Even though the catecholamines  have been detected in plants their role is poorly documented. Correlations between norepinephrine, soluble sugars and starch concentration have been recently reported for potato plants over-expressing tyrosine decarboxylase, the enzyme mediating the first step of catecholamine synthesis. More recently norepinephrine level was shown to significantly increase after osmotic stress, abscisic acid treatment and wounding. Therefore, it is possible that catecholamines might play a role in plant stress responses by modulating primary carbon metabolism, possibly by a mechanism similar to that in animal cells. Since to date no catecholamine receptor has been identified in plants we transformed potato plants with a cDNA encoding human dopamine receptor (HD1).Tuber analysis of transgenic plants revealed changes in the activities of key enzymes mediating sucrose to starch conversion (ADP-glucose phosphorylase and sucrose synthase) and sucrose synthesis (sucrose phosphate synthase) leading to altered content of both soluble sugars and starch. Surprisingly the catecholamine level measured in transgenic plants was significantly increased; the reason for this is as yet unknown. However the presence of the receptor affected a broader range of enzyme activities than those affected by the massive accumulation of norepinephrine reported for plants over-expressing tyrosine decarboxylase. Therefore, it is suggested that the presence of the exogenous receptor activates catecholamine cAMP signalling in plants.via cAMP signalling pathway.Our data support the possible involvement of catecholamines in regulating plant carbon metabolism  The catecholamines  are a group of biogenic amines possessing a substituted 3, 4-dihydroxy phenyl ring that are widespread in the animal kingdom; but they have also been detected in plants ,2. The rSolanum tuberosum plants transformed with pHD1-BinAR, a plasmid carrying a cDNA for the human dopamine receptor under the control of the CaMV 35S promoter  was recently investigated. A sensitive GCMS method based on the analysis of the trimethylsilylated catecholamine derivatives was developed to monitor the presence and concentration of these compounds and related metabolites. Based on the retention times and the mass spectra of standards the presence of dopamine, norepinephrine and a new compound normetanephrine in potato leaves and tubers was clearly detected .In contrast to the previous studies performed on plants over-expressing tyrosine decarboxylase , which cFollowing our recent finding that the action of dopamine and norepinephrine in potato is on starch mobilization, we decided to analyze transgenic tubers expressing dopamine receptor for soluble sugars and starch content. All transgenic plants showed decreased starch content, with levels that ranged from 20 to 60% percent of the wild type. This was accompanied by a significant increase in soluble sugar concentration ranging from 2.7 to 1.15 fold in comparison to Desiree Figure . ConcentChanges in carbohydrate are most likely responsible for the altered phenotype of transgenic HD1 tubers. Reduced tuber mass can be explained by decreased starch content whereas increased tuber number by the increase of soluble sugar concentration.Under normal growth conditions the major flux in potato tuber carbon metabolism is the conversion of sucrose through hexose phosphates to starch .Since HD1 plants were characterized by changed concentrations of both soluble sugars and starch we measured the activities of enzymes involved in this pathway. Sucrose transported from leaves is symplastically unloaded from the phloem and degraded by sucrose synthase (SuSy). ADP-glucose phosphorylase (AGPase) converts glucose-1-phosphate (Glu-1-P) into ADP-glucose, an immediate precursor of starch. Both SuSy and AGPase are considered as key enzymes for starch synthesis .Activities of AGPase and SuSy were significantly decreased in HD1 plants to 56% and 68% of the wild type level, respectively Figure . In agrePhosphoglucomutase (PGM) catalyzes the conversion of glucose-1-phosphate to glucose-6-phosphate. Tubers are characterized by the presence of cytosolic and plastidial isoforms of phosphoglucomutase. Repression of either of them results in plants with decreased starch levels pointing out the importance of the enzyme for starch accumulation ,19. The To establish whether enhanced starch mobilization also contributed to the observed decreases in starch content we measured the activity of starch phosphorylase. In two out of the four examined transgenic lines the activity of starch phosphorylase was significantly increased, further contributing to decreased starch content of HD1 plants Figure Moreover HD1 expression led to activation of sucrose phosphate synthase (SPS), responsible for sucrose production. Maximum SPS activity  only changed in two lines, whilst activity of the enzyme measured in the assay that contained limiting substrate concentration (Vmax/Km) and as a consequence 1/Km increased in all the lines.1/Km, correlated well with the sucrose content of transgenic tubers (cor -0.81) Figure .via the TCA cycle we measured the content of TCA intermediates. In all transgenic lines citric acid, isocitric acid and malate were significantly reduced, while fumarate showed a significant increase  were not changed. The only exception was pyruvate kinase, which showed a significant decrease of activity in all transgenic lines Figure . To invese Table .In contrast to the vast knowledge concerning the role and action of catecholamines in mammals, very little is known about the physiological significance of catecholamines in plants. Since most of the components of animal catecholamine signaling pathway have been also identified in plants  the involvement of catecholamines in plant signalling pathways is possible. Recently, the analysis of transgenic plants over-expressing tyrosine decarboxylase, which accumulate a high quantity of catecholamines, suggested a possible signalling effect on plant primary metabolism. The increase of catecholamines resulted in decreased starch concentration but increased soluble sugars .The only component of mammalian catecholamine signaling pathway that to date has not been identified in plants is the catecholamine receptor. We transformed potato plants with a cDNA encoding human dopamine receptor (HD1) in order to analyze whether the presence of a receptor affects the endogenous catecholamine action. Western blot analysis showed that the protein was produced in transgenic plants and biochemical analysis of transgenic tubers revealed vast changes in carbohydrate metabolism and carbohydrate content. Surprisingly the catecholamine level was changed as well. It has to be pointed out that in contrast to plants over-expressing tyrosine decarboxylase, those expressing human dopamine receptor are characterized by increases of all known tuber catecholamines . Whereas norepinephrine content was positively correlated with soluble sugars and negatively with starch, normetanephrine was considered as the product of norepinephrine turnover. Increased catecholamine content was accompanied by an increase of their precursors, tyramine and L-DOPA, suggesting upregulation of the biosynthetic pathway, mediated by tyrosine decarboxylase and tyrosine hydroxylase, respectively. It is hard to explain how expression of a human receptor triggers a positive loop leading to enhanced catecholamine synthesis and turnover. It is interesting to compare data on tuber carbohydrate levels from plants over-expressing tyrosine decarboxylase (TD) with those expressing human dopamine receptor. In both cases starch content is strongly decreased, this decrease was larger for HD1 plants (from 40% to 80%) than for TD tubers (from 12% to 60%) although the norepinephrine content was higher in TD plants  to the observed changes. Increased content of hexose-6-phosphates demonstrates that a direct inhibition of AGPase, rather than a substrate shortage may cause inhibition of starch synthesis. Alternatively the increased hexose phosphate levels may be due to increased starch degradation in response to elevated catecholamine levels. This is supported by the increased activity of starch phosphorylase in two of four transgenic lines. The inhibition of starch synthesis and accumulation of hexose phosphates was accompanied by an increase of sucrose synthesis. Two factors should be taken into consideration. First, that SPS is subject to allosteric activation by Glc-6-P and inhibition by Pi. Second, elevated catecholamine content led to a decrease of SPS Km suggesting increase of the enzyme catalytic activty. Sucrose phosphate synthase has many potential sites of phosphorylation and three of them were shown to influence its catalytic activity. In spinach leaf, phosphorylation of Ser 158 is responsible for enzyme downregulation in darkness, phosphorylation of Ser 229 enables binding of 14-3-3 proteins and down- regulates the enzyme whereas phosphorylation of Ser 424 under stress conditions stimulates SPS activity. There is a growing body of correlative evidence that the potato tuber SPS is regulated in an analogous manner to the leaf enzyme .Since the level of 14-3-3 proteins was not changed in any of the transgenic lines (data not shown) it is thus suggested that enzyme phosphorylation targeted to the stress site is responsible for its activity enhancement in HD1 plants.In mammals the action of epinephrine and norepinephrine is mediated by phosphorylation of enzymes involved in glycogen mobilization and synthesis. Very recent studies reported direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation in the control of starch anabolism and catabolism . TherefoIn mammalian systems, catecholamines serve as stress hormones increasing as a result of stress. In order to see whether or not a similar response occurs in plants, leaves of potato plants were wounded and catecholamines levels prior to and 5, 10 and 13 min after wounding were determined. Although the data varied, there was a consistent increasing trend in concentration of dopamine, norepinephrine and normetanephrine . Very reIntroducing humane dopamine receptor into plant cells can be considered as controversial but the obtained data would argue for the value of our approach.Vast changes in the activities of key enzymes mediating carbon metabolism of potato tuber (in HD plants) led to a dramatic reduction of starch but increased sucrose content. The relation between catecholamine, primary carbon metabolism and stress seems possible. We speculate that similarly to situation in animal cells expression of HD1 in potato resulted in activation of the cAMP mediated signalling pathway. This can be supported by the result obtained for potato plants expressing another isoform of human dopamine receptor, HD2. In contrast to HD1, HD2 receptor does not affect activity of adenylate cyclase in animal cells.Similarly plants expressing HD2 showed no changes in carbohydrate metabolism (data not shown). The obvious next step would be further investigation of our plants with respect to their kinase activity as well as cAMP levels. In parallel we have made efforts to identify a plant dopamine receptor.Solanum tuberosum L. cv. Desiree) obtained from "Saatzucht Fritz Lange KG"  were cultivated in a greenhouse in soil under 16 h light (22°C) and 8 h dark (15°C) regime. Plants were grown in individual pots and watered daily. For analysis, the leaves were harvested at noon from 30-day-old greenhouse grown plants and the tubers were harvested in September, 3 months after the transfer of the tissue culture plants to the greenhouse.Potato plants  were transformed with A. tumefaciens by immersing leaf explants in bacterial suspension. A. tumefaciens inoculated leaf explants were subsequently transferred to callus induction medium and shoot regeneration medium. Transgenic plants were pre-selected by using PCR with the primers for the respective phosphotransferase (kanamycin resistance) gene and then selected by northern blot analysis with a HD1 specific cDNA fragment as probe.The 1.3 kb SmaI, XbaI cDNA encoding HD1 from Homo sapiens ; [EMB: XX55760])), was ligated in the sense orientation into the same restriction site of the plant binary vector under the control of the 35S CaMV promoter and Nos terminator. The vector was introduced into the Total RNA was prepared from frozen plant material using the guanidinium hydrochloride method. Following electrophoresis (1.5% (w/v) agarose, 15% formaldehyde (w/v)), RNA was transferred to nylon membranes . Membranes were hybridised overnight at 42°C in 250 mmol sodium phosphate buffer (pH 7.2) containing 7% (w/v) SDS, 1% (w/v) bovine serum albumin (BSA) and 1 mM EDTA. Radioactively labelled full-length cDNA was used as a hybridisation probe. Filters were washed three times in 1 × SSC containing 0.5% (w/v) SDS at 65°C (highly stringent condition) or in the same buffer but at 42°C (medium stringent condition) for 30 min.2, 1 mM EDTA, 1 mM EGTA, 20%glycerol (v/v), 0.5 mM PMSF, 70 mM beta-mercaptoethanol) supplemented either with 0.1% or 1% TritonX- 100 (v/v). The assessment of the expression of HD1 gene by means of western blot analysis using rabbit IgG anti HD1 protein was conducted as described previously. Briefly, solubilised protein was run on 12% SDS polyacrylamide gels (w/v) and blotted electrophoretically onto nitrocellulose membranes (Schleicher and Schuell). Following transfer, the membrane was sequentially incubated with blocking buffer (5% (w/v) dry milk), and then with antibody directed against the HD1 protein (1:2000 dilution). Formation and detection of immune complexes were performed as previously described [Proteins were extracted from frozen plant material using extraction buffer E , heated for 15 min at 70°C, and centrifuged . Samples were diluted with water to 50% methanol concentration and extracted with chloroform (1:1 v/v). A portion of the water phase was dried under vacuum and used for derivatisation [-1 fresh weight).Frozen plant tissue (400 mg) was powdered in liquid nitrogen and extracted with methanol  at 37°C for 90 min and then acidic protons were derivatised with N-Methyl- N-trimethylsilyltrifluoroacetamide (MSTFA) at 37°C for 90 min. 2 μl of sample was used for analysis [-1. The temperature program was 5 min isothermal heating at 70°C, followed by a 5°C min-1 oven temperature ramp to 310°C and final 1 min heating at 310°C. The system was then temperature equilibrated for 6 min at 70°C prior to injection of the next sample. Mass spectra were recorded at 2 scan s-1 with an m/z 50 – 600 scanning range. The chromatograms and mass spectra were evaluated using the MSD ChemStation program . As standards, dopamine, norepinephrine, normetanephrine, L-Dopa, tyramine and tyrosine (Sigma) were used. The recovery samples were spiked with dopamine, norepinephrine, normetanephrine, L-Dopa, tyramine and tyrosine; the estimated recoveries were 80, 93, 85, 95, 82, 90%, respectively. The following ions were used for quantification: ribitol 307; 319, L-Dopa 218; 267; 368, dopamine 174; 338; 426, norepinephrine 174; 355, 514, normetanephrine 174; 297; 456, tyramine 174; 264; 338, tyrosine 218; 280, 354. The amounts of catecholamines were determined from the ratio of peak areas of catecholamines to peak area of the internal standard (ribitol).The dried extracts were incubated in pyridine/methoxyamine 20 mg mL0 mg mL-12. 0.5 g ± 0.1 g tissue was homogenised in a chilled mortar in 2 ml of an extraction buffer containing 30 mM HEPES-NaOH, pH 6.9, 10 mM DTT, 1 mM MgSO4, 0.5 mM EDTA, 0.5% (w/v) BSA and 0.5% (w/v) PVP at 4°C. The homogenate was centrifuged at 16000 g for 10 min and the supernatant desalted using Sephadex G-25 columns. Enzyme activities were determined using the following published methods; SPS [Tissue was harvested, weighed and immediately frozen in liquid Nods; SPS ; SuSy, Pods; SPS ; PGM [33ods; SPS ; PGI, enods; SPS ; UGPase ods; SPS ; hexokinods; SPS  and starods; SPS .The t-tests were performed using the algorithm embedded into Microsoft Excel. The term significant is used when P < 0.05.AS carried out the metabolic analysis of transgenic plants and drafted the manuscript. AŚ carried out the construction and selection of transgenic plants and performed the statistical analysis. JS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Sustainable DNA resources and reliable high-throughput genotyping methods are required for large-scale, long-term genetic association studies. In the genetic dissection of common disease it is now recognised that thousands of samples and hundreds of thousands of markers, mostly single nucleotide polymorphisms (SNPs), will have to be analysed. In order to achieve these aims, both an ability to boost quantities of archived DNA and to genotype at low costs are highly desirable. We have investigated Φ29 polymerase Multiple Displacement Amplification (MDA)-generated DNA product (MDA product), in combination with highly multiplexed BeadArray™ genotyping technology. As part of a large-scale BeadArray genotyping experiment we made a direct comparison of genotyping data generated from MDA product with that from genomic DNA (gDNA) templates.Eighty-six MDA product and the corresponding 86 gDNA samples were genotyped at 345 SNPs and a concordance rate of 98.8% was achieved. The BeadArray sample exclusion rate, blind to sample type, was 10.5% for MDA product compared to 5.8% for gDNA.We conclude that the BeadArray technology successfully produces high quality genotyping data from MDA product. The combination of these technologies improves the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples. In order to locate the disease variants involved in complex common disease it is now generally accepted that very large sample numbers will be required -4. Not oAt present, the gold standard method for generating gDNA from whole blood samples is through the process of immortalisation by transformation of the peripheral blood lymphocytes with Epstein-Barr Virus (EBV) . AlthougMolecular Staging, Inc. (MSI)  have developed a method for whole genome amplification by Φ29 polymerase Multiple Displacement Amplification (MDA). It has been reported by the company that this method can reliably amplify the whole genome from gDNA, whole blood and other clinical samples -8. Each Recent studies have been conducted using MDA product from Amersham ,10, and ®  and Invader®  with gDNA.The BeadArray genotyping platform of Illumina™ Inc.  offers a high-throughput, highly multiplexed and highly automated genotyping service facility . The BeaHLA-DRB1 genotype of each MDA sample was entirely concordant with the corresponding gDNA template, verifying the identity of each MDA sample and ruling out the possibility of contamination.We selected and sent to MSI 20 ng of 88 gDNA samples for amplification, from which an average of 200 μg of MDA product was yielded in 100 μl reactions. The yields ranged from 85 μg to 280 μg, with 61% of samples yielding between 100 μg and 200 μg. The When 100 ng of 448 gDNA samples were amplified using reagents supplied by MSI in kit form and the amplification carried out in-house, an average of 155 μg of MDA product was yielded in 150 μl reactions. The yields ranged from 31 μg to 260 μg, with 80% of samples yielding between 100 μg and 200 μg.INS) -23HphI (rs689), an allelic bias was observed in the MDA process, which resulted in the merging of the heterozygote cluster with the homozygote cluster of the major allele, making the correct assignments of genotype impossible, shown in Figure 2 [see INS in PCR reactions designed for the Pyrosequencing method  [INS may be situated in a sequence region that is predisposed to such allelic bias and the INS variable number of tandem repeats polymorphism, only 580 bp 5' to the -23HphI SNP, is a candidate for such an effect.Of 88 MDA products and their corresponding gDNAs tested at 95 SNPs using the TaqMan method of genotyping there were no samples that consistently failed to produce any data. This confirmed that, for all samples, amplification had been sufficiently successful for the TaqMan chemistry to perform at most SNPs. Genotype concordance rates between MDA product and gDNA and genotype failure rates are given in Table re 2 see . MDA prore 2 see , indicat Sweden) . These rFor 13 additional SNPs for which Invader genotyping was performed, comparison of genotypes generated from MDA product with those from gDNA are shown in Table Two SNPs were genotyped by TaqMan on the 448 samples amplified using reagents supplied by MSI in kit form (and the amplification carried out in-house), and on the corresponding gDNAs. The gDNA samples used for amplification had been extracted from whole blood. Genotype concordance rates between MDA product and gDNA and genotype failure rates are given in Table in silico assay design. These SNPs were selected for their relevance to a range of ongoing projects in our laboratory, located at genes of strong functional candidacy and within regions of linkage to type 1 diabetes e.g. the putative IDDM10 locus on chromosome 10p14-11. All SNPs were validated, having been identified either from empirically confirmed SNPs in dbSNP or from our own re-sequencing efforts. Based on ranking from the in silico design criteria [We commissioned Illumina to conduct a large-scale project using BeadArray genotyping technology involving 3,036 samples  and 384 SNPs i.e. >1.1 million genotypes. In the first instance, 757 SNP sequences were sent to Illumina for criteria , 404 SNPAs well as excluding SNPs that fail to produce robust genotypes, the Illumina protocol excludes samples that do not consistently perform. Of the total number of samples 10.5% were excluded and as a consequence very few data points were missing from the data set, resulting in an apparently low genotype failure rate Table . Within As our samples were family-based, a quality control check of misinheritance rates was possible using PedCheck . Of the Within the panel of 384 SNPs attempted by Illumina, 17 were controls for which we had already produced genotyping data by either TaqMan or Invader methods, enabling an evaluation of the BeadArray data for concordance. Two of these 17 control SNPs failed to be converted to a working assay, giving 15 SNPs and a maximum of 2,503 samples that were genotyped in common. Excluding failed duplicates noted above, comparison of BeadArray genotypes with existing data revealed a concordance rate of 99.6% , indicating the compatibility of the non-excluded gDNA samples with BeadArray, and the quality of existing data. Of the 15 control SNPs, 11 had been genotyped using TaqMan and four using Invader. The concordance rates for each platform were 99.7% using TaqMan  and 99.6% using Invader  when compared with BeadArray data, showing no significant difference between the two platforms.t-test P-value >0.05 for every SNP).Within the BeadArray experiment described above were 86 MDA products and their corresponding gDNA samples. These data were directly compared for sample failure rate and genotype failure rate as shown in Table In this study we have evaluated the Φ29 polymerase MDA whole genome amplification method from MSI by assessing the compatibility of its product with the established TaqMan and Invader genotyping chemistries and with the highly multiplexed BeadArray genotyping platform. We have also evaluated Illumina's BeadArray genotyping platform for a large-scale experiment using gDNA.n = 88 in order to be confirmed. This result is comparable to the smaller study by Tranah et al. [et al. with fluorescent minisequencing on Amersham MDA products compared to gDNA [INS -23HphI, rs689).At 95 SNPs, comparison of TaqMan genotypes generated from MDA product and gDNA templates revealed a very good concordance rate but a higher failure rate for MDA product compared to gDNA. This would need be estimated in a sample size larger than the current  to gDNA . One maret al. [et al. [et al. [Compared to the yields indicated in Dean et al. , our ave [et al. . Further [et al.  protocolet al. using 2,320 SNPs and five samples [In the evaluation of MDA product in conjunction with BeadArray technology, the high concordance rate between genotypes obtained from MDA product and gDNA templates is encouraging. A concordance rate of 99.86% has been reported by Barker  samples . However samples . As withThe performance of MDA product is continuously being monitored in our laboratory. In a study blinded to all genotypers and database administrators, 288 family-based gDNA samples , were replaced with MDA product and left in continual use in our genotyping pipeline for 12 months. The change went undetected by all users. The failure rate for MDA was 3.34% for 15,921 genotypes, compared to 2.39% for 19,272 gDNA genotypes. Therefore, this improvement in the MDA performance for TaqMan is likely to be applicable to BeadArray, which improves the feasibility of mapping susceptibility loci in complex traits.When using a highly multiplexed, highly automated genotyping platform, slight reductions in the quality of template material are likely to have a greater adverse effect on data than in scenarios in which markers are assessed individually and manual scoring is undertaken. Our results indicate that MDA is an adequate solution for the vast majority of SNP markers, even in this highly multiplexed allelic assay platform.It is noted that 5.8% of markers that passed the Illumina acceptable scoring threshold were in fact showing high misinheritance rates in our family samples. This problem was at the same magnitude as TaqMan for individually genotyped markers. This highlights the importance of checking potential positive results with a second genotyping technology.MDA should allow the continuation of genetic analysis on archived DNA in researchers' freezers worldwide, providing the very necessary increases in sample sizes so urgently required ,2,17.The combination of BeadArray high throughput, multiplex genotyping and amplified DNA (MDA product) successfully produced high quality genotype data thereby improving the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples.For both the validation experiments (MDA product as a template for TaqMan and Invader genotyping) and for the combined experiment (MDA product as a template for BeadArray genotyping) the same MDA samples were tested. We sent to MSI 20 ng (5 μl at 4 ng/μl) of 88 gDNA samples for amplification, which was performed as a service according to the protocol for human gDNA with the omission of the denaturing step . These gHLA-DRB1 and comparison made with data generated from the corresponding gDNA. HLA-DRB1 genotyping was performed using the Dynal Auto RELI™ SSO HLA-DRB Test system  for each gDNA sample and their MDA products. Although, these samples were amplified by MSI as a service, the reagents are also available from MSI in a kit form for amplification in-house. Following the amplification by MSI we have amplified 448 DNA samples by using the reagents in kit form and 100 ng (25 μl at 4 ng/μl) gDNA template in 100 μl reactions. In the interim, one major change to the MDA protocol had taken place, the inclusion of a denaturation of the DNA template prior to amplification. Previously no denaturation step took place. Two TaqMan markers were tested on these 448 samples and the genotype failure rate calculated.In order to verify the identity of each MDA-produced sample, genotyping was performed at SNP TaqMan assays were carried out for allelic discrimination, 8 ng of DNA (2 μl at 4 ng/μl) used in a 5 μl total reaction volume. TaqMan genotypes from the 88 MDA samples, described above, were compared with TaqMan genotypes generated from their corresponding gDNAs, at 95 SNPs, with a broad range of allele frequencies. The Invader method was used to genotype 13 additional SNPs on the same samples. Comparison was made between data generated from both templates by the measurement of genotype failure and genotype concordance rates.Of the 384 SNPs selected for genotyping 3,036 samples, 17 were control SNPs for which we had existing genotype data, generated by either TaqMan or Invader methods, with which comparison of genotype failure and genotype concordance rates were made. These 384 SNPs covered a broad range of allele frequencies.Incorporated into this experiment was an assessment of suitability and compatibility of the BeadArray genotyping method with MDA product. This involved 86 of the 88 amplified samples, described above, for which genotyping was attempted at all 384 SNPS. Concordance between genotypes generated from MDA product and gDNA templates, together with the genotype and sample failure rates of each template type were measured.In our laboratory we store genotyping data in a MySQL database on a Sun server. The volume of data expected from Illumina was the equivalent of 6 months' in-house genotyping. We separated phenotypic and pedigree information, which is associated with a sample, from genotype data, which is associated with a DNA plate and well position, with a link table to relate the two. Sample aliases are also supported, so that no recoding of identifiers is required, either to export or import Illumina data .RP participated in the design of the study, performed in-house genotyping experiments, and assisted in preparing the manuscript. HER prepared DNA and MDA amplified samples. BJB participated in the design of the study and assisted in the preparation of the manuscript. SN prepared DNA and MDA amplified samples. DS performed in-house genotyping experiments. MS prepared DNA and MDA amplified samples. RCJT participated in the design of the study and assisted in the preparation of the manuscript. AS participated in the design of the study. ACL and LJS performed bioinformatics. NMW managed the data and participated in its interpretation. JAT participated in the design of the study and assisted in the preparation of the manuscript. All authors read and approved the final version of the manuscript.INS -23 HphI SNP. Both the MDA product plots could not be scored. All plots represent the same individual samples with gDNA plots containing 8 additional samples.Figure 2 TaqMan and Invader fluorescence data plotted for the Click here for file


Little data exists on the effectiveness of psychological interventions for children with posttraumatic stress disorder (PTSD) that has resulted from exposure to war or conflict-related violence, especially in non-industrialized countries. We created and evaluated the efficacy of KIDNET, a child-friendly version of Narrative Exposure Therapy (NET), as a short-term treatment for children.Six Somali children suffering from PTSD aged 12–17 years resident in a refugee settlement in Uganda were treated with four to six individual sessions of KIDNET by expert clinicians. Symptoms of PTSD and depression were assessed pre-treatment, post-treatment and at nine months follow-up using the CIDI Sections K and E.Important symptom reduction was evident immediately after treatment and treatment outcomes were sustained at the 9-month follow-up. All patients completed therapy, reported functioning gains and could be helped to reconstruct their traumatic experiences into a narrative with the use of illustrative material.NET may be safe and effective to treat children with war related PTSD in the setting of refugee settlements in developing countries. In the wars and armed conflicts of the past decades, children have been among the survivors who have been exposed to war or conflict-related violence. The United Nations High Commissioner for Refugees (UNHCR) recently stated that 43% of its population of concern are children under the age of 18 -5.An increasingly important field of research addresses the wide-ranging negative sequelae that children and adolescents in modern post-conflict populations such as in Iraq, Kuwait, Bosnia, Rwanda, Croatia, South Africa and others may develop consequent to war and conflict violence -17. CurrGiven the pervasiveness of war and conflict-related trauma, especially in resource poor countries, interventions tailored to suit the circumstances of the overwhelming number of such survivors are especially in demand. However, treatment outcome studies in this field are still few. Many such interventions are derived from interventions initially developed for adults, such as cognitive behavioural therapy. Cognitive-behavioural interventions have been successfully used with school children exposed to violence, after single-incident stressors, after natural disasters as well as to treat sexually abused children ,30-33.Other interventions currently in use with children include psycho-pharmacological treatments, play therapy, psychological debriefing and testimony therapy ,26,34-39vivo developed Narrative Exposure Therapy as a standardized short-term approach based on the principles of cognitive behavioural exposure therapy by adapting the classical form of exposure therapy to meet the needs of traumatized survivors of war and torture [Narrative Exposure Therapy (NET) is a treatment approach that was developed for the treatment of PTSD resulting from organized violence.  torture -42. In eAs most victims of organized violence have experienced many traumatic events, it is often impossible to identify the worst event before treatment. To overcome this difficulty in NET, the patient constructs a narration of his whole life from early childhood up to the present date while focusing on the detailed report of traumatic experiences. The focus of NET is therefore two-fold. As with exposure therapy, one goal is to reduce the symptoms of PTSD by 1) confronting the patient with memories of the traumatic event. However, recent theories of PTSD and emotional processing suggest that the habituation of the emotional processes is only one of the mechanisms that improve symptoms . Other tIn narrative exposure procedures, children are asked to describe what happened to them in great detail, paying attention to what they experienced in terms of what they saw, heard, smelled, felt, the movements they recall and how they felt and thought at the time. Initially, the session is distressing, but as it is long enough to allow habituation, distress levels diminish towards the end and more and more details are recalled. After only four sessions of exposure, scores on intrusion and avoidance may drop significantly .This study investigated the effectiveness of NET when applied to child refugees. The investigation was carried out in the context of the Nakivale mental health project, which aimed at the examination of mental health symptoms as well as the evaluation of different treatment approaches in the Nakivale refugee settlement in Uganda .The first aim of this paper is to present and illustrate the procedure of KIDNET as a child-friendly treatment approach for traumatized children in post-conflict populations. In addition, we present the results of a small sample pilot test to allow the examination of the feasibility and potential efficacy of the method in a field context.The study protocol was approved by the Ethical Review Board of the University of Konstanz and by the Ugandan National Council for Science and Technology, Kampala.Six child refugees  of Somali ethnic origin screened as having PTSD from a larger epidemiological survey in Uganda's Nakivale refugee settlement .As instruments need to go through a work-intensive process of translation and validation and interpreters as well as interviewers need extensive training so that instruments are properly applied, we used the same already-validated instruments as in the adult epidemiological survey . The PosThis was done within the context of a clinical interview during which the clinicians clarified the questions for the children. The clinicians employed child-appropriate language to make sure the children understood the questions. In all six cases, the initial diagnosis was confirmed.All six were assigned to a Narrative Exposure Therapy (KIDNET) treatment group, with treatment being offered by expert clinicians experienced in the use of NET. The post-tests were also conducted by expert clinicians and trained interpreters, again using the CIDI.All six child patients gave verbal assent for the screening. They were then formally offered individual treatment after diagnosis, along with a brief psycho-education describing the nature and prevalence of PTSD symptoms, and what treatment would entail. A standard written rationale that has been developed for this purpose was used, the goal being to explain that PTSD-related symptoms and dysfunction are frequently consequent to multiple traumatic experiences. All six gave their assent for treatment, which only began after informed consent from the parents or guardians was granted. It was made clear that both assessment and treatment would be entirely voluntary, and no monetary or food-item inducements would be offered. In all cases, both the patients and their parents or guardians were relieved that treatment was offered.The patients were tested again with the CIDI approximately four weeks (post-test) and nine months (follow-up) after the end of treatment.Narrative Exposure Therapy treatment sessions lasted between 4 to 6 sessions, of between 1 – 2 hours in duration. The treatment involved one-to-one sessions with a clinician attending to an single child patient at a time. At the beginning of Session 1, the patient was requested to draw any picture that came into their minds, as the very first step. Next, the patient was given a lifeline (length of rope) and a selection of stones of varying characteristics and sizes, as well as fresh flowers of varying sizes and colours. He or she was then asked to construct his lifeline, outlining the major events using flowers for positive events and stones for negative events in a chronological order.When he or she was quite sure about the sequence and magnitude of events, the patient was then asked to make a drawing of this lifeline, with brief titles for each event. The narrative session then began, with the patient narrating the events of his or her life starting with his or her birth, with the aid of the lifeline and the drawings. The patient used a representative object, which he or she moved at will to indicate where he or she had reached on the lifeline.In the following narration procedure, the participant constructed a detailed chronological account of his own biography in cooperation with the therapist. The therapist's task was to document the patient's autobiography, which was corrected with each subsequent reading. Special focus of the therapy was on the transformation of the generally fragmented report of traumatic experiences into a coherent chronological narrative, and working through emotions, sensations and reactions relevant to the traumatic events. During the discussion of the traumatic life experiences, the therapist asked for current emotional, physiological, cognitive and behavioural reactions, while accompanying the patient back into the details and emotions surrounding each event, and helping the patient to reconstruct the trauma memory. The participant was encouraged to relive these reactions and emotions while reporting the events. The discussion of a traumatic event was not terminated until a habituation of the emotional reactions presented and reported by the patient occurred.During the session and in subsequent sessions, the testimony was read back to the patients, who was asked to correct, modify or add to it until a complete document has been made of the patient's experiences. During the last session, the participant received a complete written document of his biography. The precepts of Narrative Exposure Therapy are described in detail in a manual .The main innovation of KIDNET as compared to the adult version of NET is the use of illustrative material such as a lifeline , stones and flowers, as well as coloured drawings and role-play, to help the child reconstruct the memories of his or her experiences. Unlike with adult NET thus far, the patients were encouraged to extend the narration beyond the present, to describe their hopes and aspirations for the future, mainly with the use of flowers. This is done at the end of therapy. These hopes and aspirations were included in the drawings, lifeline and narrations as an integral part of the document. The therapist also highlighted the length of rope still left over, to illustrate opportunities and possibly improved life circumstances, despite negative past experiences. The patient was requested to construct his or her lifeline at the beginning of each session, after which narration was resumed. The therapist was also alert to detect any connection between the initial picture and the traumatic events in the storyline, especially the worst-ever event. As a final task, the therapist requested the child to draw any picture that came to their minds after handing over the document to the patient at the end of the last session, in order to compare this with the initial picture drawn.AWH is a very slim young person of middle height, with active uneasy eyes that never rest. He was interviewed in March 2003 when he was 15 years old by a locally trained interviewer using the PDS. This PTSD diagnosis was confirmed within a two-week period by an expert using the CIDI, after which AWH was invited for therapy. He was very shy when he came. In fact, he approached the house then ran back into the street. After some time, he slowly approached again. Both the therapist and the interpreter put in a lot of effort to make him feel welcome and comfortable. As we later learnt, he lost both parents during the civil war in Somalia. This was without question his "worst event".He has lived by himself since the age of 14 in the camp, but fended for himself since the age of 9. He had no family in the camp or anywhere else, as far as he knew. At the time of his parent's death in Somalia, he recalled a younger brother and sister, but he had never heard of them again. He has been a registered refugee in Uganda since 1998.His initial drawing at the beginning of therapy shows the tiny lonely figure of a child, placed in the middle of the large white sheet of paper, nothing else. He named it " a boy".The beginning of NET was challenging, since he seemed to recall very little of his early years as a child in Somalia. He was also very economical with words and upon being asked to place flowers for good events in his life, he found there was no event that would deserve a flower. He simply placed two stones on his lifeline, one for the day his parents were shot in front of him, one for the time he reached Nairobi as a refugee, a time when he had to struggle hard to stay alive in the absence of any aid.After NET 1 (session 1), he did recall his early years in quite some detail and even remembered some joyful events; for one of which he later placed a flower: 'the time I used to play football with my dad in the evenings in our compound'.After having witnessed the death of his parents and being able to escape through the back of the house, he was completely on his own. He escaped Mogadisho with a group of strangers, survived Nairobi by himself, eating left-overs at hotel garbage dumps and finally smuggled himself into Uganda by hiding in the back of a bus.Excerpts of his story read:I was born in Mogadisho, Somalia. I do not know my exact birthyear, I think it is 1986. I grew up with both my parents. I have a sister who is 2 years younger called Halimo and a 4 years younger brother called Mohamed. We lived in a part of town called ...My mother had fair brown hair and skin. She was young and I loved her a lot. I was her first-born and her favourite. She even told me so. My father was of darker complexion. He was also a young man in those days. He was hard working. He had a shop close by in the market. He would usually leave in the morning and return home in the evening. Sometimes when he came home, he played with us in the evening. We played football together. Those were good times. I do not know how old I was then; I just remember that I was very young...When I remember those days I get sad. All these memories come back and I only know what I have lost. The years went by and I used to live like this until the war broke out. I don't remember the year, but I was still young... One day we fled from home in the late afternoon...We went with a car to a place called Bal'aad, about 30 km out of town...Eventually we went back home to Mogadisho. I must have been about 10 years by then. A few months later, the war reached us again. It was early in the morning. A group of about 10 civilian men reached came to our house. They were armed with guns...I stood very near to my parents. I was so scared. Suddenly I heard the sound of bullets. One of the soldiers had started shooting. The moment I saw that he pulled the trigger and heard the first bullet, I panicked. I started running. I felt such great fear. I ran inside the house and tried to hide myself behind a door in one of the rooms. I was shivering, fearing, thinking, they will also come for me, they will come and kill me'. I still have a heart beat now, when I recall that moment. After some time it went quiet outside. I still stood behind the door, silent, not moving. After a while I slowly moved towards the window and peeped out. What I saw was terrible. My mother and my father had been hit by the bullets. They were both lying on the ground. My mother had fallen on top of my father. They both had blood on their clothes. My mother had blood on her face and her stomach. They were not moving anymore, they had died. Until that day, I had never seen a dead person. I felt horror. I was so afraid of them, shocked by what I saw. I only thought of running away, leaving this place. I escaped through the back of the house and jumped over the fence. This was the last time I have seen my parents and also the last time I had been in our home in Mogadisho...While fleeing, I joined strangers in the street. So many people were trying to flee. I simply ran with them. When these people reached their destination, they branched off from the road. It was night time by then. I was alone. I hated my life. I followed the road and finally fell asleep under a bush. I had given up about life by then. I felt like I had died as well. I knew about the danger of wild animals and lions, but I did not care...This is how I came to Uganda. When we reached Kampala I saw a group of Somalis and went to greet them. They took me in and I lived with them for a few weeks. They also showed me how to register as a refugee with UNHCR. I remember the day I came to Nakivale Refugee Camp. I was so surprised how people can live in a place like this. I stayed with the Somali family that found me in Kampala for about two years in the camp. Finally, Red Cross helped me to build my own house in 2000, I was about 14 years then. Since then I live alone. I started going to school when I came to Nakivale. I will graduate from P7 at the end of this year. I have learnt how to live by myself; I can do everything by myself. I never ask for help. No one can help me anyway. I have never heard about my brother and sister again. Whether they are still alive and if so, where I could find them. But now I am ready to look for them.As mentioned before, he has been surviving on his own since the age of 9 years. This has probably led to his enormous shyness but also an amazing sense of self-reliability. 'I can do everything by myself. I never ask for help. Anyway who could help me?' There is also a strong feeling of sadness and loneliness around him that is very moving. Our local translator Haji, who is also trained as a NET therapist, was moved to free flowing tears at several moments of AWH's life recall, specially when he spoke about his loneliness and desperation.AWH is a smart boy, so he frequently challenged the concept of therapy in the beginning. 'It won't make my parents come back, and it won't change my life situation'. AWH has never really entered a state of expressing strong emotions like crying or anger during therapy. He said he could not cry, also not by himself. There were however visible signs of emotional processing like tears and strong heartbeat especially during NET 1 and 2.AWH reported having strong PTSD symptoms when he started therapy, especially continuous nightmares and flashbacks, mainly related to the day his parents were killed. Re-experiencing actually increased, he said, once therapy was started. After NET 4 he talked of having fewer symptoms. In his last session AWH talked about wanting to try and trace his family with the help of the Red Cross. He was especially interested in finding his two younger siblings.When the sum of symptoms were counted according the CIDI-K section, AWH had a sum score of 12 in the CIDI pre-test. This had decreased to 8 in the CIDI post-test immediately after the end of therapy, and 9 at the nine-month follow-up. At the nine-month follow-up, AWH was looking well-dressed, and had put on both height and weight. He had completed his primary school education at the camp primary school and spoke confidently of his plans. He had also joined the camp soccer team and played along with the group now every evening, a significant behavioural change, given that he previously would not even talk or socialize with others. He was not more friendly than usual, but admitted significant symptom reduction to an expert evaluator who had never seen him before.UG was a pretty seventeen-year old girl in March 2003 when she was interviewed by a locally trained interviewer. She looked, however, visibly ill and strained -quite unlike a normal happy young woman. She complained of constant headache and pain in her eyes. UG's PTSD diagnosis was confirmed within a two-week period by an expert using the CIDI.UG did not wait to be offered treatment, but came herself to the project centre and requested for help. In her own words, "I have been to all the doctors but they have done nothing for me. All they give me are painkillers. I had to drop out of school because of my headaches and pain in my eyes." When the treatment protocol was explained to her, she was enthusiastic about relating her experiences and readily gave her assent. Her mother was quick to give her consent. She said she would try anything that would help her daughter. In her own words, ' My daughter has not been her real self for a long time.'At the beginning of NET 1, UG drew a picture of the Somali flag. Asked to explain, she said she loved her homeland and hoped to go back there one day when there was peace. While laying her lifeline, UG included a happy family life in Somalia till the age of 4, her arrival in Uganda after a long and difficult flight itinerary and being accepted as a refugee by the UNHCR as the flowers in her life. The stones in her life represented the worst event, when her older brother was killed, her sister was severely injured and her two younger brothers were lost, never to be seen again, to date. Other stones symbolised flight difficulties such as extreme hunger, and a failure to access a solution for her headaches and eye pain, which she has had since the worst event.In subsequent NET sessions, UG talked in detail about these events. Excerpts from her story read:I was born on August 26th 1986 in Mogadishu.... I had six brothers, two younger than me and two sisters older than me. We were a very happy family. As a big family, we conversed a lot and made jokes. We were very happy with our father. He made us laugh and brought us presents whenever he went anywhere. This was a happy time till I was 4 years old...One day,... we heard guns and bullets firing and we were excited. My father ran from work and picked up the other children from school and came back home with them. Some people came to chase us away from our house because we were a minority clan (Madiban). They were from the Hawie (Habrigidir). They were men, very many. They told us to leave everything and flee from the house. They did not beat us. I was still a child, with a soft head. I heard the bullets and started vomiting and fell down. My mother picked me up and put me on her back. We all left the house...On the way, we met very many militia men dressed as army men. They told us to lie down on the ground. I was still crying. The rest were silent. One of them knocked me with the butt of the gun on the soft part of my head. Then I kept quiet. They wanted to kill everyone...They put us in a house for security purposes. A heavy gun was shot near the house. There was another house near our house. They shot this house and the fragments reached our house. Some people were killed in our group, including my brother. He was older than me. He was called Mahad. He was in his school uniform. He fell down on his stomach. I did not see him fall but I saw the blood. My sister Khadija was lying down when the fragments hit her. I did not see any blood, but the fragments went into her stomach and she was hurt in her stomach. My father thought she was dead, but people said she was still alive and she was still talking. He went to her and told her to get up. She could not get up. My father carried her to the hospital. Two of my brothers, Abdirahman aged four years, and Abdirasaq aged three years, disappeared in that group as everyone ran away. They were my two younger brothers. My dead brother Mahad was left there. We never saw my two brothers who disappeared again. So many other people died as well...After one year, we came back to Mogadishu. We found our father and sister in the hospital. They first stayed in the hospital for some time, then moved to a house. My sister had been operated on. My father had become a bit deaf because of the gunshot.We decided to leave Somalia and go somewhere else more secure. We left Somalia with nothing. My mother, my father, my injured sister Khadija, and the other six children...We went to Kampala. My mother was advised to go to the UNHCR and got a mandate. My sister Shamso and my cousin sister Fardosa also got separate mandates.... In 1997, we were resettled in Nakivale camp...I hit my head down and I felt the pain of the gun again. I was not hurt but I got a terrible headache for three days. I went to the doctor but I did not get any assistance except Panadols and eyedrops. Since then, I was unable to go to school because the headaches increased. My eyes had begun paining from birth, but they increased with the headaches.I am still here but I hope for a better future and a happier life e.g. to be resettled elsewhere, to have an education, to have a happy family of my own and to make contact with my lost loved ones.Among her hopes and aspirations for the future, UG hoped to trace her missing brothers under the auspices of the Red Cross, as well as re-enter the education system. She also hoped to one day have a happy family of her ownIn the final picture at the end of therapy, she drew a happy family inside a nice house, living in peace.UG had a CIDI pre-test sum-score of 16; immediately after treatment, this reduced to 12 in the CIDI post-test and 1 at the nine-months follow-up. The nine months follow-up found UG no longer in the camp but in Kampala, the capital city of Uganda. She looked happy and cared about her appearance. She said she had no more headaches, and little eye problems. In her own words, "I feel like a newborn child," she told the blind evaluator. She had moved away from the camp to explore possibilities for further education, resettlement and to seriously try to trace her brothers. She also reported joining other adolescents when they were out to socialize. "My biggest dream is to play soccer myself, but as a girl, I would never be allowed. But at least I go and cheer to the boys when they play." She says her family members, especially her mother, have all noticed the difference in her and keep commenting on the improvements she has made. She is more like any healthy young woman.M = 14.3, SD = 1.9). Scores dropped to M = 9.0 (SD = 2.2) at post-test and again to M = 6.2 (SD = 3.3) at 9-months follow-up  = 6.50, p = 0.039. The analysis at individual level showed that the symptom scores of each individual patient decreased in the period between the pre-test and the follow-up period.All the patients accepted and completed treatment. Two patients were unavailable for the post-test as they had left the settlement after treatment. At the 9-months follow-up, all children could be tested again, as the investigation included a testing in other regions to which Somali refugees had moved. At baseline, all six had moderate to severe PTSD according to the CIDI , it seeNeither the clinical impression nor the symptom scores indicated a worsening of the symptoms in any of the patients. Therefore, this pilot study suggests that NET might be used effectively as a short-term treatment with child patients even in the unsafe conditions of a refugee camp in an African country. A possible adjustment would be to allocate more sessions where needed, such as where traumatic events were particularly severe or numerous.The case reports illustrated that the child patients were able and willing to narrate their traumatic experiences, especially with the aid of illustrative material. There is also evidence that children can recall details of traumatic events that occurred when they were younger. Teenaged children are also able to comply with short-term treatment approaches such as the version of NET presented here.It is noteworthy that this group of children had multiple and very severe war events and yet showed a clear benefit from treatment with NET. This encourages research into the effectiveness of KIDNET with other child trauma populations such as child PTSD after single stressors, after natural disasters or after accidents. More pressing of course, are randomized controlled trials comparing KIDNET to existing therapies for children. Any such comparisons between different groups of children has to await further research.It is often a matter of survival that mental function is restored to an extent that the survivor can cope with the daily stressors and that children actively take advantage of scholastic opportunities and ultimately strive for finding ways out of the camp.In this study, the therapies were carried out by highly trained experts. Given the large numbers of possibly traumatised youth in post-conflict low-income areas worldwide, treatment costs would be prohibitive unless further research can increase the ease with which non-professional paramedics can acquire adequate therapy skills through rigorous short-term training and supervision.KIDNET proved to be a feasible method for the treatment of traumatized children in an African refugee settlement. The clinical observations and the assessed reduction in symptoms should approve KIDNET to be tested in further controlled trials.The author(s) declare that they have no competing interests.The study was designed by LPO, FN, MO, MS and TE. PLO, VE, and ES carried out the treatments and the assessments, FN and MS supervised the treatments. Data was analyzed by FN and LPO. LPO drafted the manuscript, all authors revised the manuscript and approved the final version.The pre-publication history for this paper can be accessed here:


ADAMTS20).The ADAMTS  proteins are a family of metalloproteases with sequence similarity to the ADAM proteases, that contain the thrombospondin type 1 sequence repeat motifs (TSRs) common to extracellular matrix proteins. ADAMTS proteins have recently gained attention with the discovery of their role in a variety of diseases, including tissue and blood disorders, cancer, osteoarthritis, Alzheimer's and the genetic syndromes Weill-Marchesani syndrome (ADAMTS10), thrombotic thrombocytopenic purpura (ADAMTS13), and Ehlers-Danlos syndrome type VIIC (ADAMTS2) in humans and belted white-spotting mutation in mice , chordate (Ciona) and invertebrate (Drosophila and Caenorhabditis) ADAMTS homologs has elucidated the evolutionary relationships of this important gene family, which comprises 19 members in humans.Phylogenetic analysis and comparison of the exon/intron organization of vertebrate  that may have distinct aggrecanase and angiogenesis functions.The evolutionary history of  ADAMTS1-20; there is no ADAMTS11 because early reports of an ADAMTS11 [ADAMTS5. Many of these genes have been implicated in a variety of diseases, including connective tissue disorders [ADAMTS10 gene [ADAMTS  proteins have homology with the metalloprotease region of the ADAM proteases, but also have at least one of the Thrombospondin type 1 Sequence Repeat (TSR) motifs that are common in extracellular matrix proteins. Since the discovery of a gene encoding ADAMTS1 in 1997 , numbereADAMTS11  were latisorders , cancer isorders ,8, and pisorders ,9. RecenADAMTS proteins are characterized by a pro-domain, a metalloprotease domain, the so-called disintegrin-like and spacer domains, and a tail of TSR repeats. The pro-domain of ADAMTS1 and -4 is cleaved at the RX(K/R)R furin cleavage site  in the GThe metalloprotease domain of ADAMTS proteins is shared with the related ADAM proteins, and the catalytic Zn2+-binding motif HEXGHXXXXXHD  is well L. Iruela-Arispe, personal communication). Further proteolytic processing within this domain has been demonstrated for ADAMTS1, -2, -5, and -12 [via proteolytic removal of their TSR domains may be a common theme, as we see similar proteolytic removal of the C-terminal TSRs of the unrelated neuronal guidance protein F-spondin by plasmin, releasing it from ECM binding [vs. free ADAMTS protein could be mediated by MMPs. The region containing these sites is conserved to varying degrees in the newly discovered ADAMTS proteins, suggesting variable (perhaps tissue-specific) MMP processing of these proteins. ADAMTS4, which lacks a TSR tail, may not have an ECM-bound form.There are four matrix metalloprotease (MMP) cleavage sites in the spacer domain of ADAMTS1 ,18, incl and -12 ,6,7,19.  and -12 . Release binding . While tgon-1, a Caenorhabditis ADAMTS homolog, to mediate cell migration during gonadogenesis [Manduca sexta lacunin protein which plays a role in basal lamina remodeling during morphogenesis [The region between the metalloprotease domain and the TSR repeat tail was demonstrated to be necessary for ogenesis . A variaogenesis  and in togenesis . It willADAMTS20 gene have been found to cause the belted white-spotting mutation, resulting from a defect in melanocyte development or migration during embryogenesis [There is evidence that several mammalian ADAMTS proteins are expressed organogenesis. For example, mutations in the mouse ogenesis , the ADAogenesis , ADAMTS1ogenesis , and sevogenesis  are exprADAMTS proteins contain a single "classic" TSR motif (WXXWXXW) in the disintegrin-like domain, and a variable number of variant TSRs within the C-terminal tail of the protein, which contain 4 amino acids between tryptophan residues (W4XW) rather than two. TSRs can be divided in several structural groups, based on the presence and spacing of cysteines . The preADAMTS13 gene result in inappropriate platelet activation, leading to the blood disorder thrombotic thrombocytopenic purpura (TTP) [ADAMTS1 and -8 inhibit angiogenesis , and genra (TTP) -40.ADAMTS4. TGFβ also induces ADAMTS2 [ADAMTS1, ADAMTS6, and ADAMTS9 in ocular cells [ADAMTS1 in endothelial cells [ADAMTS genes may ultimately provide similar benefits with fewer risks.Recently, an intriguing link has been discovered between ADAMTS metalloproteases. The proinflammatory cytokines IL17 , IL1β 4 and TGFβ ADAMTS2  and TNFαar cells , suggestal cells . As seveal cells , we specal cells ,49, tranADAMTS genes, including their comparison with invertebrate and chordate ADAMTS homologs. Prior analyses included fewer species and did not address the sequence of genomic events that resulted in the current ADAMTS genomic structure [ADAMTS1, -4, -5, -8, and -15 genes all derive from a retrotransposition event that occurred prior to the divergence of vertebrates and the urochordate Ciona intestinalis, and from subsequent gene duplications that occurred prior to the divergence of mammals and the pufferfish, Fugu rubripes. This ADAMTS protein subfamily encompasses proteins that share aggrecanase and angiogenesis-related activities.To better understand the multiple functions of the ADAMTS proteins, we carried out the most detailed and comprehensive analysis to date of the phylogenetic relatedness and intron/exon organization of all human tructure ,28,50. OADAMTS genes based on several sequence databases  and found them to be identical to those subsequently published by Cal, et al [ADAMTS9 has three known splice variants, of which the long variant that we used for analysis was NM_182920. ADAMTS20 has two known splice variants, of which we used NM_025003. ADAMTS18 has two known variants, of which we used NM_199355, minus the final exon. ADAMTS13 has four known variants, of which we used NM_139025.To perform a phylogenetic analysis of the ADAMTS gene family we first examined several genome databases to have a comprehensive survey of all ADAMTS genes in humans and other species. When we started this analysis in 2002 only 12 ADAMTS proteins were known. We predicted coding sequences of the human l, et al  based onl, et al . ADAMTS9ADAMTS10 and ADAMTS6 each have a single known coding sequence, and we found evidence of others. The variant of ADAMTS6 that is published (NM_014273) is a short form, and contains a non-consensus exon immediately following the metalloprotease catalytic site, while the variant of ADAMTS10 that is published (NM_030957) has a consensus exon at this location, and is in the long form. We predict a long form of ADAMTS6, as well as a consensus exon for ADAMTS6 and a non-consensus exon for ADAMTS10. We used the consensus exons of both genes in our analyses.Drosophila and Caenorhabditis) from which entire genome sequences were recently determined, to elucidate their evolutionary relationships  ADAMTS orthologs that were unannotated at the time of our initial work have since been identified by others [ADAMTS genes, and those most closely related to the vertebrate ADAMTS orthologs were retained in the analyses presented herein. The most divergent Drosophila and Caenorhabditis ADAMTS homologs represented in Figure Ciona, Fugu and Mus orthologs, annotated with intron positions and phases, is available [To perform this analysis we inferred the coding sequence of sixteen ADAMTS proteins in the s genome , and drey others ,53, and y others . Howevervailable .Homo, Fugu, Ciona, Drosophila and Caenorhabditis homologs of ADAMTS genes, in attempt to corroborate and further elucidate their evolutionary relationships, as shown in Figure ADAMTS1, 4, 5, 8 and 15 of vertebrates and Ciona reveals that this subgroup of genes evolved by retrotransposition early during chordate evolution, was repopulated by new introns , and subsequently underwent gene duplication during the evolution of vertebrates.We compared the positions of introns and their phases between The phylogenetic analysis of animal ADAMTS homologs reveals that proteins that are known to have similar biological activities are closely related, and that they have evolved by a series of gene duplication events Figure . Since tADAMTS homologs have undergone several gene duplications. Assuming that the Ecdysozoa hypothesis is true and arthropods and nematodes are united as a group [ADAMTS gene duplication preceded the divergences of Ecdysozoa and chordates. At least 3–4 ADAMTS gene duplications occurred in chordates prior to the divergence of Ciona and vertebrates, followed by additional gene duplications in vertebrates prior to the divergence of Fugu and mammals .ADAMTS1, -4, -5, -8 and -15 and their single sister in Ciona. Vertebrate members of this group share unique intron positions and lack all of the intron positions held by other ADAMTS genes and their invertebrate homologs  encode proteins with aggrecanase and angiogenesis-related functions, which suggests the examination of ADAMTS5 and -15 for similar biological activities. This putative "angiogenesis/aggrecanase group" appears most closely related to ADAMTS20 and -9. Further, the unique intron positions shared by ADAMTS1, -4, -5, -8, and -15, and lack of invertebrate orthologs in this putative "angiogenesis/aggrecanase group" suggest that this group's progenitor arose within chordates via a retrotransposition event from the common ancestor of the group comprised by ADAMTS20 and -9  we see evidence of acquisition of a new intron following the process of duplication, but prior to the divergence of mammals and pufferfish.A second evolutionarily related group is comprised of s Figure . Three mTS1 gene , and ourADAMTS20 and -9 are most closely related and, along with the members of the angiogenesis/aggrecanase clade, are most closely related to invertebrate gon-1 (Caenorhabditis) and CG6107 (Drosophila). The finding that ADAMTS9 and -20 together as a group have a single sister gene in Ciona indicates that they evolved by gene duplication in the vertebrate lineage, after their divergence from urochordates. However, the results of our phylogenetic analyses demonstrate that neither ADAMTS9 nor ADAMTS20 can alone be rightfully dubbed as being orthologous to gon-1, as has been recently proposed [gon-1 and CG6107 are likely orthologs, the chordate ortholog of these genes was the common ancestor of Ciona ADAMTS9 and -15, i.e. also the common ancestor of the later-duplicated vertebrate ADAMTS genes ADAMTS9, -20, -15, -5, -8, -4, and -1  was found that grouped with the remainder of the human ADAMTS homologs. The placement of this gene with or within a group of these remaining ADAMTS genes would suggest the number of gene duplication events in this gene family that occurred prior to the divergence of vertebrates and invertebrates from a common ancestor. The Kishino-Hasegawa test revealed that the likelihood of Drosophila CG4096 being most closely related to an ancestor of the group of all remaining mammalian ADAMTS proteins, or of the various groups nested within it, was not significantly different from the likelihood that Drosophila CG4096 is most closely related to ADAMTS 7 and -12 (data not shown). The most parsimonious explanation of this result is that a single gene duplication of an ancient ADAMTS homolog occurred early during the evolution of animals, prior to the divergence of chordates from invertebrates, followed by lineage-specific gene loss and gene duplications .Only one invertebrate sequence XCG motif 5' and the W4XW motif at the 3' end. This exon structure may facilitate the formation of alternately spliced isoforms, such as we describe here, but it would also lend itself to duplication or loss of individual repeats. However, the number of variant TSRs in the tails of these proteins has been conserved for the gene pairs ADAMTS17 or -19 in the pufferfish genome  This gene was involved in a duplicative chromosomal inversion, and later a duplication of the chromosomal segment containing both ADAMTS genes. (v) Another intron was gained, in ADAMTS8, prior to the divergence of the pufferfish and mammalian lineages. In the other branch of the ADAMTS family, we see a remarkable frequency of genes located on chromosome 5, suggesting it as the location of the ancestor of these genes. We can speculate that a similar scenario of within-chromosome duplication followed by duplication of chromosomal segments took place, although none are in as close physical proximity as the ADAMTS1-subfamily genes.Thus, by combining the phylogenetic analysis and intron/exon structure determinations, we are able to propose the following series of events leading to the ADAMTS protein family Figure : (i) Theears ago . (ii) Inears ago , multiplThis comprehensive bioinformatic survey of the human genome affirms the widely held belief, derived from experimental work, that the nineteen known human ADAMTS proteins constitute the complete gene family. By examining both the amino acid sequences using rigorous phylogenetic methods and comparing the exon structure of these proteins, we were able to draw conclusions about the evolutionary history of this family of proteins which, in turn, provides a framework for further analysis of the functions of these clinically-relevant genes.ADAMTS homolog sequences presented here were identified from 2001 to 2004 using genomic sequences cataloged in the NCBI, JGI , with reference to protein multiple sequence alignments. We used the same method to identify Caenorhabditis elegans, Drosophila melanogaster, and Fugu rubripes homologs of ADAMTS genes, and their genomic structure. Multiple sequence alignments of the inferred amino acid sequences were prepared using Multalin [Sequences of nd Ciona ,70) and nd Ciona . We searMultalin  and ClusMultalin  and manuMultalin  and MacCMultalin .Drosophila melanogaster and four from Caenorhabditis elegans ADAMTS ADAM (Thrombospondin type 1 Sequence Repeat)TSR MMP (extracellular matrix)ECM SAGE (thrombotic thrombocytopenic purpura)TTP (interleukin)IL (transforming growth factor)TGF (tumor necrosis factor)TNF BLAST (ACN and EGVM initiated the project and all authors were involved in the design phases. ACN, SBM and JML inferred sequences of previously un-annotated ADAMTS genes from Genbank, Celera, and JGI, and determined intron positions. SBM performed the phylogenetic analyses. ACN and SBM drafted the manuscript and JML and EGVM contributed to writing the paper and advised throughout.Alignment used for phylogenetic analyses of animal ADAMTS homologs. Unambiguously aligned amino acid sites, indicated by the "mask" line in the alignment, were used for phylogenetic analyses (Figure s Figure . Accessis Figure .Click here for fileAlignment used for phylogenetic analyses of human ADAMTS proteins and invertebrate homologs. Unambiguously aligned amino acid sites, indicated by the "mask" line in the alignment, were used for phylogenetic analyses (figure s figure . Intron s figure  and 4.Click here for file


Xenoestrogens and antifungal azoles probably share a common route of metabolism, through hepatic cytochrome P450 (CYP) enzymes. Chemical interactions with metabolic pathways may affect clearance of both xenobiotics and endobiotics. This study was carried out to identify possible chemical interactions by those substances on CYP1A and CYP3A, in Atlantic cod liver. We investigated effects of two xenoestrogens (nonylphenol and ethynylestradiol) and of the model imidazole ketoconazole, alone and in combination.O-deethylase (EROD) activity. Treatment with nonylphenol resulted in 40% reduction of CYP1A activity. Combined exposure to ketoconazole and nonylphenol resulted in 70% induction of CYP1A activities and 93% increase in CYP1A protein levels. Ketoconazole and nonylphenol alone or in combination had no effect on CYP3A expression, as analyzed by western blots. However, 2-dimensional (2D) gel electrophoresis revealed the presence of two CYP3A-immunoreactive proteins, with a more basic isoform induced by ketoconazole. Treatment with ketoconazole and nonylphenol alone resulted in 54% and 35% reduction of the CYP3A-mediated benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD) activity. Combined exposure of ketoconazole and nonylphenol resulted in 98% decrease in CYP3A activity. This decrease was greater than the additive effect of each compound alone. In vitro studies revealed that ketoconazole was a potent non-competitive inhibitor of both CYP1A and CYP3A activities and that nonylphenol selectively non-competitively inhibited CYP1A activity. Treatment with ethynylestradiol resulted in 46% decrease in CYP3A activity and 22% decrease in protein expression in vivo. In vitro inhibition studies in liver microsomes showed that ethynylestradiol acted as a non-competitive inhibitor of CYP1A activity and as an uncompetitive inhibitor of CYP3A activity.Treatment with ketoconazole resulted in 60% increase in CYP1A-mediated ethoxyresorufin-Ketoconazole, nonylphenol and ethynylestradiol all interacted with CYP1A and CYP3A activities and protein expression in Atlantic cod. However, mechanisms of interactions on CYP1A and CYP3A differ between theses substances and combined exposure had different effects than exposure to single compounds. Thus, CYP1A and CYP3A mediated clearance may be impaired in situations of mixed exposure to those types of compounds. Gadus morhua).A great challenge in pharmacology and toxicology is to understand the molecular mechanisms behind how mixtures of compounds affect living organisms. This study focuses on two classes of substances, imidazoles and xenoestrogens, and how these chemicals alone and in combination affect hepatic drug-metabolizing hepatic cytochrome P450 (CYP) enzymes – specifically, CYP1A and CYP3A enzymes, in juvenile Atlantic cod (a]pyrene in gizzard shad (Dorosoma cepedianum) -1-propanesulfonate (CHAPS), precision protein standards (low range) and supported nitrocellulose membrane (0.45 μm) were purchased from BioRad . The 17β-estradiol and testosterone enzyme immuno assay (EIA) kits were purchased from Cayman Chemical . The 11-keto-testosterone EIA kit and the Atlantic cod vitellogenin Enzyme Linked ImmunoSorbent Assay (ELISA) kit were obtained from Biosense Laboratories AS . HRP-conjugated donkey-anti-rabbit IgG, the ECL™ Western blotting detection reagents and Immobiline™ DryStrip 7 cm ranging from pH 4 to 7 were from Amersham Biosciences . Ampholytes for isoelectric focusing  was purchased from Serva Feinbiochemica . Dithiothreitol (DTT), Kodak X-Omat AR-ray film, X-ray developer and fix were from VWR International . The 7-benzyloxy-4-[trifluoromethyl] coumarin (BFC), 7-hydroxy-4-[trifluoromethyl] coumarin (HFC) and the CYP3A4 inhibition kit were from BD Biosciences Company, Gentest™ . All other chemicals used were of the purest grade available in Sweden or Norway, from Sigma-Aldrich, BioRad and VWR international.The 4-nonylphenol and the 17α-ethynylestradiol, for the 3 indoor glass fibre tanks, at Industrial Laboratory (ILAB), Bergen High Technology Centre , provided with continuously flowing seawater at a temperature of 8 ± 0.5°C and a salinity of 3.4%. Throughout the experimental period, the fish were subjected to continuous 24 h artificial light . The fish were acclimated to these conditions for five days prior to the experimental period. During the experimental period the fish were starved and i.p. injected with either 12 mg ketoconazole/kg b.w. resuspended in peanut oil (2.5 mg/ml); 25 mg nonylphenol/kg b.w. dissolved in peanut oil (5.0 mg/ml); 5 mg ethynylestradiol/kg b.w. dissolved in peanut oil (1.0 mg/ml) or a mixture of ketoconazole and nonylphenol (12 mg ketoconazole + 25 mg nonylphenol/kg b.w. in peanut oil). Control fish were injected with 5 ml peanut oil/kg b.w. (vehicle). There were eight to nine fish in each treatment group. When designing the experiment, we could only test one combination due to limited fish numbers. We selected nonylphenol to combine with ketoconazole because a previous study showed that, in Atlantic cod, alkylphenols affect CYP1A/3A more strongly than the natural estrogen 17β-estradiol [Hatchery reared juvenile Atlantic cod of both sexes with an average body weight (b.w.) around 400 g were supplied by Sekkingstad, Preserving AS, Hordaland, Norway. The fish were kept in 0.5 mstradiol . The ketstradiol . The nonstradiol .After five days exposure, the fish were sacrificed by a sharp blow to the head. Blood samples were collected from the dorsal vein using a heparinized syringe and the liver was quickly dissected out and placed in ice-cold homogenization buffer . Liver microsomes were prepared according to the published protocol by Goksøyr , and stoin vitro inhibition studies, feral Atlantic cod of both sexes were caught off the West coast of Sweden and placed in concrete basins provided with recirculating aerated seawater at 10 ± 2°C and a salinity of 3.0% and alternative light/dark photoperiods of 12 hours. Prior to sampling, the animals were starved and acclimated to these conditions for three weeks. Eight fish were injected i.p. with β-naphthoflavone (BNF), 50 mg/kg b.w. dissolved in peanut oil (5.0 mg/ml). The fish were placed in a 100 l glass aquarium provided with aerated seawater (above) and 30% of the water volume was replaced each day. To eliminate visual stress, the sides of the aquaria were covered with black plastic sheets. After 3 days exposure, the fish were sacrificed. Livers were quickly dissected out and placed in ice-cold homogenization buffer. Livers were pooled from twenty untreated Atlantic cod and from eight BNF treated Atlantic cod, respectively. Microsomal fractions were isolated (above) and stored in aliquots at -80°C. Ethical approval from the Ethical committee of Göteborg license number (99–2003). The duration of exposure was decided according to results from previous time-course studies showing maximal CYP1A protein and EROD activities in rainbow trout and in the marine viviparous blenny (Zoarces viviparous), 3 days post-injection with either the prototypical CYP1A inducers BNF or 3-methylcholanthrene [For anthrene -54.Western blot analyses of 40 μg hepatic microsomal protein were carried out using enhanced chemoluminescence (ECL), based on the protocol previously described  and PAb The 2D gel electrophoresis was performed using immobilised pH gradient gels with linear gradient from pH 4 to 7. The samples were concentrated by acetone precipitation and pellets dissolved in rehydration buffer  to a final protein concentration of 20 μg/μl or 80 μg/μl. The samples were rehydrated overnight followed by isoelectric focusing for 2.5 h. The rehydration was passive and carried out overnight in an Immobiline Dry Strip reswelling tray (Amersham Biosciences). First-dimension isoelectric focusing (IEF) was performed on a Multiphor II unit (Amersham Biosciences) at 20°C using a MultiDrive XL power supply (Pharmacia LKB). Settings for IEF were 30 min at 100 V and 3 h at 3500 V for a total of 10,520 Vh. Amperage and wattage were set to 2 mA and 5 W. The proteins were resolved on 9% continuous acrylamide gel in Tris:HCl, including sodium dodecyl sulphate polyacrylamide using a mini-gel apparatus from BioRad at 200 V for 45 min. Each sample consisted of pooled liver microsomes from eight to nine fish from each treatment group. Gels loaded with 25 μg microsomal protein were initially stained with 0.1 % (w/v) Coomassie brilliant blue, and then destained, followed by silver staining. The latter was performed according to Heukeshoven and Dernick . Stainedet al. [et al. [The CYP1A activity was determined as EROD activity, using resorufin as standard in a SpectraFluor plate reader according to the protocol provided by Nilsen et al. . The CYP [et al.  and optiIn vitro inhibition studies were carried out in 96-multiwell plates using a VICTOR™ 1420 Multilabel Counter. The IC50 values were determined for nonylphenol, ethynylestradiol, ketoconazole and the ketoconazole:nonylphenol (1:5) mixture on CYP1A and CYP3A activities. The substances were dissolved in DMSO and diluted with ethanol. The final concentrations never exceeded 0.01% (v/v) DMSO and 0.001% ethanol (v/v). For CYP1A and CYP3A inhibition studies, pooled liver microsomes from BNF-treated and from untreated Atlantic cod, respectively, were used. For comparison, the IC50 values for ketoconazole, nonylphenol and ethynylestradiol were determined in cDNA expressed human CYP3A4 baculovirus supersomes using the CYP3A4 inhibition kit from BD Gentest.Pooled liver microsomes from untreated Atlantic cod were pre-incubated for 10, 30 and 60 min, at room temperature, with ethynylestradiol and ketoconazole following CYP3A western blot analysis. The reaction mixture consisted of microsomes (2.5 or 5.0 mg protein/ml) and various concentrations of ethynylestradiol  or ketoconazole (0.3 and 1.0 μM) ± 3 μM NADPH in a total volume of 50 μl in homogenization buffer, containing 20% (v/v) glycerol. The CYP3A western blot analysis was performed as described above. Ethynylestradiol and ketoconazole were dissolved in acetonitrile (vehicle) and the final acetonitrile concentration in the reaction mixture was 0.02% (v/v).492 after 15 min incubation with substrate solution, using a VICTOR™ 1420 Multilabel Counter.Plasma levels of vitellogenin protein were determined using a non-competitive sandwich ELISA kit and employing rabbit PAb against Atlantic cod vitellogenin from Biosense Laboratories AS  . Each pl405 after 40 min (17β-estradiol), 60 min (testosterone) or 80 min (11-keto-testosterone) incubation with substrate solution, using a VICTOR™ 1420 Multilabel Counter.Plasma levels of 17β-estradiol and testosterone were determined using competitive EIA kits from Cayman Chemical . Plasma levels of 11-keto-testosterone were analyzed using a competitive EIA kit, from Biosense Laboratories AS . Plasma from each fish was concentrated (2:1) by extraction once with six volumes diethyl ether and 50 μl was analyzed and compared to purified standard substances. The signals were detected at AData were tested for homogeneity of variances using the Levene's test. When there was homogeneity of variances we used a parametric one-way ANOVA, followed by Scheffé post hoc test. When there was no homogeneity of variances we used the non-parametric Kruskal-Wallis ANOVA, followed by the two-tailed Mann-Whitney U test. No values were log transformed. Data are presented as means (n = 6–9 fish) accompanied with the standard deviations (SD). The significance level (α) was set at 0.05. The statistical analyses were performed using SPSS 11.0 software, from SPSS Sweden AB .LH performed most of the analyses, participated in fish exposure, sampling, experimental design and drafted the manuscript. BEG assisted with experimental design, 2D-analysis and writing. AG participated in experimental design and writing. MCC rose funding, coordinated, participated in fish exposure, sampling, experimental design and writing.


In 1995, the first complete bacterial genome sequence was published. Now, nearly 200 bacterial genomes have been completed, and a new one hits the scientific press most weeks. This burgeoning industry is not just scientific “stamp collecting,” however. Having all these genome sequences may provide useful clues about why some bacteria cause human disease, how to control their spread, and how to treat the infections caused by them. By comparing genome sequences, scientists can learn much more about what makes a bacteria tick than they can learn from a single sequence.Campylobacter. Infection with a Campylobacter species is one of the most common causes of human bacterial gastroenteritis. In the US, 15 out of every 100,000 people are diagnosed with campylobacteriosis every year, and with many cases going unreported, up to 0.5% of the general population may unknowingly harbor Campylobacter in their gut annually. Diarrhea, cramps, abdominal pain, and fever develop within 2–5 days of picking up a pathogenic Campylobacter species, and in most people, the illness lasts for 7–10 days. But the infection can sometimes be fatal, and some individuals develop Guillain-Barré syndrome, in which the nerves that join the spinal cord and brain to the rest of the body are damaged, sometimes permanently.Derrick Fouts and his colleagues have taken this comparative approach with C. jejuni, a spiral-shaped bacterium normally found in cattle, swine, and birds, where it causes no problems. But the illness can also be caused by C. coli , C. upsaliensis (found in cats and dogs), and C. lari (present in seabirds in particular). Disease-causing bacteria generally get into people via contaminated food, often undercooked or poorly handled poultry, although contact with contaminated water, livestock, or household pets can also cause disease.Campylobacteriosis is usually caused by C. jejuni was the first food-borne pathogen to be completely sequenced, but we still know little about how Campylobacter species cause disease. In their search for clues, Derrick Fouts and coworkers have completely sequenced the genome of C. jejuni strain RM1221 (isolated from a chicken carcass) and compared it with the previously sequenced C. jejuni strain NCTC 11168 and with the unfinished sequences of C. coli strain RM2228 (a multi-drug-resistant chicken isolate), C. lari strain RM2100 , and C. upsaliensis strain RM3195 (taken from a patient with Guillain-Barré syndrome).In 2000, Campylobacter strains and species. For example, there are major structural differences between the genomes caused by the insertion of new stretches of DNA. Some of these pieces of DNA may carry genes that improve bacterial virulence or fitness, so their presence could help to explain the different biological behaviors of these strains. There are also major variations in the genes responsible for synthesis of molecules that are important for the interaction of Campylobacter with the environment. Such differences could underlie the host specificity of the different species.The researchers describe numerous differences and similarities between these different Campylobacter species in genes that are likely to be involved in aspects of bacterial virulence, such as adherence, motility, and toxin formation, are all detailed by Fouts et al., who also describe a new putative Campylobacter virulence locus. Further work is needed to relate these genomic differences to functional differences, but this detailed comparative genomic analysis provides the core blueprint for this important family of human pathogens. And in doing so, it lays the foundation for the development of new ways to monitor and control Campylobacter in the food chain and in human infection.Differences between the


Immunity & Ageing is a new Open Access, peer reviewed journal that aims to cover all the topics dealing with innate and clonotypic immunity which are relevant to ageing. The journal will provide an opportunity to focus on this topic, which is emerging as one of the critical mechanisms of ageing. Furthermore, as an online, Open Access journal, Immunity & Ageing will promote immediate accessibility to research, which is generally not possible for articles published in printed journals. We hope this forum, concentrating on the themes of ageing and immunology with a strong focus on human studies, will create a new perspective for viewing a world that is inevitably becoming older.In the elderly, many alterations of both innate and clonotypic immunity have been described. Alterations to the immune system in the elderly are generally viewed as a deterioration of immunity, leading to the use of the term immunosenescence. However, although many immunological parameters are often notably reduced in the elderly, retained function of both innate and clonotypic immunity in the elderly is tightly correlated to health status. Recognising the important role of the immune system in ageing, over the last few years, journals oriented towards gerontology and geriatric sciences have increasingly published articles dealing with the immunology of ageing, but a specialised journal in this area does not exist.  Immunity & Ageing is a new Open Access, peer reviewed journal that aims to provide a forum for articles examining ageing from an immunological point of view.During the past century, humans have gained more years of average life expectancy than in the last 10,000 years; we are now living in a rapidly ageing world. The sharp rise in life expectancy, coupled to a steady decline in birth rates in all developed countries, has led to an unprecedented demographic revolution characterized by an explosive growth in the number and proportion of older people. The number of people aged 60 years or older exceeded 635 million in 2002, and is expected to grow to nearly 2 billion by 2050. The proportion of people aged 60 and over stands about 1 in 4 in many Western European countries as well as in Japan. Should the present trend continues, this ratio is expected to reach 1 in 3 by 2050 [th century, mortality was described as increasing exponentially with respect to progression through the lifespan [Ageing is a post-maturational process that, due to a diminished homeostatic capacity and increased vulnerability, reduces responsiveness to environmental stimuli and is generally associated with an increased predisposition to illness and death. At the beginning of the 19lifespan . This trlifespan . Thus falifespan ,5. So, aIn the elderly, many alterations in innate and clonotypic immunity have been described and viewed as deleterious, hence the term immunosenescence. In 1969, Roy Walford published his landmark book, "The Immunologic Theory of Aging", and first coined the term immunosenescence ,8. HowevThe process of maintaining life for the individual is a constant struggle to preserve his/her integrity. This can come at a price when immunity is involved, namely systemic inflammation . InflammRecently, longitudinal studies have shown that a cluster of immunological parameters can be used to evaluate the expectation and quality of life, i.e. the immunological risk phenotype ,18. SeneIn contrast to T cells, no evidence for a loss of B cell function has been found as neither the total number of B cells or immunoglobulin secreting cells have been shown to be profoundly decreased with age. However, the B-cell repertoire is influenced by ageing during an actual immune response, where the spectrum of expressed immunoglobulin genes, as well as the frequency of somatic mutations, affects the quality, though not necessarily the quantity, of the antibody response, which is highly relevant in clinical practice ,21.Immunity & Ageing was conceived to cover all topics relevant to immunity and ageing in an interdisciplinary manner. It is clear that immunological mechanisms are involved in process and manifestation of ageing in many systems. An example is displayed by the increase in serum and tissue levels of circulation pro-inflammatory cytokines which is accompanied by typical cellular ageing phenomena such as telomeric loss, oxidative damage, DNA defects, accumulation of advanced glycosylation products, cellular loss and others. These defects may be stimulators of cytokine secretion, and subsequently, cytokines are released into the systemic circulation. This results in a slowly progressive endo-crinosenescence and neurosenescence. The different factors may influence each other in form of a vicious spiral [Considering the paramount function of the immune system during ageing, journals oriented towards gerontology and geriatric sciences are now publishing an increasing number of articles dealing with immunology and ageing, but a specialised journal in this area does not exist. s spiral .The journal will provide an opportunity to focus the topic of immunology of ageing, which is emerging as one of the critical mechanisms of ageing which aspects should get to all scientists physicians and other professions who are involved in the different inter-related disciplines, and the bridge that is emerging between these fields.Immunity & Ageing will promote "immediate" accessibility to this fast moving area of research, which is generally not possible for articles published in printed journals. From a modern perspective, electronic publishing is obviously attractive for its speed, easy global access and low cost, which all present considerable advantages over print and are seen as attractive factors by authors, readers and publishers. Open Access appears to provide exactly what people would like: rapid publication for authors, free access to information for readers, and inexpensive, global availability to readers for publishers [Immunity & Ageing will take advantage of the Open Access policy to make peer-reviewed information widely and almost immediately available. We hope this forum, focussing on the themes of ageing and immunology, will create a new perspective for looking at a world that is inevitably becoming older. Creating and pushing forward a research knowledge base in Immunity & Ageing provides a unique opportunity to dissect out some parameters which might make health span equate with increasing lifespan.Furthermore, as an online, Open Access journal, blishers . We beli