing a full description of the cell interactome. By combining phage display and SPOT selection, the binding partners of any peptide recognition motif can be identified, thus facilitating the identification of all protein-protein interactions within a proteome Saccharomyces cerevisiae emphasized the importance of confirming any interaction by different methods  that act as receptors to accommodate, in their binding pockets, short peptides in an extended conformation .We have recently shown that the peptide sequences, obtained by panning phage-displayed random peptide libraries with SH3 domains, can be used to derive position-specific scoring matrices to computationally scan the entire proteome in search of putative partners . This ap7. This figure is far beyond the limits of the current technology for peptide synthesis. On the other hand, one could use the information obtained from screening random peptide repertoires to filter out the amino acid sequences that are highly unlikely to bind, thereby decreasing the peptide sequence space to be tested experimentally. We will refer to this approach with the acronym WISE (whole interactome scanning experiment)  .It should be pointed out that WISE only addresses the problem of identifying natural peptides with the potential for binding to any given recognition domain. Although we use this information to infer the formation of protein complexes in vivo, there are a number of reasons why this inference could turn out to be incorrect. For instance, a peptide could be unavailable for interaction in the native protein structural context. Alternatively, the two inferred partners could be located in different cell compartments or expressed in different tissues or at different times during development. Finally, all the interactions that are mediated by an extended region of a protein surface and that cannot be supported by a relatively short peptide will be missed by this approach.S. cerevisaie proteins that contain SH3 domains belonging to five different specificity classes, as determined by phage display experiments . A detailed description of the relaxed patterns can be found in For each SH3 domain, we have defined a “relaxed pattern,” less selective than the pattern identified by comparing the most frequent ligands discovered by phage display. We have then used this to scan the whole  TM instrument)This approach was repeated for the eight SH3 domains. For each domain, approximately 1,500 peptides, matching the relaxed patterns, were selected for synthesis see . The pep−4 M or above give a positive signal in the assay  that the intersection of a protein list with any given functional category occurs by chance. The inferred ligands of the Ysc84, Yfr024c, and Rvs167 SH3 domains were significantly enriched for the GO biological process term “actin cytoskeleton organization and biogenesis” . By contrast, the partners of Abp1 and Myo5 SH3 domains were found to be enriched for the GO terms “actin cortical patch assembly” (p < 3.49 × 10−7) and “actin cytoskeleton” (p < 7.14 × 10−6). These results are in accord with the available information about the participation of these bait proteins in the organization of the yeast cytoskeleton, whereas arbitrarily selected gene groups of similar size showed no comparable enrichments for any of the GO terms .Interacting proteins often share similar functions and participate in common processes. Hence, we examined whether the proteins, found in our approach to bind to a specific SH3 domain, could be preferentially associated to a biological process. For this analysis we considered, as putative ligands, all the proteins containing at least one peptide with an intensity higher than an arbitrarily chosen threshold of 20,000 . We then used the FunSpec software  to identWe have previously shown that the information obtained by panning random peptide libraries can be used to draw an interaction network that recapitulates a fraction of the SH3-mediated interaction network determined by the two-hybrid approach . By usinThe Abp1 SH3 domain, compared to most of the remaining SH3 domains that we have studied, has a narrower peptide recognition specificity and, as a consequence, fewer inferred partners. Our analysis confirmed that Srv2 and Ark1, previously identified Abp1 SH3 functional partners , containIn order to assess how accurately peptide binding affinity permits us to infer physiological partners, we investigated whether the putative partners can be copurified with Abp1 in vivo. We used the tandem affinity purification (TAP) technology  to tag tTo establish whether Abp1 forms a complex with these proteins in vivo, we next affinity-purified the four tagged proteins on an IgG resin. We next probed the purified complexes with an anti-Abp1 antibody. As shown in We have also investigated whether the fraction of coimmunoprecpitated Abp1 protein correlates with either the BLU intensity or the dissociation constant of the corresponding SH3–peptide interaction. The observed lack of correlation indicates that other factors, as, for instance, local protein concentration (mediated by different interactions), are important in determining the efficiency of complex formation.Finally, we asked whether this approach could be extended to the analysis of a mammalian proteome that is approximately five to six times more complex than the yeast one. To this end, we selected two proteins involved in membrane recycling, amphiphysin-1 (P49418) and endophilin-1 (Q99962), and whose SH3 domains we had previously characterized by phage display . These tDynamin and synaptojanin, two proteins involved in endocytosis, form an SH3-mediated complex with amphiphysin and endophilin in vivo . Our appThe WISE strategy described here has the merit of combining the strengths of a selective approach  with a quantitative analysis that can be achieved by screening a large number of peptides arrayed at high density on a solid support. This makes it possible to identify rapidly and directly the tighest ligands of a peptide-binding receptor among all the peptides in an entire proteome. We have demonstrated the approach by applying it to the family of SH3 domains. However, WISE can also be extended to all those domain families  that mostly recognize short peptides in their partner proteins.Our approach, as any in vitro approach, suffers from some simplifications when it comes to inferring the physiological partners from the domain–peptide interaction data. According to a naive strategy, we would assimilate the cell to a cellulose membrane, where all the peptides are equally represented and accessible to the bait domain, and we would be tempted to conclude that all the proteins containing the identified peptide ligands were likely to be physiological partners. In the real cell, however, the target peptides may be hidden inside the core of the folded proteins, and the protein partners may not be equally represented. Furthermore, the partner proteins may be expressed in different cell types or segregated in different macromolecular complexes or cell compartments. In order to obtain more reliable inferences, the peptide interaction information obtained by a WISE approach should be complemented by information about peptide accessibility obtained by structural predictors  and dataNevertheless, the average number of peptides in the yeast proteome that have the potential to bind SH3 domains with an affinity that may have physiological relevance was found to be surprisingly high, ranging from a few peptides, in the case of the Abp1 and Boi2 SH3 domains, to several tenths, in the case, for instance, of the Yfr024w SH3 domain. Given the hypothesis that all (or most of) these peptides are equally expressed inside the cell and exposed to the solvent in the folded protein structure as most Pro-rich peptides are, these findings raise the question of whether the observed binding promiscuity has any physiological implication. Recent proteome-wide analyses of yeast protein complexes have revealed that many proteins are organized in discrete complexes . Yet thiAll the same, we have identified a larger number of target peptides that bind with affinities that are comparable to the ones of the validated physiological targets. Some of these peptides may never encounter the cognate SH3 domain, while some will only meet partners in specific physiological conditions. Others may contribute to add specificity to the formation of a complex by cooperating with other associated low-specificity binding domains. Finally, we have to consider a new scenario in which proteins, even when not forming stable complexes, are seldom isolated in solution, but navigate in the cell by moving from one weak partner to another. These weak interactions may be important in modulating cell architecture even when they are not instrumental in the nucleation of a stable complex. Although this is difficult to prove, the semiquantitative data provided by our approach, complemented with the results of large-scale expression and localization studies, may eventually allow one to model these different settings.The in vitro approach that we have described, albeit limited to interactions in which one of the partners can be reduced to a relatively short peptide, presents a number of interesting features that complement other strategies aimed at revealing the details of the protein interaction network within cells.First, it takes full advantage both of the genomic information that is being accumulated and of the array format in which all the possible targets are equally represented. Second, it is comprehensive and provides a high level of detail on the interaction topology. Third, it is not affected by protein concentrations inside the cell and is very sensitive (interactions up to 100 μM can be detected). Fourth, interactions that depend on peptide modifications, for instance, phosphorylation, can also be studied. Fifth, the output is semiquantitative. Finally, the identified target peptide can be used as a lead to develop tighter binding molecules in order to interfere with complex formation in vivo.We have shown that the current implementation of the SPOT synthesis technology is sufficient to carry out WISE screening of a proteome as complex as the one of a mammalian organism. Foreseeable technological improvements of the SPOT synthesis technology will permit the assembly of relatively cheap microarrays containing up to 15,000 peptides. This will extend the approach's power by relieving, in some cases, the requirement for an experimental filtering step, as performed here by the phage display approach, thereby allowing more freedom in the design of the relaxed pattern.Yeast PJ694a strains expressing TAP-tagged ORFs were constructed as described . PrimersThe polymerase chain reaction (PCR) fragments were used to transform the yeast strain. Positive clones were selected on selective plates and checked by PCR analysis and Western blot analysis. For the PCR analysis, we used a new forward primer together with the reverse ones used for the construction: forward for Yir003, AGCAGATGGAGGACCAAATGGAGGTTG; forward for Scp1, CGGTTATATGAAAGGTGCATCTCAGGC; forward for Prk1, CGTTTACAATCAAAGAAACTGCCGATTG; and forward for Ynl094w, GGACTCAATTCAAAAATTGAGCAATCAAG.Yeast strains expressing TAP-tagged Yir003w, Scp1, Prk1, Ynl094c, or Bmh1 as a control, were cultured at 30°C in 5-l flasks containing 2 l of YPD medium, collected in the exponential growth phase, and lysed mechanically with glass beads in 5 ml of IPP-150 buffer  in the presence of protease inhibitors . Half of the extract was incubated for 2 h at 4°C with 100 μg of GST–Abp1SH3 (bound to glutathione–sepharose), while the remaining half was incubated with 100 μg of GST as a control. The resins were washed four times with 5 ml of IPP-150 buffer and the bound proteins recovered (by boiling in SDS–BLU–dye) and analyzed on a 10% SDS–polyacrylamide gel. They were transferred onto nitrocellulose membranes. Filters were blocked overnight at 4°C in PBS containing 5% milk powder (blocking solution), and then incubated with peroxidase (POD)-conjugated anti-POD antibody (PAP) antibody  diluted 1:1,000 for 2 h at room temperature (RT), washed five times for 15 min with PBS–0.05% Tween, and revealed by chemoluminescence. GST fusion proteins were expressed and purified by standard procedures.Yeast cultures expressing TAP-tagged Yir003, Scp1, Prk1, Ynl094c, or Bmh1 were cultured, collected, and lysed as described for the pulldown experiments. Each extract was incubated with 500 μl of IgG–sepharose  for 2 h at 4°C. The resins were washed four times in 5 ml of IPP-150 buffer, resuspended in 300 ml of 50 mM Tris–HCl (pH 8), 0.5 mM EDTA, 5 mM DTT, transferred to Eppendorf tubes, and incubated with 30 U of recombinant TEV protease  for 1 h at 20°C. After centrifugation for 2 min at 2,300 rpm, the supernatants were loaded on 10% SDS–polyacrylamide gels and then transferred onto nitrocellulose membranes. Filters were blocked overnight at 4°C in blocking solution, incubated for 2 h at RT with anti-Abp1 antibody , and washed five times with PBS–0.05% Tween. They were then incubated for 1 h at RT with anti-rabbit POD coniugated, washed ten times with PBS–0.05% Tween, and detected by chemoluminescence.Cellulose membrane-bound peptides were automatically prepared according to standard SPOT synthesis protocols  using a p-toluenesulfonic acid in methanol (50 ml) and shaken for 3 min. The membrane was removed from the tray and air-dried. Meanwhile a solution of 7.8 g of N--phathalimid in dioxane (60 ml) was heated up to 80°C in a covered stainless steel dish placed on a shaking platform using a heater mat. Then, a solution of 400 mg of p-toluenesulfonic acid in 5 ml of dioxane was added. Immediately, the membrane was placed in this solution and shaken at 80°C for 3–5 h. Afterwards, the membrane was washed three times with 50 ml of dioxane and ethanol  and subsequently incubated with a 10% (v/v) solution (50 ml) of hydrazine hydrate (80%) in ethanol for approximately 6 h. Finally, the membrane was washed twice with ethanol, three times with dimethylacetamide, and once again with ethanol , and dried. The loading of this type of amino-functionalized cellulose membrane is about 120–200 nmol/cm2.A 18 × 28 cm Whatman 50 paper  was immersed in a stainless steel dish containing a solution of 400 mg of TM instrument . For quantification, the SPOT signal intensities were measured in BLUs. To exclude false-positive results, in the SH3-incubation experiment, each membrane was preexamined with GST/anti-GST mAb/anti-mouse mAb. The data obtained with the different membranes were normalized by using as a reference the intensity of three control peptides that bind to the anti-GST antibody. The sequence of these peptides were QRALAKDLIVPRRP, LAKDLIVPRRPEWN, and DLVIRPPRPPKVLGL.Generally, all incubations and washing steps were carried out under gentle shaking. After washing the membrane once with ethanol (10 min) and three times for 10 min with Tris-buffered saline (TBS: 50 mM Tris-(hydroxymethyl)-aminomethane, 137 mM NaCl, 2.7 mM KCl, adjusted to pH 8 with HCl), the membrane-bound peptide arrays were blocked (3 h) with blocking buffer. Blocking reagent  was diluted 1:10 in TBS containing 5% (w/v) sucrose. After washing with TBS (10 min), 10 μg/ml of the corresponding GST-fused SH3 domain (in blocking buffer) was added and incubated overnight at 4°C. After washing three times for 10 min with TBS, the anti-GST monoclonal antibody (mAb)  was added at a concentration of 1 μg/ml in blocking buffer for 2 h at RT. Subsequently, the membrane was washed three times with TBS (10 min each) and the POD-labeled anti-mouse mAb (1 μg/ml in blocking buffer) was applied for 1.5 h at RT, followed by washing three times with TBS. Analysis and quantification of peptide-bound SH3 domains were carried out using a chemoluminescence substrate and the Lumi-ImagerSurface plasmon resonance measurements were carried out with a BIAcoreX instrument . Experiments were carried out on sensor chips with GST-fused SH3 domains and GST as a control. GST-fused SH3 domains and the GST were coupled to CM5 sensor chips using the EDC/NHS (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide)amine-coupling kit, yielding approximately 4,350 resonance units in the case of the GST-fused SH3 domain and 4,330 resonance units for GST. Interaction analysis was performed at 25°C with the peptides dissolved in 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20 (pH 7.4), at 15 μl/min flow rate, using six to seven dilutions, ranging from 500 μM to 65 nM. Dissociation constant values were evaluated by applying the steady-state model using BIAcore evalution 3.1 software.Dataset S1(102 KB TXT).Click here for additional data file.Dataset S2(104 KB TXT).Click here for additional data file.Dataset S3(103 KB TXT).Click here for additional data file.Dataset S4(86 KB TXT).Click here for additional data file.Dataset S5(104 KB TXT).Click here for additional data file.Dataset S6(104 KB TXT).Click here for additional data file.Dataset S7(104 KB TXT).Click here for additional data file.Dataset S8(76 KB TXT).Click here for additional data file.Dataset S9(183 KB TXT).Click here for additional data file.Dataset S10(184 KB TXT).Click here for additional data file.Table S1(66 KB PDF).Click here for additional data file.


Melanoma inhibitory activity (MIA) is a small secreted protein that interacts with extracellular matrix proteins. Its over-expression promotes the metastatic behavior of malignant melanoma, thus making it a potential prognostic marker in this disease. In the present study, the expression and functional role of MIA was analyzed in pancreatic cancer by quantitative real-time PCR (QRT-PCR), immunohistochemistry, immunoblot analysis and ELISA. To determine the effects of MIA on tumor cell growth and invasion, MTT cell growth assays and modified Boyden chamber invasion assays were used.The mRNA expression of MIA was 42-fold increased in pancreatic cancers in comparison to normal pancreatic tissues (p < 0.01). In contrast, MIA serum levels were not significantly different between healthy donors and pancreatic cancer patients. In pancreatic tissues, MIA was predominantly localized in malignant cells and in tubular complexes of cancer specimens, whereas normal ductal cells, acinar cells and islets were devoid of MIA immunoreactivity. MIA significantly promoted the invasiveness of cultured pancreatic cancer cells without influencing cell proliferation.MIA is over-expressed in pancreatic cancer and has the potential of promoting the invasiveness of pancreatic cancer cells. Despite improvements in diagnosis and treatment, pancreatic cancer remains one of the most common causes of cancer-related deaths in the world . One of To quantify the mRNA expression of MIA in pancreatic tissues, QRT-PCR was performed using RNA from pancreatic cancer tissues (n = 23) and normal pancreatic tissue samples (n = 17). The analysis revealed a 42 ± 28-fold increase (p = 0.0013) of MIA mRNA levels in pancreatic cancer tissues compared to the normal pancreas Fig. . To deteTo determine the functional role of MIA in pancreatic cancer, we first investigated MIA expression in pancreatic cell lines. QRT-PCR analysis revealed relatively high MIA mRNA levels in Mia PaCa-2, Panc-1, and SU8686 pancreatic cancer cells compared to the other cell lines Fig. . ImmunobMethods section, MIA significantly increased the invasion of Aspc-1 by 2.4-fold (p < 0.001) and the invasion of T3M4 by 3.1-fold (p < 0.0001) . To analyze whether MIA may promote invasiveness of pancreatic cancer cells, Matrigel-based invasion assays using a modified Boyden chamber were carried out in T3M4 and Aspc-1 pancreatic cancer cell lines that exhibited relatively low MIA expression levels according to QRT-PCR and Western blot data. Added to the top chamber as described in 01) Fig. . To inveOne of the most devastating aspects of malignant growth is the emergence of cancer foci in organs distant from the primary tumor, with most cancer mortality being related to metastases. Thus, understanding the molecular mechanisms underlying the metastatic process is one of the most important issues in cancer research.Pancreatic cancer is characterized by aggressive local tumor growth and early systemic tumor spread -10. ManyAnother important aspect of the metastasis process is neo-angiogenesis. Angiogenesis itself encompasses a cascade of sequential processes emanating from microvascular endothelial cells, which are stimulated to proliferate, degrade the endothelial basement membranes of parental vessels, migrate, penetrate host stroma, and initiate a capillary sprout . NumerouMelanoma inhibitory activity (MIA) increases cell motility by decreasing the attachment of the cells to the extracellular matrix (ECM). Overexpression of MIA leads to increased metastasis of malignant melanoma cells by enhancing invasion and extravasation ,23. In tIn contrast to observations in malignant melanoma, where MIA has been established as a reliable marker for prognosis ,24, we cThe reason why high MIA mRNA levels lead to high serum levels in malignant melanoma, but not in pancreatic cancer, is currently not known. As to the possible role of MIA in pancreatic cancer pathogenesis, MIA had no effect on the proliferation of pancreatic cancer cells, similar to previous experiments employing fibroblasts, keratinocytes, endothelial cells and lymphocytes. The only cellular system in which MIA has been found to influence cell growth is melanoma cells; in these cells, MIA exerts anti-proliferative effects .Although MIA did not affect the growth of pancreatic cancer cells in vitro, its impact on the invasion was striking. Interestingly, promotion of invasiveness of pancreatic cancer contrasts the previously demonstrated decrease in the invasion of MIA-treated malignant melanoma cells . A possiIn conclusion, our study shows a striking overexpression of MIA in cancerous pancreatic tissues without consequent elevation of MIA in the circulation. Involvement of MIA in regulation of invasiveness of pancreatic cancer cells indicates that this protein may serve as a novel therapeutic target in the search for anti-metastatic drugs.Pancreatic cancer tissue samples were collected from 55 patients who underwent pancreatic cancer resection in the Department of General Surgery at the University of Heidelberg, Germany, and in the Department of Visceral and Transplantation Surgery at the University of Bern, Switzerland. Twelve cases were stage I, 13 cases stage II, 23 cases stage III, and 7 cases stage IV pancreatic adenocarcinomas, according to the Union International Contre Le Cancer (UICC) system. According to routine pathological grading, 16 cases were well-differentiated, 22 moderately differentiated, and 17 poorly differentiated. Normal pancreatic tissues were collected from 34 healthy organ donors. Pancreatic tissues were either frozen in liquid nitrogen and stored at -80°C (for RNA and protein extraction) or immediately fixed in 4% paraformaldehyde solution and subsequently embedded in paraffin. In order to determine MIA serum concentrations, sera from 50 pancreatic cancer patients  and healthy volunteers  were collected at the Department of General Surgery, University of Heidelberg, Germany. Written informed consent was obtained from all patients. The study was approved by the Ethics Committees of the Universities of Bern, Switzerland, and Heidelberg, Germany.2. For TGF-β1 induction experiments, pancreatic cancer cells were seeded in 10 cm dishes in 10% FBS growth medium and allowed to attach for 12 hrs. Growth medium was replaced by serum-reduced medium (0.5% FBS), supplemented with 200 pM TGF-β1 for the indicated time periods. For experimental hypoxia, cells were subjected to a hypoxic microenvironment by one hour-long flushing in a special incubator chamber with an anoxic gas mixture  and sealing of the unit.Mia PaCa-2, T3M4, Aspc-1, Bxpc-3, Capan-1, Colo-357, SU8686 and Panc-1 pancreatic cancer cells and B16 (cloneB78/H1) mouse melanoma cells were grown in RPMI 1640 medium containing 10% FBS , 100 U/ml penicillin and 100 μg/ml streptomycin . Cells were maintained in a 37°C humidified atmosphere saturated with 5% COAll reagents and equipment for mRNA/cDNA preparation were purchased from Roche Applied Science . mRNA was prepared by automated isolation using MagNA Pure LC instrument and isolation kits I (for cells) and II (for tissue). cDNA was prepared using a 1st strand cDNA synthesis kit for RT-PCR according to the manufacturer's instructions. Real-time PCR was performed with the Light Cycler Fast Start DNA SYBR Green kit . The numBriefly, consecutive paraffin-embedded tissue sections (5 μm thick) were deparaffinized and rehydrated. Antigen retrieval was performed by pretreatment of the slides in citrate buffer (pH 6.0) in a microwave oven for 10 min. Thereafter, slides were cooled to room temperature in deionized water for 5 min. After blocking of endogenous peroxidase activity with 0.3% hydrogen peroxide and washing in deionized water 3 times for 10 min, the sections were blocked for 1 h at room temperature with normal rabbit serum , then incubated with primary goat polyclonal anti-MIA antibody  overnight at 4°C. The slides were rinsed with washing buffer  and incubated with secondary rabbit anti-goat HRPO-labeled IgG , diluted 1:200 for 45 min at room temperature. After color reaction, tissues were counterstained with Mayer's hematoxylin. For negative control, appropriately diluted goat IgG was used instead of the primary antibody.The amount of secreted MIA protein in cell culture supernatants and serum samples was determined using a one-step MIA ELISA  according to the manufacturer's instructions.Cells were washed with ice-cold PBS and collected in lysis buffer  containing the Complete mini-EDTA-free protease inhibitor cocktail tablets from Roche . Lysates were centrifuged at 13,000 rpm at 4°C for 30 min, the supernatants were collected, and protein concentrations were measured with the BCA protein assay  using BSA as protein standard. 20 μg of protein were mixed with loading buffer, heated at 95°C for 5 min, separated on 12% SDS polyacrylamide gels, and transferred onto nitrocellulose membrane at 100 V for 90 min. Membranes were blocked in 5% non-fat milk in TBS-T  for 1 h, incubated overnight at 4°C with anti-MIA antibody  and exposed to secondary HRPO-labeled donkey anti-goat antibody (Santa Cruz) for 1 h at room temperature. The signal detection was performed using the ECL system .For immunoprecipitation, pancreatic cell lines (Mia PaCa-2 and SU8686) were suspended in lysis buffer  supplemented with the Complete-TM mixture of proteinase inhibitors  and incubated for 30 min on ice. After centrifugation, the supernatant was transferred into a fresh vial, pre-cleared with protein A-Sepharose beads (Santa Cruz) and incubated with 50 μl anti-MIA antibody  overnight at 4°C. Following addition of 30 μl of protein A-Sepharose for 1 h at 4°C, the mixture was pelleted, washed three times with lysis buffer, and resuspended in Laemmli sample buffer.Cell growth experiments were performed using the 3--2, 5-diphenyltetrazolium bromide (MTT) assay. Pancreatic cancer cells were seeded at a density of 5000 cells/well in 96-well plates, grown overnight, and then exposed to different concentrations of recombinant MIA protein as indicated. After 24 h, MTT was added (50 μg/well) for 4 hours. Formazan products were solubilized with acidic isopropanol, and the optical density was measured at 570 nm.4 cells were incubated for 24 h and subsequently treated with MIA (100 ng/ml) [Invasion assays were performed in a BD Biocoat Matrigel Invasion Chamber with 8-μm pore size  according to the manufacturer's instructions. The Matrigel was rehydrated with 500 μl DMEM (serum-free) and incubated in a 37°C, 5% CO2 atmosphere for 2 h. 5 × 100 ng/ml) , which wThe author(s) declare that they have no competing interests.JEF, NG, and AG carried out all the experiments, and participated in data analysis and interpretation. JK, MWB, and HF conceived of the study, and participated in its design and coordination. JK, NG, and AKB analyzed and interpreted the data and drafted the manuscript. All authors read and approved the final version.


Arabidopsis genome revealed that Pseudoviridae insert randomly along the chromosome and have been recently active whereas Metaviridae were more active in the past and preferentially target heterochromatin.An analysis of the distribution and age of LTR retrotransposons in the  Genome evolution and size variation in multicellular organisms are profoundly influenced by the activity of retrotransposons. In higher eukaryotes with compact genomes retrotransposons are found in lower copy numbers than in larger genomes, which could be due to either suppression of transposition or to elimination of insertions, and are non-randomly distributed along the chromosomes. The evolutionary mechanisms constraining retrotransposon copy number and chromosomal distribution are still poorly understood.Arabidopsis thaliana genome, using an automated method for obtaining genome-wide, age and physical distribution profiles for different groups of elements, and then comparing the distributions of young and old insertions. Elements of the Pseudoviridae family insert randomly along the chromosomes and have been recently active, but insertions tend to be lost from euchromatic regions where they are less likely to fix, with a half-life estimated at approximately 470,000 years. In contrast, members of the Metaviridae (particularly Athila) preferentially target heterochromatin, and were more active in the past.I investigated the evolutionary dynamics of long terminal repeat (LTR)-retrotransposons in the compact A. thaliana, their non-random genomic distribution is due to both selection against insertions in euchromatin and preferential targeting of heterochromatin. Constant turnover of euchromatic insertions and a decline in activity for the elements that target heterochromatin have both limited the contribution of retrotransposon DNA to genome size expansion in A. thaliana.Diverse evolutionary mechanisms have constrained both the copy number and chromosomal distribution of retrotransposons within a single genome. In  Drosophila melanogaster [Ty1/Copia elements) and Metaviridae (Ty3/Gypsy).It has become increasingly clear that the activity of transposable elements (TEs) is a major cause of genome evolution. TEs are ubiquitous components of eukaryotic genomes. For example, 22% of the nogaster , 45% of nogaster  genomes nogaster . In plannogaster  and/or anogaster ,8-10. LTThe evolutionary forces that control copy number and shape the chromosomal distribution of different kinds of TEs in eukaryotic genomes are still poorly understood. Some large plant and animal genomes have expanded owing to an ability to tolerate massive amplification of retrotransposons, whereas in more compact genomes these elements are found in lower copy numbers, non-randomly distributed and mainly confined to heterochromatic regions -14. TEs It is often hypothesized that the non-random genomic distribution of TEs in some species reflects the action of purifying selection on the host against the deleterious effects of TE insertions in certain regions. Models differ in the kind of deleterious effects they propose: chromosomal rearrangements due to 'ectopic'  recombination ; disruptArabidopsis thaliana genome. The total TE content of the compact genome of A. thaliana, with a haploid size of approximately 150 Mbp (million base-pairs), has been previously estimated as around 10%, and is known to cluster around the pericentromeric heterochromatin [A. thaliana [A. thaliana. By comparing the age and chromosomal distributions of young and old insertions it is possible to distinguish between preferential targeting and passive accumulation of elements into heterochromatin. I show that members of the Pseudoviridae have recently been active, that they integrate randomly into the genome (relative to centromere location) and only passively accumulate in proximal regions, as purifying selection eliminates euchromatic insertions. In contrast, the Metaviridae (particularly members of the Athila group) preferentially insert into the pericentromeric heterochromatin, and their transpositional activity has declined in the last million years.To evaluate these hypotheses, I investigated the evolutionary history of different groups of LTR-RTs in the hromatin . Despitethaliana ,23. I haAthila superfamily, though the core centromere sequences themselves were not available. In fact, the size of the A. thaliana genome has been recently estimated as approximately 157 Mbp (around 20% larger than the estimate published with the genome sequence), and the additional size appears to be due to (unsequenced) heterochromatic repetitive DNA in the centromeres, telomeres and nucleolar-organizing regions [Athila is the most abundant superfamily, followed by the Copia-like, Gypsy-like, and TRIM . The ratio of solo-LTRs to complete elements is around 2:1. In addition to solo-LTR formation, deletion and fragmentation of retrotransposon DNA in A. thaliana also occur via other mechanisms: 36% of the DNA in the Athila, 38% in the Gypsy-like, 32% in the Copia-like, and 21% in the TRIM superfamilies correspond to degraded insertions that are neither 'complete' elements nor solo-LTRs.Most of the retrieved elements are fragmented and truncated, and nested insertions are common particularly among pericentromeric elements belonging to the  regions .  Table Katydid-At1 and Katydid-At2, most intra-element LTR pairs did not cluster. In contrast, when trees were constructed for representatives of the Athila (athila2), Gypsy-like (atlantys2), and Copia-like  superfamilies, intra-element LTR pairs always clustered (data not shown), providing evidence for the lack of inter-element recombination in those 'families'.To obtain the genome-wide age distribution of each superfamily (except TRIM), 564 pairs of intra-element LTRs were (pairwise) aligned and their sequence divergence estimated. Many of the complete TRIM elements have highly divergent LTRs, and I suspect that extensive recombination between inter-element LTRs has occurred. In neighbor-joining trees of LTR sequences (of both complete and solo elements) from the TRIM families Athila insertions are significantly older than the Gypsy-like , Gypsy-like older than Copia-like (p < 0.0001). Age distributions are summarized in Figure The superfamilies differ significantly in their average age of insertions. p < 0.000. Age dis-8 substitutions per site per year [Copia-like elements are younger than 3 million years (Myr), 90% younger than 2 Myr, and only two insertions estimated to be older than 4 Myr. This shows that complete insertions from the known Copia-like families in the A. thaliana genome are younger than the species itself, whose time of divergence from its closest relatives, such as A. lyrata has been estimated (with the same rate of evolution) to be 5.1-5.4 Myr ago [Athila (and the Gypsy-like TEs), as 7% of 219 intra-element LTR pairs were estimated to be older than 5 Myr (3% of the Gypsy-like). Furthermore, the Athila and Gypsy-like superfamilies have an excess of degraded insertions relative to Copia-like  has been shown to occur in A. thaliana [Arabidopsis are not evolving in a completely neutral fashion [Chs) and alcohol dehydrogenase (Adh) loci, their synonymous sites would be evolving more slowly than LTR-RT fossils, and the dating method described above would systematically overestimate the ages of their insertion events.Using the rate of 1.5 × 10per year , 97% of  Myr ago . The sitthaliana , and the fashion . If thisCopia-like elements appears to show a recent burst of activity  in  regions . HoweverAthila elements are almost exclusively inserted in the pericentromeric regions, and the other superfamilies in significantly and progressively less proximal regions of the chromosome arms . Furthermore, except for TRIM, within each superfamily the solo-LTRs are significantly more distal than the complete elements , suggesting that formation of solo-LTRs is more likely to occur in distal regions. The distribution of complete TRIM elements relative to the centromere is not significantly different from random , although sample size is small, while their solo-LTRs are significantly clustered .A. thaliana would affect age comparisons among different groups of elements, as they show different degrees of clustering into the pericentromeric heterochromatin. However, older estimates for the age of heterochromatic elements are consistent with the hypothesis that heterochromatin is a 'safe haven' where TE insertions persist for longer periods of time. Here I show that the mechanisms that led to the accumulation of LTR-RTs in proximal regions are distinct for different groups: elements of the youngest superfamily (Copia-like) insert randomly into the genome (relative to the location of the pericentromeric heterochromatin), but there is negative selection (on the host genome) against their insertions in euchromatin; elements of the older superfamilies  preferentially insert into the pericentromeric regions. These distinct mechanisms become apparent when temporal and spatial data are combined  regions are more likely to persist for longer periods of time. This interpretation assumes that the neutral mutation rate is the same for both the distal (euchromatic) and proximal (heterochromatic) portions of the genome. Intra-genomic variation in the per-replication mutation rate has been reported between the two sex chromosomes of a flowering plant  . Furthermore, the distribution along the chromosome arms of 34 Copia-like insertions with no divergence between their intra-element LTRs is not significantly different from random . This is evidence that Copia-like elements integrate randomly relative to the location of the centromeres, but tend to get eliminated from distal, and passively accumulate in proximal regions.For complete t) for a neutral allele is given by t = 4Ne, where Ne is the effective population size. For A. thaliana t can be estimated using an average of estimates of nucleotide diversity (θ) for 8 different A. thaliana genes, θ = 9 × 10-3 [μ = 1.5 × 10-8 [t = 2θ/μ, yielding an estimate of t ≈ 1.2 Myr. This value for t is consistent with an independent estimate that placed the time since the divergence between A. thaliana and A. lyrata between 3.45t and 5.6t [Copia-like insertions are younger than 1.2 Myr, most of them are likely to be polymorphic. Taken together with the highly significant negative correlation between age and distance from the pericentromeric regions, these results indicate that complete Copia-like insertions are less likely to get fixed in the distal, euchromatic portions of the chromosome arms than in the pericentromeric heterochromatin.The average time to fixation , as both young and old insertions are found only in proximal regions . If the arm is excluded from the analysis there is no significant correlation . This suggests that for the Gypsy-like also there is an insertional bias towards proximal regions. This bias is not as strong as for Athila, as complete Gypsy-like insertions are not exclusively found around the centromeres, and they cluster (to a much lesser extent) in at least one other heterochromatic region (the telomere of the short arm of chromosome II). Included in the Gypsy-like 'superfamily' is a clade of elements, known as Tat, which is a sister group to Athila to the exclusion of the remaining Gypsy-like elements [Tat does not differ from those of the remaining Gypsy-like elements ; Tat show insertion bias towards the pericentromeric regions, but again to a lesser degree than Athila.elements . The ageCopia-like elements have been active until recently but tend to be eliminated by purifying selection, their age distribution  = NoeaK-, where N(K) is the number of elements observed with intra-element LTR divergence K, and No and a are constants to be fitted. The rate of elimination can then be estimated by linear regression of the log-transformed data . Figure Copia-like insertions (R2 = 0.94), and for complete insertions outside the proximal regions . Complete Copia-like elements are eliminated from the genome with a half-life of 648,000 years . Insertions exclusively outside the proximal (heterochromatic) regions are lost more rapidly, with a half-life of 472,000 years .Given that n Figure , bottom A. thaliana the accumulation of insertions in the pericentromeric heterochromatin is the result of both insertion bias (for Metaviridae elements) and a lower probability of fixation in euchromatin (Pseudoviridae).The results above indicate that within a single genome, distinct evolutionary mechanisms can lead to the non-random distribution of retrotransposons, as in A. thaliana were already present in its common ancestor with Brassica oleracea (the two species diverged around 15-20 Myr ago), and that copy numbers are generally higher in B. oleracea [A. thaliana and B. oleracea was responsible for the larger genome of the latter. Here I have shown that the major LTR-RT families have been active in A. thaliana since its divergence from its closest relatives, such as A. lyrata. The transpositional activity of Metaviridae elements has declined relative to its level between 1 and 2 Myr ago, perhaps suggesting that the host genome has more efficiently suppressed their transposition since. However, Pseudoviridae (Copia-like) elements in A. thaliana have been subject to constant turnover. They have been recently active and show no insertion bias, and I estimate that the half-life of a complete element inserted in the euchromatic (non-coding) regions of the chromosome arms as around 470,000 years. Most of these Pseudoviridae insertions are lost before they reach fixation, and the half-life estimate provides a measure of the pace at which natural selection on the host constrains the genomic distribution and copy number of Pseudoviridae insertions. Turnover of Pseudoviridae insertions, in contrast to the longer persistence of Metaviridae elements that have declined in activity, is consistent with the higher sequence diversity among the Pseudoviridae than the Metaviridae in A. thaliana (107 Repbase update (RU) 'families' represented in 215 complete Pseudoviridaeelements, 25 RU 'families' in 349 complete Metaviridae elements, where 'families' were defined on the basis of sequence divergence); frequent reverse transcription during transposition would be likely to lead to faster evolution than that generated by the host genome DNA polymerase error rate on chromosomal insertions.It has recently been shown that most TE lineages in oleracea . The autA. thaliana genome does not correlate with local recombination rate [The lower probability of fixation in euchromatin relative to heterochromatin implies that insertions into euchromatin are more deleterious to the host (and perhaps that purifying selection is less efficient in heterochromatin due to a much reduced rate of recombination). TE density in the ion rate , providiion rate .Copia-like elements, if such a process occurred more often in the euchromatic than in the pericentromeric regions of the genome, and if it were frequent enough. One mechanism that removes LTR-RT DNA from the genome is solo-LTR formation via unequal homologous recombination between intra-element LTRs. However, this mechanism cannot be the driving force shaping the distribution of complete Copia-like elements because Copia-like solo-LTRs are also non-randomly distributed and clustered in proximal regions . Copia-like solo-LTRs are either eliminated faster from distal than proximal regions, like complete elements, or solo-LTR formation on average occurs more slowly than extinction for euchromatic insertions. Despite clustering around the centromeres, Copia-like solo-LTRs are significantly more dispersed than complete elements. This suggests that solo-LTRs do form before extinction for distal insertions, but are probably less efficiently eliminated (possibly because they are less deleterious to the host genome) than complete elements. Another known mechanism of  DNA loss operates via small deletions due to illegitimate recombination (between short repeats); this has been shown to occur in the A. thaliana genome by an analysis of internal deletions in LTR-RTs [Drosophila, rates of spontaneous deletions in euchromatin and heterochromatin do not seem to differ [A. thaliana the relative rates between the two chromatin domains are unknown, but fragmented  Copia-like insertions are as clustered around the centromeres as complete ones . Therefore small, spontaneous deletions cannot account for the genomic distribution of complete elements. Larger deletions (that remove the entire LTR-RT sequence) occurring primarily in euchromatin would be necessary to explain the observed accumulation pattern; if such a mechanism existed it would be an important force for genome size contraction. As there is no evidence for such mechanism, and given that I estimate that the half-life of (complete) insertions to be less than half the average time to fixation for a neutral allele, a lower probability of fixation in euchromatin relative to the pericentromeric heterochromatin is more likely to be driving the genomic distribution of Pseudoviridae elements.As an alternative to selection, a neutral mutational process that deletes (part of the) insertions could in principle be driving the distribution of  LTR-RTs . In Droso differ . In A. tAthila elements have been known to be present in the A. thaliana core centromeric arrays of the 180-bp satellite repeats and are abundant in pericentromeric heterochromatin [Copia-like elements, the striking compartmentalization of both recent and older Athila insertions in the pericentromeric heterochromatin indicates that these elements actively target those regions, and represents an example of a group of retrotransposons that have evolved to colonize a particular 'genomic niche'. Passive accumulation could not explain the distribution of Athila insertions unless they were generally much more deleterious to their host than Copia-like ones. Given the absence of complete Athila insertions from euchromatin, any one insertion would have to be so deleterious as to be almost immediately eliminated by purifying selection, even from intergenic DNA. Rather, it is likely that Athila elements preferentially insert into the pericentromeric heterochromatin and it is possible that this group of elements has been co-opted to play a part in centromere function. There is some evidence that such hypothetical role cannot be that of cis-acting sequences [A. thaliana [It is interesting to note that the integrase proteins encoded by LTR-RTs differ between the Pseudoviridae and the Metaviridae in their carboxy-terminal domains, as they have different characteristic motifs ,36. Thishromatin ,41. In tequences , but it equences -45 pointequences , centromequences -51, chicequences  and A. tthaliana .A. thaliana. The rapid and recent genome evolution inferred for A. thaliana may be a feature common to other higher eukaryotes, in particular those with compact genomes. High turnover of TE insertions in euchromatin also occurs in Drosophila and pufferfish [A. thaliana, be due to diverse evolutionary mechanisms.Both purifying selection (at the host level) against insertions (in euchromatin) and a decline in transpositional activity (of Metaviridae elements) appear to have limited the recent contribution of retrotransposon DNA to genome size expansion in fferfish , for exaA. thaliana.A methodology was developed for the automated mining of sequence data to retrieve the sequence and chromosomal location of genomic 'fossils' of LTR-RTs, identifying complete elements and solo-LTRs among the retrieved sequence fragments, and estimating the age of the insertion events that gave origin to these elements. This methodology was applied to the genome sequence of A. thaliana genome (around 116 Mbp of available sequence) was performed using sequence-similarity search algorithms [A. thaliana 'families' of LTR-RTs. The programs RepeatMasker [Sequences of the organellar and the five nuclear chromosomes (version 200303) were obtained from the Munich Information Center for Protein Sequences (MIPS) . Computagorithms  against gorithms ,57 sequeatMasker  and WU-BatMasker  were useAthila, Gypsy-like , and Copia-like . The Metaviridae was split into two groups (Athila and Gypsy-like), as initial mining of the A. thaliana genome revealed that Athila elements have been particularly successful in colonizing it. Their copy number is roughly double the number of all other members of the Metaviridae, and higher than the total of all Pseudoviridae elements. Athila form a clade and are retroviral-like elements that are likely to have an envelope (env) gene [Copia- and Gypsy-like elements are typical LTR-RTs, although one of the Copia-like 'families' (metaI) comprises non-autonomous elements [endovir1 [atcopia41-43 [env gene. A fourth 'superfamily' was used to include TRIMs. These are short, non-autonomous elements that feature LTRs but no coding genes and cannot currently be classified into either the Pseudoviridae or the Metaviridae; they are described in ['Families' of LTR-retrotransposons (as classified in Repbase update) are present in low copy numbers; therefore, for the purpose of this analysis they were grouped into three 'superfamilies': nv) gene . Most ofelements  and a feendovir1 , atcopiapia41-43 ) are retribed in .Athila (10 families): athila2 - 5, athila4A - D, athila6A, athila7, athila8A and B; Gypsy-like (15 families): atgagpol1, atgp2 and 3, atgp2N, atgp5 - 10, atgp9B, atlantys1 - 3, tat1; Copia-like (107 families): atcopia1 - 97, atcopia8A and B, atcopia18A, atcopia32B, atcopia38A and B, atcopia65A, endovir1, TA1-2, meta1; TRIM (3 families): katydid-At1, katydid-At2, katydid-At3.The four superfamilies comprise the following 'families'. Perl script, LTR_MINER (available on request), was written to parse all the chromosomal LTR-RT fragments reported by RepeatMasker (WU-BLAST hits of similarity to reference sequences) and identify complete elements and solo-LTRs. LTR_MINER performs the pattern-recognition function of assembling hits that originated from single LTR-RT insertion events. The algorithm involves: 'defragmentation' of LTR hits. If a chromosomal LTR fossil contains insertions/deletions (indels) relative to the most similar library sequence, it may be reported as multiple hits (fragments). Defragmentation is the identification of multiple hits that correspond to the same LTR. Parameters were set so that LTR hits were defragmented only when they were separated by no more than 550 bp, belonged to the same family, had the same orientation on the chromosome, and their combined length did not exceed the length of the corresponding family reference sequence by more than 20 bp.A LTR, IR, LTR. In order to check whether the pattern could be straddling a nested insertion of the same family, the search is then recursively extended from each end of the pattern for further contiguous hits to an IR and a LTR (of same family and orientation). The two LTRs of the innermost pattern are classified as a pair of intra-element LTRs.An intact LTR-RT insertion consists of at least three hits: LTR-IR-LTR . After LTR defragmentation, LTR_MINER searches for contiguous patterns of LTR_MINER also identifies such elements provided an IR is present between the LTRs. A maximum pairing distance between LTRs was set at 30 kb.LTR_MINER was set to classify a LTR fragment as a solo-LTR if no other LTR or IR (of same family and orientation) is present within a 5 kbp radius from the fragment's ends. The aim was the identification of elements resulting from deletion (of the IR and one LTR) events via homologous recombination between intra-element LTRs, and not to classify as solo-LTRs sequences that are separated from IRs because of insertions.K) between each intra-element LTR pair was then estimated using Kimura's two-parameter model [Nucleotide sequence divergence between pairs of intra-element LTRs was used as a molecular clock, as these pairs are identical at the time of insertion . All miner model . To reduK greater than 0.2 they were inspected by eye and manually edited if necessary . Estimates of the ages of insertion were obtained by using the equation t = K/2r, where t is the age, and r is nucleotide substitution rate for the host genome DNA polymerase. The value of 1.5 × 10-8 substitutions per site per year was used for r , estimated in [Chs and Adh loci in Arabidopsis/Arabis species.Alignments with fewer than 80 nucleotides were discarded. As CLUSTAL-W alignments could be poor if LTR sequences were only partially overlapping, for all LTR pairs with mated in  for the Finally, if recombination between LTRs from different insertions had occurred frequently, the dating method above would be invalid for obtaining the age profiles of different families. To detect possible recombination events, multiple alignments of all LTRs (including solos) of certain families were generated using BLASTALIGN , a progrA. thaliana. Additional data file A. thaliana. Additional data file Perl script LTR_MINER, used to de-fragment sequence similarity hits to LTR-retrotransposons, and identify complete and solo-LTR elements. Additional data file Perl script in Additional data file Perl script used in conjunction with LTR_MINER, used to divide long sequences into smaller chunks labeled by their coordinate range. Additional file data Perl script in Additional data file The following additional data files are available with the online version of this article. Additional data file A. thalianaThe entire dataset of chromosomal coordinates and ages of complete LTR-retrotransposons in Click here for additional data fileA file describing the data fields in Additional data file 1Click here for additional data fileA. thalianaThe entire dataset of chromosomal coordinates of solo-LTRs in Click here for additional data fileA file describing the data fields in Additional data file 3Click here for additional data filePerl script LTR_MINER, used to de-fragment sequence similarity hits to LTR-retrotransposons, and identify complete and solo-LTR elementsThe Click here for additional data filePerl script in Additional data file 5A file describing the utility and usage of the Click here for additional data filePerl script used in conjunction with LTR_MINER, used to divide long sequences into smaller chunks labeled by their coordinate rangeThe Click here for additional data filePerl script in Additional data file 7A file describing the utility and usage of the Click here for additional data file


Epidemiological studies have found reduced health-related quality of life (QoL) in patients with personality disorders (PDs), but few clinical studies have examined QoL in PDs, and none of them are from an ordinary psychiatric outpatient clinic (POC). We wanted to examine QoL in patients with PDs seen at a POC, to explore the associations of QoL with established psychiatric measures, and to evaluate QoL as an outcome measure in PD patients.72 patients with PDs at a POC filled in the MOS Short Form 36 (SF-36), and two established psychiatric self-rating measures. A national norm sample was compared on the SF-36. An independent psychiatrist diagnosed PDs and Axis-I disorders by structured interviews and rated the Global Assessment of Functioning (GAF). All measurements were repeated in the 39 PD patients that attended the 2 years follow-up examination.PD patients showed high co-morbidity with other PDs and Axis I mental disorders, and they scored significantly lower on all the SF-36 dimensions than age- and gender-adjusted norms. Adjustment for co-morbid Axis I disorders had some influence, however. The SF-36 mental health, vitality, and social functioning were significantly associated with the GAF and the self-rated psychiatric measures. Significant changes at follow-up were found in the psychiatric measures, but only on the mental health and role-physical of the SF-36.Patients with PDs seen for treatment at a POC have globally poor QoL. Both physical and mental dimensions of the SF-36 are correlated with established psychiatric measures in such patients, but significant changes in these measures are only partly associated with changes in the SF-36 dimensions. According to the DSM-IV  personalSince the DSM-IV included the Global Assessment of Functioning Scale (GAF) as Axis V, it is reasonable to consider if the GAF should be used for evaluations of "significant distress or impairment". Two problems are implicit in such an approach: at what GAF cut-off score should "significant distress or impairment" be set, and the evaluation is only done by a professional. As to the first problem, Kessler et al  suggesteMajor treatment outcome studies of PDs like the Collaborative Longitudinal Personality Disorders Study , the NorIn contrast, two epidemiological studies of PDs have used the SF-12 as a measure of disability. In a national study from Australia, Jackson and Burgess  reportedA review of the literature showed that the QoL had only been used as a disability measure in a few clinical research studies of PD patients. In depressed elderly patients, Abrams et al  found thNone of these clinical studies takes QoL as observed in PD patients seen at an ordinary psychiatric outpatient clinic (POC) as their point of departure. However PD patients are frequent at POC, and the QoL is an important self-rated measure of "clinically significant distress or impairment". Since QoL data on PD patients seen at at POC seems to be lacking from the literature, we found it relevant to study a consecutive sample of PD patients from a POC and collect QoL data with SF-36. We posed the following research questions: 1) How are the SF-36 dimensions mean scores in PD patients compared to age- and gender-adjusted norm data? 2) To what extent are the SF-36 scores in PD patients associated with co-morbid Axis I disorders? 3) How is the association between the SF-36 dimension and established patient- and professional-rated psychiatric measures in PD patients? and 4) What changes in the SF-36 dimensions of treated PD patients are observed from baseline to follow-up, and how are they related to changes in the psychiatric measures?Furuset POC serves a communality of Oslo City, Norway with a population of 28.000 people. At the time of the study, the staff consisted of three psychiatrists, three clinical psychologists, two psychiatric nurses, and two social workers. The intake rate was approximately 400 new patients a year. The first author (KN) invited the staff to take part in the study by referring to her new patients with probable PDs. Six professionals were willing to participate, while four declined due to heavy clinical burden, or lack of interest.At the start of the study in 1996, Furuset was a new suburb of Oslo, and the inhabitants were characterized by lower socio-economic conditions, high mobility, and a considerable prevalence of immigrants from Asian countries. The suburb had a high proportion of municipal housings, and the criterion for allotment to them was severe mental disorder and/or severe socio-economic problems. Many patients seen at Furuset POC were out of work due to mental disorders, and/or due to socio-economic circumstances.Patients aged from 18 to 75 years were consecutively recruited from January 1, 1996 to June 30, 1998. The patients were referred from the local GPs, and physical examination and adequate treatment and follow-up of physical diseases were the responsibility of the GPs. The six therapists screened for probable PDs among new patients scheduled for treatment. Exclusion criteria were mental retardation, lifetime psychosis and bipolar disorder, organic mental disorders, current strong suicidal ideation, and insufficient knowledge of the Norwegian language. Eligible patients received oral and written information about the study from their therapists. Then the patients were invited to take part in the study, and they all gave written informed consent. The Ethical Review Board of Department of Psychiatry, Aker University Hospital approved the project.The six therapists did not miss out any patients at screening, but 5 (4%) eligible patients declined to take part in the study. Among 110 eligible patients referred to the study, only 91 filled in the SF-36 at baseline due to administrative misunderstandings. However, when they were compared to the 19 who did not fill in, the non-attenders only had significantly fewer co-morbid Axis I-disorders (data not shown).In order to answer the research questions, the sample was divided into three groups: cluster A+B PDs (n = 39), cluster C (n = 33), and Axis I-disorders (n = 19). The cluster A+B group could also contain co-morbid cluster C PDs and Axis I-disorders, and the cluster C co-morbid Axis I-disorders.Two years after baseline, the patients received a mailed written appointment for a follow-up interview. Those who did not show up were sent a written reminder. If they still did not meet, they were called by phone, and if there was no answer, their addresses and phone numbers were checked at the Census register. Appointments were mailed to new addresses, and phone-calls were made in case of non-response. Only a few patients responded to these extended search procedures.Norm data on the SF-36 was obtained from the Survey of Level of Living in Norway 1998  comprisiAt baseline, diagnoses of PDs were made with the use of the Personality Disorder Examination, and Axis I-disorders were diagnosed by the MINI-International Neuropsychiatric Interview. Anamnestic data were collected, and global assessment of function was rated. The professional-based interviews and examinations of all patients at baseline and follow-up were carried out by a single experienced psychiatrist (KN), who did not take part in any treatment given.All patients also filled in the following self-rating instruments at baseline: the SF-36, the Social Adjustment Scale, and the Symptom Checklist 90-Revised Personality Severity Index.At follow-up all these assessments were repeated, and additional information about treatment as well as job/education, social- and family changes was collected.Personality Disorder Examination (PDE) .This result could indicate that the other dimensions of the SF-36 are less valid as measures of changes in mental health of PD patients, or alternatively that most aspects of QoL measured by the SF-36 do not change in PD patients even if established psychiatric measures do.The main strength of our study was that we were able examine systematically various aspects of the QoL measured by the SF-36 in a clinically relevant sample of PD patients at a POC which is a common setting for such patients in psychiatry.Our study had a number of weaknesses. The study groups were small with limited statistical power, and there was a considerable risk of type II errors. More significant differences as to the SF-36 dimensions could turn up in larger samples. Although we put considerable efforts into location of patients, we had a lower follow-up rate than we had expected. However, the PD patients who did not show up at follow-up did not differ much from those who did. We cannot, therefore, generalize the discrepancy observed between significant changes in established psychiatric measures and lack of such changes in most of the SF-36 dimensions of PD patients treated at a POC to widely.The same experienced psychiatrists did all the interviews at baseline and follow-up. Although she was not involved in any treatments, we cannot exclude an expectation bias from her side.We think that our study has to be considered an exploratory one. Our finding of a generally strongly reduced QoL should be replicated in a PD sample with less comorbid Axis I disorders, although their influence was limited. The same is true for QoL as a valid measure for change in PD patient, since it was not recommended as part of a standard outcome battery and was not used by major treatment studies of PD patients. However, our study confirmed that the SF-36 MH dimension seemed to be a valid psychiatric measure in our PD patient sample.In this study of the QoL in PD patients seen at an ordinary POC, we found that the PD patients had significantly lower mean scores on all the SF-36 dimensions compared to age- and gender-adjusted norm data. This is in accordance with the SF-36 measurements of other major diagnostic groups of mental disorders. Although the SF-36 dimensions correlated considerably with established psychiatric measures in our PD patients, they did not show the same significant changes over time as the established measures. The use of QoL measures like the SF-36 as an outcome measure in PD patients is in need of further investigation.BP: SF-36 Bodily painGAF: The Global Assessment of FunctioningGH: SF-36 General healthMH: SF-36 Mental healthPD: Personality disorderPDs: Personality disordersPF: SF-36 Physical functioningPOC: Psychiatric outpatient clinicPSI: Personality severity index of SCL-90-RQOL: Health-related quality of lifeRE: SF-36 Emotional role functioningRP: SF-36 Physical roleSAS: The Social Adjustment ScaleSCL-90-R: The Symptom Checklist 90-RevisedSF-36: MOS Short Form 36SF: SF-36 Social functioningVT: SF-36 VitalityThe author(s) declare that the have no competing interests.KN conceived and planned the study, prepared the therapists at Furuset Outpatient Department, did all the psychiatric interviews at baseline and follow-up, and drafted the manuscript. AM helped designing of the study, supervised the statistic calculations, and drafted the manuscript. AAD participated in the design and coordination of the study, and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Escherichia coli a model organism provides information for annotation of other genomes. Our analysis of its genome has shown that proteins encoded by fused genes need special attention. Such composite (multimodular) proteins consist of two or more components (modules) encoding distinct functions. Multimodular proteins have been found to complicate both annotation and generation of sequence similar groups. Previous work overstated the number of multimodular proteins in E. coli. This work corrects the identification of modules by including sequence information from proteins in 50 sequenced microbial genomes.E. coli K-12 proteins were identified from sequence similarities between their component modules and non-fused proteins in 50 genomes and from the literature. We found 109 multimodular proteins in E. coli containing either two or three modules. Most modules had standalone sequence relatives in other genomes. The separated modules together with all the single (un-fused) proteins constitute the sum of all unimodular proteins of E. coli. Pairwise sequence relationships among all E. coli unimodular proteins generated 490 sequence similar, paralogous groups. Groups ranged in size from 92 to 2 members and had varying degrees of relatedness among their members. Some E. coli enzyme groups were compared to homologs in other bacterial genomes.Multimodular E. coli paralogous groups correlates with known physiological and taxonomic relationships between the organisms.The deleterious effects of multimodular proteins on annotation and on the formation of groups of paralogs are emphasized. To improve annotation results, all multimodular proteins in an organism should be detected and when known each function should be connected with its location in the sequence of the protein. When transferring functions by sequence similarity, alignment locations must be noted, particularly when alignments cover only part of the sequences, in order to enable transfer of the correct function. Separating multimodular proteins into module units makes it possible to generate protein groups related by both sequence and function, avoiding mixing of unrelated sequences. Organisms differ in sizes of groups of sequence-related proteins. A sample comparison of orthologs to selected  Eschericia coli remains a useful resource to the genomic community as it provides important knowledge which can be applied to the analysis of most microbial genomes. Its central role devolves from two facts; first, the accumulated results of seven decades of laboratory experimentation have identified the function(s) of over half of its gene products; second being a metabolic generalist, E. coli's metabolic functions are widely shared among other organisms.E. coli's genes. While searches for sequence matches to unknown genes usually yield a large number of matches, chances are high that firm functional information comes only from experimental studies on E. coli. Because annotations of genes do not always indicate that the assignments are derived, and because derived annotations are used serially for further annotation without experimental confirmation, many genes carry original E. coli annotations. It is therefore important to the entire genome-analyzing community that the data on E. coli gene products be as accurate as possible. Since the original GenBank deposit of E. coli K-12 (U00096), new and updated annotations are available at NCBI (U00096.2) and at more specialized databases including, ASAP '. Altogether 1946 unimodular proteins belonged to one of the groups. Modules from 94 of the multimodular proteins were present in 61 of the groups. Table We identified 490 sequence-similar or paralogous groups in E. coli with known or predicted function, they tend to be present in smaller paralogous groups as compared to the transporters and regulators. Among the larger groups the oxidoreductases and the subunits of oxidoreductases are most common, making up 8 of the top 20 enzyme groups -binding oxidoreductases of E. coli. The number of homologous sugar kinases, respiratory reductase subunits, and nucleoside diphosphate (Nudix) hydrolases appeared overall to be lower in B. subtilis.In addition to using paralogous groups for intra-genomic analyses, the groups were also used in cross genome comparisons , while in Xylella fastidiosa, an aspartokinase is fused to a diaminopimelate decarboxylase (gi9106073). One needs to be alert to partial alignments. In this case, the annotation is correct for both activities of the Xylella protein, although the description does not follow the convention of stating the N-terminal activity first, raising the potential for misidentification of the activity of a partial homolog.As shown in Figure E. coli and X. fastidiosa connected proteins acting in the same pathway. However, the pathways are different for the two organisms, threonine biosynthesis for E. coli and lysine biosynthesis in X. fastidiosa. Fusions of genes in a pathway have long been known and also the fusions of different genes in different organisms. In the tryptophan biosynthesis pathway of E. coli both the trpC gene (formerly trpC(F)) and the trpD gene (formerly trpG(D)) encode two enzymes as indicated in their former names. In contrast Rhizobium meliloti has a fusion between the trpE and trpG genes, trpE(G) [The details of gene duplication and divergence and of gene fusions have followed different courses in separate lines of descent of bacteria. The fusions of different gene partners to aspartokinase in  trpE(G) . Such diTo promote awareness of fused proteins, databases should list such proteins with their separate component activities and the approximate locations of these; either by start and end residues, or by module location . Such a format has been implemented in GenProtEC . When anE. coli proteins similar in sequence vary in size from two (simple pairs) up to 92 members -binding oxidoreductases is much larger in B. subtilis (31 proteins) than in the enterics (18 proteins). The B. subtilis enzymes in this group are fatty acid biosynthesis enzymes, agreeing with the known fact that this organism synthesizes a greater variety of fatty acids and has dedicated more of its proteome towards diversifying its fatty acid biosynthetic capabilities [In another example, bilities ,34. Thus• Proteins encoded by fused genes, multimodular proteins, require special attention in genome analysis. Such multimodular proteins contain two or more functional components that are located at separate parts of the protein and that may exist as independent proteins in other genomes. Annotation of the multimodular proteins should include the separate functions and their corresponding locations in the gene product. This will improve transfer of function between the fused proteins and sequences matching their entire length or only the length of one of their module components. Current annotation errors involving fused genes can be remedied by introducing this approach.E. coli was improved by making use of sequence information from 50 genomes to detect alignments between the fused proteins and smaller, un-fused homologs corresponding to the component modules. The more common multidomain proteins, proteins containing fused sequence domains or motifs that together make up one overall function, were not detected as multimodular proteins by this approach. As a result the current number of fused E. coli proteins was reduced to 109 proteins with 8 containing three modules and 101 containing two modules. The multimodular E. coli proteins consist mainly of enzymes, regulators and transport proteins. Their component modules are often not related by sequence but many are related in that they function in a common pathway or cell role. Components of fused genes appear to vary from genome to genome hence complicating their detection and function assignment.• The identification of multimodular proteins in • Multimodular proteins are different from multifunctional proteins in that the latter catalyze more than one reaction in the same region of the protein.E. coli proteins ranging in size from 2 to 92 were generated from the new module data. The smaller groups range widely in degree of relatedness while the larger groups have diverged from one another to about the same extent. Transport proteins and regulatory proteins were found in the larger groups while enzyme groups tended to have fewer members.• The generation of paralogous or sequence related groups is improved when the modules of multimodular proteins are separated and treated as independent proteins for the grouping process. 490 groups of sequence related E. coli proteins belong to paralogous groups, reflecting the prominent role of duplication and divergence in the evolution of the genome. The number and sizes of paralogous groups reflect the distinctiveness of the organisms and they can be used in cross genome comparisons.• Over half of the Aquifex aeolicus, (AE000657); Archaeoglobus fulgidus, (AE000782); Aeropyrum pernix, (BA000002); Agrobacterium tumefaciens, (AE007869/AE007870); Borrelia burgdorferi, (AE000783); Bacillus halodurans, (BA000004); Bacillus subtilis, (AL009126); Buchnera sp. APS, (BA000003); Campylobacter jejuni, (AL111168); Clostridium acetobutylicum, (AE001437); Chlamydia muridarum, (AE002160); Chlamydophila pneumoniae CWL029, (AE001363); Deinococcus radiodurans, (AE000513/AE001823); Escherichia coli K-12, (U00096); Escherichia coli O157:H7 EDL933, (AE005174); Escherichia coli O157:H7, (BA000007); Haemophilus influenzae, (L42023); Helicobacter pylori 26695, (AE000511); Halobacterium sp. NRC-1, (AE004437); Lactococcus lactis subsp.lactis, (AE005176); Mycobacterium leprae, (AL450380); Mycoplasma genitalium, (L43967); Mycobacterium tuberculosis H37Rv, (AL123456); Methanococcus jannaschii, (LL77117); Mesorhizobium loti, (BA000012); Mycoplasma pneumoniae, (U00089); Mycoplasma pulmonis, (AL445566); Methanobacterium thermoautotrophicum, (AE000666); Neisseria meningitidis MC58, (AE002098); Pseudomonas aeruginosa, (AE004091); Pyrococcus horikoshii, (BA000001); Pasteurella multocida, (AE004439); Pyrococcus abyssi, (AL096836); Rickettsia prowazekii, (AJ235269); Salmonella enterica subsp. enterica serovar Typhi, (NC_003198); Salmonella typhimurium LT2, (AE006468); Shewanella oneidensis MR-1, (NC004347); Sinorhizobium meliloti, (AL591688); Staphylococcus aureus subsp.aureus Mu50, (BA000017); Streptococcus pneumoniae TIGR4, (AE005672); Streptococcus pyogenes M1 GAS, (AE004092); Sulfolobus solfataricus, (AE006641); Synechocystis PCC6803, (AB001339); Thermoplasma volcanium, (BA000011); Thermotoga maritima, (AE000512); Treponema pallidum, (AE000520); Ureaplasma urealyticum, (AF222894); Vibrio cholerae, (AE003852/EC003853); Xylella fastidiosa 9a5c, (AE003849); Yersinia pestis, (AL590842).Protein coding sequences were obtained from GenBank and included the following genomes: Pairwise sequence alignments and scores were generated using the AllAllDb program of Darwin , version 2.0, developed at the ETHZ in Zurich . MaximumFor the work reported here, sequence pairs were collected that had alignment lengths of at least 83 amino acids and distances of 200 PAM units or less. We chose the length requirement of 83 residues as it improves the significance of the sequence alignments for the more distantly related protein pairs . The reqE. coli literature and from unequal sequence alignments. The literature was searched for E. coli proteins with more than one function encoded at separate parts of the protein. The locations of the alignment regions in the proteins were analyzed for orthologous and paralogous protein pairs. We identified proteins with two or more non-overlapping alignment regions where each region aligned separately to smaller homologs. Figure Proteins encoded by fused genes were identified from the E. coli were aligned against themselves. Protein pairs aligning with >45% of the length of the peptides were used in a transitive grouping process as previously described [The sum of the separated modules from the multimodular proteins and the naturally occurring unimodular proteins of escribed . The traMS designed the study, performed the sequence analysis, and participated in the data analysis and in writing the manuscript. MR participated in the data analysis and in writing the manuscript.E. coli proteins. The table contains a complete list of the multimodular proteins in E. coli. Each module is described by its Gene name, Module Id, Module Start and End positions, Gene type, and Module Product.Multimodular Click here for fileE. coli. The members of the 409 paralogous groups are indicated by their Group Membership, Module Id, Module Start and End Position, Module Product.E. coli paralogous groups and their members. The table contains a complete list of the paralogous protein groups in Click here for file


Acute myocardial infarction (MI) following anaphylaxis is rare, especially in subjects with normal coronary arteries. The exact pathogenetic mechanism of MI in anaphylaxis remains unclear.The case of a 32-year-old asthmatic male with systemic anaphylaxis, due to oral intake of 500 mg amoxycillin, complicated by acute ST-elevation MI is the subject of this report. Following admission to the local Health Center and almost simultaneously with the second dose of subcutaneous epinephrine (0.2 mg), the patient developed acute myocardial injury. Coronary arteriography, performed before discharge, showed no evidence of obstructive coronary artery disease. In vivo allergological evaluation disclosed strong sensitivity to amoxycillin and the minor  determinants of penicillin.Acute ST-elevation MI is a rare but potential complication of anaphylactic reactions, even in young adults with normal coronary arteries. Coronary artery spasm appears to be the main causative mechanism of MI in the setting of "cardiac anaphylaxis". However, on top of the vasoactive reaction, a thrombotic occlusion, induced by mast cell-derived mediators and facilitated by prolonged hypotension, cannot be excluded as a possible contributory factor. Acute myocardial infarction (MI) complicating anaphylaxis induced by drugs or other chemicals is uncommon and only sporadic cases have been reported -9. The uA 32-year-old man was admitted to the emergency room of the local Health Center because of anaphylaxis, which developed 2 hours and 15 minutes after the ingestion of amoxycillin (500 mg), prescribed by his dentist. Prodromal signs of anaphylaxis  reportedly occurred about 15 minutes before the onset of symptoms from other systems. Ten days earlier the patient, an asthmatic since childhood, had completed a 4-day course of amoxycillin (500 mg TID) without any side effects. Apart from obesity, there were no other risk factors for coronary artery disease.2) with generalized erythema, angioedema, cyanosis and diffuse wheezing; the systolic blood pressure was 70 mmHg and the pulse rate 120 bpm. The patient was then connected to a cardiac monitor, which showed sinus tachycardia (approximately 140 bpm) without ST-segment and T wave abnormalities  of 90%.On admission the patient was in acute distress. He was complaining of dizziness, blurred vision, dyspnea and abdominal pain. Initial examination revealed an obese man  was injected subcutaneously (SC); dimethindene (4 mg) and hydrocortizone (500 mg) were administered intravenously (IV) in slow infusion. Nebulized salbutamol and supplemental oxygen were given as well. Normal saline with 50 mg ranitidine hydrochloride and Ringer's solution were infused through separate intravenous lines.3 to V6  a second dose of epinephrine 0.2 mg) was given SC 20 minutes later. Almost simultaneously with the administration of the second dose of epinephrine, ST-segment elevation appeared on the monitor Fig.  and the  mg was gth hospital day showed preserved systolic function (ejection fraction 60%) without wall motion abnormalities. Left  were performed using penicilloyl polylysine (PPL), and minor determinant mixute (MDM) [supplied as Allergopen by Allergopharma ]; amoxycillin, ampicillin and cefamandole solutions were also used (concentration 20 mg/ml) for PST. PST were strongly positive  to amoxycillin and ampicillin. Intradermal tests were performed only to PPL, MDM (dilution 1/10) and cefamandole (0.2 mg/ml and 2 mg/ml) and resulted in strongly positive reaction (4+) only to the MDM. Circulating specific IgE to penicillin V, penicillin G, amoxycillin, ampicillin and cefaclor was not demonstrable by CAP .Anaphylactic reactions may trigger cardiovascular events, including MI -9, primaAcute coronary syndromes following antibiotic-induced anaphylaxis is uncommon and only few cases have been reported so far ,4,12-14.Evidence that the human heart is the target as well as the site of anaphylaxis is constantly growing. A number of studies by Marone et al ,16 and oIn the present case, the temporal sequence of events suggests that cardiac anaphylaxis was the triggering factor of MI. We propose that prolonged coronary vasospasm induced by vasoactive and inflammatory mediators, released during anaphylaxis, was the main causative mechanism. However, in view of the persistence of chest pain following IV nitroglycerin infusion, and its complete resolution promptly after IV thrombolysis, a thrombotic vascular occlusion, on top of the vasospastic reaction, cannot be excluded. The latter is also supported by the prolonged systemic hypotension, which, as it has been emphasized in previously reported cases , probablThe administration of epinephrine – a life saving agent in cases of anaphylaxis – has been implicated as a cause of acute MI in a limited number of reports ,28. In tThe allergological evaluation performed in our patient showed strong sensitivity to amoxycillin and to the minor determinants, which are the allergenic epitopes associated with systemic anaphylaxis. The negative CAP results do not negate the in vivo findings, since it is well established that the in vitro techniques are not as sensitive as skin tests. Furthermore, CAP detects antibodies against the major determinants of penicillins  and not to the minor ones, which are implicated in systemic anaphylaxis. For technical reasons, relating to both the Allergology Center and the patient's professional obligations as well as residence distance, the allergological work up was completed 6 months later; such a delay might also have contributed to the negative in vitro findings.Acute ST-elevation MI is a rare but potential complication of anaphylactic reactions, even in young adults with normal coronary arteries. Physicians should be alert for such a complication in order to diagnose it early and treat properly.It is fairly well established that that human heart can be both, the site and the target of severe anaphylaxis; in this setting cardiac mast cells – activated and releasing multiple vasoactive mediators – play an important role in pathogenesis of cardiac complications -18.In the above case, mediator-induced coronary artery spasm was the main, but probably not the exclusive causative mechanism of anaphylaxis-related MI. The thrombotic vascular occlusion, induced by inflammatory mediators and facilitated by prolonged hypotension, cannot be excluded as a possible contributory factor.ECG = electrocardiogram; IV = intravenously; MDM = minor determinant mixute; MI = myocardial infarction; NV = normal values; PPL = penicilloyl polylysine; PST = prick skin tests; SC = subcutaneously.The author(s) declare that they have no competing interests.AG was responsible for the initial evaluation and management of the patient, GL was involved in the cardiological evaluation and management and, KK-F performed the allergological evaluation. All authors have equally contributed in the preparation and revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: