E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes.Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin  with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands.C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide modified antigens for immunological studies and vaccination, including live subunit vaccines, and might be useful to co-express MOMP with other chlamydial membrane proteins. Chlamydiales is an obligate intracellular pathogen  undeC. trachomatis MOMP is highly immunogenic. Antibodies to the protein neutralised EB infectivity . Howectivity , with THecretion . C. tracCh. psittaci (formerly known as C. psittaci) OmcA is a 9 kDa lipid-anchored protein with 14 cysteine residues )pAb Fig. .The absence of a signal from control cells and cells expressing MOMP in its non-leadered, mature form confirmed the incubation and blotting conditions did not cause cell lysis and expose unincorporated protein. Both OmpT- and native-leadered MOMP were detected on the cell surface Fig.  showed ME. coli, cross the inner membrane, and be processed in the periplasm. Furthermore, under modified incubation and induction conditions , MOMP could be folded and incorporated into the outer membrane.We concluded that MOMP constructs encoding appropriate leaders could be expressed in C. trachomatis MOMP was inserted into the OM of E. coli cells, we set out to investigate how the protein was organized in the membrane. While noting that predictive algorithms must always be deployed with care, and with reference to established findings for a given protein, we first analyzed MOMP's primary sequence for membrane crossings using a neural network trained with OM proteins of known structure [C. trachomatis MOMP generally corresponded to regions of the protein predicted to be extracellular.Having confirmed that tructure . The anaWe then reanalyzed the sequence using two β-strand prediction programs Fig. . The comC. trachomatis MOMP constructs  in which substantial regions of VS domains 1, 2, 3 or 4  buffer , or the addition of 2 mM EDTA, failed to "unmask" any immunoreactivity  was -31 ± 1.5 mV . This corresponded to a relative cation vs anion selectivity of 3.8 under these specific ionic conditions. Control preparations (detailed under Methods), including membrane proteins from control BL21omp8 cells subjected to the same experimental conditions, where OM proteins were solubilised and subjected to GE chromatography in exactly the same way, did not give rise to similar channel activity (6 experiments).Fully processed and correctly folded MOMP should function as a porin-like ion channel . We test or OmpC . We thenC. trachomatis MOMP at the single-channel (single molecule) level from cells lacking many endogenous porins provides very strong evidence that MOMP adopted its native fold when expressed in E. coli under suitable conditions. Although a leadered version of recombinant chlamydial MOMP was expressed and functionally analysed previously [E. coli porins for liposome-swelling studies. Although MOMP may have contributed additional porin-like activity, functional modification of endogenous porins could not be ruled out.Functional reconstitution of recombinant eviously , membranE. coli cells depends on the precise leader sequence, as well as on the specific protein. PorB is less "toxic" with its native leader, in contrast to MOMP, which is less "toxic" with the E. coli OmpT leader, and native-leadered C. muridarum MOMP is less deleterious to E. coli than Ch. abortus MOMP. Although a full investigation of the role of leader sequences could not be undertaken here, it is known that successful OM insertion, as well as prior transport across the inner membrane and processing, is also signal sequence-dependent. For example, a large proportion of E. coli LamB porins with signal sequence mutations remained "tethered" to the inner membrane , even though the protein was also closely associated with the OM [C. trachomatis MOMP, use of the Omp-T leader and induction at 16°C , in either "wild-type" cells or "porin knockout" cells in a supportive medium (SOC), provides improved processing and OM insertion, and there is also significant insertion at 25°C in "wild-type" E. coli.Interestingly, the successful expression and processing of recombinant chlamydial porins in h the OM . For C. C. trachomatis MOMP are consistent with previous data on bacterial [The single-channel properties of acterial  and putaacterial  porins. acterial , and pooC. trachomatis MOMP, like putative porins in the intracellular pathogens Burkholderia thailandensis and B. pseudomallei [Despite the lack of sequence similarity to known bacterial porins, a combination of different predictive approaches (none of which was entirely satisfactory in isolation), set in the context of elegant and pioneering work from many laboratories on the properties of VS domains, predicted that domallei , could bdomallei ,40. The C. trachomatis MOMP differs in some significant respects from the recent prediction for C. muridarum MOMP [C. muridarum prediction. This would preclude the significant interactions with OmcB and OmcA, described in the Background. We also assigned L2, 4, 6 and 7 as C. trachomatis VS domains, not L2, 3, 5 and 6, the homologous regions in C. muridarum MOMP. Experimental tests of the predicted membrane topology of C. trachomatis MOMP are consistent with our model, because individual VS domains can be substantially truncated without preventing incorporation of the protein into the bacterial OM. If MOMP is a β-barrel porin, as suggested, and VS domains are confined to specific extracellular loops, it is conceivable that MOMP can continue to fold into a β-barrel in the absence of one of these domains. On the other hand, the removal of β-strands would disrupt folding. Removal of a single strand, bringing periplasmic residues into direct contact with external residues, is predicted to be particularly destructive to the global fold. Removal of more than one strand might be better tolerated, provided the β-barrel can form with a significantly reduced diameter. In practice, it appears that C. trachomatis MOMP cannot accommodate either type of strand modification.Our working model for the membrane topology of rum MOMP  . HoweveCh. abortus MOMP forms SDS-resistant oligomers of ~100 K [E. coli OMs. We speculate that this may be because native MOMP oligomers are stabilised by interactions with other chlamydial components , and poond Fig. .C. trachomatis MOMP, an immunodominant, cysteine-rich, chlamydial surface protein of crucial importance in the immune response to infection, is a major subunit vaccine target. However, unlike many other bacterial porins, it has been difficult to refold from inclusion bodies or to achieve and demonstrate functional surface expression. This study is the first to report unambiguous functional analysis, by single-channel recording, of recombinant chlamydial MOMP recovered from bacterial outer membranes. The modified expression system described in the present study provided a means to test specific hypotheses provided by a working model for the C. trachomatis protein. However, although our results are consistent with a working model of MOMP as a 16-stranded β-barrel, more mutations or other approaches are needed before a specific model can be accepted. The protein also formed oligomers, even in the complete absence of cysteine residues. The surface display of modified, functional MOMP in E. coli cells , together with a working topological model, could guide the removal of unwanted or harmful epitopes from engineered proteins, and it might also be possible to display external loops containing specific MOMP epitopes on other porin "scaffolds" in living cells. However, it is important to note that such approaches will be limited if essential disulphide bonds in the native chlamydial envelope, including bonds involving non-MOMP cysteines, stabilise the conformation of key immunogenic VS domains.C. trachomatis ompA  and Ch. abortus ompA were cloned without their leader sequences into the Nde-I/Nco-I sites of pET22b(+) (Novagen) after destroying an internal Nde-I site in C. trachomatis ompA by Quik-Change PCR mutagenesis (Stratagene). This did not alter the encoded protein. C. muridarum ompA and porB were amplified with and without their leaders from genomic DNA and cloned into the Nde-I/Bam-HI sites and Nde-I/Nco-I sites, respectively, of the same vector . The E. coli OmpT protease leader sequence or the native C. trachomatis MOMP leader sequence was added to the 5' end of the leaderless C. trachomatis and Ch. abortus inserts by sequential gene extension PCR using three overlapping primers. A 5' Nde-I site was again used to provide the starting methionine codon in the final full-length construct. Quik-Change PCR was also used to create pairs of unique internal restriction sites in native-leadered C. trachomatis ompA to permit the deletion of specific domains by plasmid restriction and religation [Age-I; for VS2, Bcl-I; and for VS3, VS4 and the predicted β-strands, Aat-II. Successful deletions were confirmed by hemi-nested single-colony PCR (using Taq polymerase) to identify clones that could be amplified by gene-spanning primers but not by primers complementary to regions that had been removed. We also generated C. trachomatis MOMP expression constructs containing inserts in which all 9 cysteine residues  were replaced by alanine using Quik-Change PCR. Most of the modifications were carried out in a pSTBlue-I/NovaBlue system, and the fidelity of each insert was confirmed by automated DNA sequencing (MWG Biotech).ligation . These sE. coli BL21(DE3) or BL21(DE3)omp8 [DE3)omp8  cells wevs the logarithm of the molecular mass of Precision Plus unstained protein markers (BioRad). For Western blotting, proteins were electrophoretically transferred to PVDF membranes under conditions compatible with the transfer of high-MW proteins including native MOMP oligomers [C. trachomatis MOMP antibody  for 1 hour at room temperature. Following 2 × 30 sec and 3 × 5 min washes in PBS-T, membranes were incubated in 1:10,000 HRP-conjugated anti-goat/sheep antibody (Sigma) for 1 hr at room temperature. After washing, immunoreactive proteins were detected by ECL.Unless otherwise indicated, SDS-PAGE was carried out under reducing conditions using 10–12% (w/v) gels. Molecular masses were estimated from plots of relative mobility ligomers . The memC. trachomatis MOMP polyclonal antibody as described above. Immunofluorescence was carried out as described previously [10 ml of LB medium was seeded 1:100 with cultures grown to saturation overnight, and incubated until the OD reached 0.6. The cells were pelleted by centrifugation  and resuspended in fresh medium. Following incubation at the selected temperature for 10 mins, 0.1–1 mM IPTG was added and incubation was continued for another 2–16 hrs. Intact cells were harvested by gentle centrifugation  and washed in 1 ml PBS. The pellets were resuspended in 200 μl PBS, and 10 μl was applied to a nitrocellulose membrane and allowed to dry. The membrane was blocked and probed with anti-eviously , with fiVt 320 ml) was calibrated in the presence of detergent using standard proteins. V0 (the void volume) was 115 ml, and Kav was calculated as (Ve - V0)/(Vt - V0), where Ve is the elution volume.Solublised OM proteins were separated by GE chromatography using a high resolution 26/60 HiLoad Superdex 200 prep grade column (Amersham Pharmacia Biotech) freshly equilibrated in 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl and 5 mM DTT (omitting the latter for the cysteine-less MOMP mutant). The buffer also contained either 0.05% (w/v) LDAO or 0.05% (w/v) Zwittergent 3–14. 2 ml aliquots of solubilised OM proteins  were loaded, and the column was eluted with the same buffer for 800 min. at a flow rate of 0.5 ml/min. 5 ml fractions were collected and 10 μl of each protein-containing fraction was deposited onto a pre-prepared PVDF membrane and probed for MOMP as described earlier under Western blotting. The column  were added to the cis chamber, followed by aliquots of 5 M KCl to raise the salt concentration to 500 mM. Channel incorporation usually occurred within 30 min, accelerated by switching the holding potential between +/- 60 mV. Experimental protocols were programmed and the digitised data were low-pass filtered  and recorded using pClamp8 software (Axon Instruments), and analysed offline. The bilayer potential was slowly and repeatedly ramped between -100 mV and +100 mV (each sweep taking 32 s) in the presence of an asymmetric  gradient of KCl, or with equimolar 500 mM or 1 M KCl. At least 3 voltage ramps were recorded and analysed for each experiment, and equilibrium recordings were obtained at defined holding potentials. Holding potentials refer to the cis chamber, and upgoing deflections represent net movement of cations from cis to trans or of anions from trans to cis. Relative ionic permeabilities were determined from the equilibrium solution of the Nernst-Planck flux equations [Planar bilayers were cast from diphytanoyl phosphatidylcholine (Avanti) between two 0.5 ml chambers containing 50 mM KCl, 20 mM Tris-HCl (pH 8.0) and 1 mM DTT, designated nd trans . The cisquations . When thEr is the equilibrium  potential in asymmetric KCl, and R, T and F have their usual significance.  (the permeability ratio of K+ to Cl-) was calculated using appropriate activity coefficients (a) from standard tables. Control experiments were carried out using equivalent amounts of detergent and with equivalent amounts of solubilised OM proteins purified from non-transformed bacteria, selecting identical GE column fractions.C. trachomatis MOMP and the known characteristics of antiparallel, amphipathic β-barrel strands in porins.The number of membrane crossings was predicted using a neural network based-outer membrane protein topology prediction program trained with known porins . We discHEF and HM carried out most of the experiments. HEF analysed and organised the data, and drafted the first version of the manuscript. RHA conceived the overall project, provided experimental guidance, carried out some of the experiments, and redrafted the manuscript. All the authors read and approved the final manuscript.Detergent extraction of recombinant MOMP. Immunoblot with ECL detection following SDS-PAGE of OM proteins (10 μg per lane) from BL21 cells expressing OmpT-leadered C. trachomatis MOMP, induced for 2 hrs at 37°C. OM proteins (see Methods) were solubilised in 1% (w/v or v/v) octylglucoside, Triton X-100, Zwittergent 3–14 or LDAO, as indicated. NB & B are non-boiled and boiled samples, respectively. NE = non-expressing (control) cells.Click here for fileC. trachomatis MOMP expression and processing in BL21omp8 cells. Optimisation of Growth curves of BL21omp8 cells expressing C. trachomatis MOMP with its native leader, in LB or SOC medium .Click here for fileMOMP epitopes are not unmasked by Tris buffer or EDTA in whole cell immunoblots. Control BL21 cells and cells expressing "strand-deleted" constructs were suspended in 100 mM NaCl containing 50 mM Tris-HCl (pH 7.4) with or without 2 mM EDTA, applied to nitrocellulose membranes, and probed with anti-C. trachomatis MOMP polyclonal antibody.Click here for fileRecombinant MOMP does not form SDS-resistant oligomers. SDS-PAGE and immunoblot analysis of C. trachomatis MOMP expressed with its native leader in BL21omp8 cells at 16°C (induced for 12 hrs in the presence of 0.1 mM IPTG). Lanes 1 & 2 contain 10 μg non-boiled and boiled OM proteins, respectively, solubilised in 1% (w/v) OG. Note successful transfer of high-MW proteins.Click here for file


The purpose of this prospective study was to assess the injury rates in male and female adult Canadian Taekwondo athletes relative to total number of injuries, type and body part injured.Subjects  participated in the 1997 Canadian National Taekwondo Championships in Toronto, Canada. Injuries were recorded on an injury form to documents any injury seen and treatment provided by the health care team. These data were later used for this study. The injury form describes the athlete and nature, site, severity and mechanism of the injury.The overall rate of injuries was 62.9/1,000 athlete-exposures (A-E). The males  sustained significantly more injuries than the females . The lower extremities were the most commonly injured body region in the men , followed by the head and neck . Injuries to the spine  were the third most often injured body region in males . All injuries to the women were sustained to the lower extremities. The most common type of injury in women was the contusion . However, men's most common type of injury was the sprain  followed by joint dysfunction . Concussions were only reported in males . Compared to international counterparts, the Canadian men and women recorded lower total injury rates. However, the males incurred more cerebral concussions than their American colleagues .Similar to what was found in previous studies, the current investigation seems to suggest that areas of particular concern for preventive measures involve the head and neck as well as the lower extremities. This is the first paper to identify spinal joint dysfunction. With the inclusion of Taekwondo as a medal sport in the 2000 Olympic Games interest has gained momentum from participants, national governments and scientists alike. With increased participation, the issue of safety becomes very important and timely. The rules and regulations with regards to these Championships were that of the World Taekwondo Federation . All athProspective studies on Taekwondo injuries sustained at single tournaments have been conducted before -3. For iIt is not clear why the men in the studies mentioned above sustained more injuries. Sample size may be a factor. For instance, in a prospective study covering multiple Taekwondo tournaments, Pieter and Zemper  reportedAs expected in a collision sport, the contusion was the most frequently occurring injury type in both male and female Taekwondo athletes -4. At thThe body region most frequently affected in single tournaments involving both recreational and elite Taekwondo athletes is the lower extremities, especially the (instep of the) foot -4. SinceThere is a lack of data on gender differences in injuries to body region and body part in karate. Most prospective studies combined the injury rates for males and females . Using There is also scarce information on male-female comparisons on body regions injured in judo athletes. Our own studies in judo seem to indicate that in the women, the upper extremities are mostly affected, while in the men, the head and neck as well as the lower extremities are injured most often ,12.In line with the frequent use of the legs in Taekwondo, the main injury mechanism was found to be delivering or receiving a kick ,5. FurthThe purpose of this study was to identify and compare the rates of injury in Canadian male and female Taekwondo competitors relative to total number of injuries, type, body part injured and mechanism.Subjects  participated in the 1997 Canadian National Taekwondo Championships in Toronto, Canada. Injuries were recorded on an injury form to document any injury seen and treatment provided by the health care team as it was required by law. The first author was the only person who kept the injury forms and entered the data, therefore, keeping the identity of athletes confidential. Oral consent was obtained from the athletes for assessment and providing therapy. Data describe the athlete and nature, site, severity and mechanism of the injury. No reliability and validity information for the instrument is available and this study was carried out to pre-test the injury data collection form , who has been the national team chiropractic physician for several years and is an experienced (black belt) Taekwondo athlete himself. One injury form was filled out by the attending physician for each time the athlete reported a new injury. However, at each presentation there could be more than one injury reported on the same Injury Report form. For the purposes of this study, an athlete was considered injured if any of the following conditions applied : 1) any Injury rates were calculated from matches fought using the basic rate formula: (# injuries / # athlete-exposures) × 1,000 = # injuries per 1,000 athlete-exposures (A-E). The Colorado concussion classification was utilized in management of the concussions ,17. AccoThe age range for the males was 17–34 years with a mean of 24.2 years and for the females, 16–26 years with a mean of 21 years. The age was not recorded for 3 males and 1 female. Table The lower extremities were the most commonly injured body region in the men , followed by the face , and the spine  . If the head and neck (which includes the face area) are combined, as was done in other studies ,7, this The top five injuries in the males include the sprain , followed by the joint dysfunction , contusion and laceration , and strain . The cerebral concussion is ranked sixth . There were one third degree and two first degree concussions. In the women, the contusion was the most often occurring injury , followed by the sprain and strain  and 90.1/1,000 A-E (women) . At one In view of the nature of the sport, as alluded to above, it is not surprising to find the lower extremities to sustain most of the injuries as was found in previous studies as well ,3,13. WiAs expected, the contusion was found to be the most frequently occurring injury type in other studies on Taekwondo injuries e.g., ,3,13]. T,13. T3,1Of more concern, however, is the occurrence of cerebral concussions. The Canadian males recorded a higher rate than found in American   and GreeIn accordance with what was found previously, the injury mechanisms included both receiving and delivering kicks for men and women alike ,5. The mMore research on Canadian Taekwondo athletes of different age groups and skill levels is needed. Age in the present study is not believed to have played a role in the injuries sustained. The injury profile of the Canadians is quite similar to those found in other studies with Taekwondo athletes in their early twenties ,3,5. ChiJoint dysfunction was identified as the second most common injury sustained by male athletes . Haldeman  defines The total injury rates for the Canadian Taekwondo athletes compare favourably to those reported in the literature, which is contrary to what was expected based on such a small sample size. It is hypothesized that recent rule changes may have contributed to these relatively low rates when compared to those found for other single tournaments, although more research is indicated before a definitive conclusion may be drawn. Interestingly, joint dysfunction was identified for the first time, which warrants more study. The injury data collection form should also include the technique used as a specification of the injury mechanism. General Taekwondo and competition-specific experience in addition to belt rank should also be recorded.None declared.MK collected the data, designed the Injury Report form, and wrote the initial draft of the manuscript. WP did the result part, revised and proof read the manuscript.The pre-publication history for this paper can be accessed here:


Daphnia. Commonly known as the water-flea, Daphnia's ecological merit is unequaled among metazoans, largely because of its sentinel role within freshwater ecosystems and over 200 years of biological investigations. By consequence, the Daphnia Genomics Consortium (DGC) has launched an interdisciplinary research program to create the resources needed to study genes that affect ecological and evolutionary success in natural environments.wFleaBase is a database with the necessary infrastructure to curate, archive and share genetic, molecular and functional genomic data and protocols for an emerging model organism, the microcrustacean These tools include the genome database wFleaBase, which currently contains functions to search and extract information from expressed sequenced tags, genome survey sequences and full genome sequencing projects. This new database is built primarily from core components of the Generic Model Organism Database project, and related bioinformatics tools.Daphnia pulex will be integrated into wFleaBase, including gene predictions and ortholog assignments based on sequence similarities with eukaryote genes of known function. wFleaBase aims to serve a large ecological and evolutionary research community. Our challenge is to rapidly expand its content and to ultimately integrate genetic and functional genomic information with population-level responses to environmental challenges. URL: .Over the coming year, preliminary genetic maps and the nearly complete genomic sequence of  Daphnia is a ubiquitous resident of inland waters within all continents of the globe and is the subject of study for numerous biological disciplines including limnology, ecology, physiology, toxicology, population genetics and evolution. Many attributes make this organism an ideal model for ecological and evolutionary genomics research. As the principal grazers of algae and the primary forage of fish, Daphnia are key members of aquatic food webs and are easily sampled in great numbers. These animals inhabit remarkably diverse environments and show striking patterns of convergent evolution linked to specific habitat transitions [Daphnia offer unprecedented opportunities to study historical responses to environmental change, by harvesting, dating and resurrecting annually sedimented diapausing eggs within lacustrine basins and by competing past products of evolution against their modern descendents [The micro-crustacean nsitions . Their mnsitions . Yet moscendents ,4. For tDaphnia Genomics Consortium [Daphnia specific sequences with standard bioinformatic tools to facilitate gene discovery. This function includes BLAST analyses and links to gene reports for other eukaryotic genomic models via euGenes [wFleaBase is a project of the nsortium  and is d euGenes . However and Internet file transfer at .The main sources of data for wFleaBase are direct submissions from DGC members and from research at large genome sequencing centers. The latest data can be accessed by web browser at This service is built using tested genome database components and open source software that are shared in common with several other databases. Middleware in Perl and Java are added to bring together BLAST, sequence reports, searches and other bioinformatics programs for web access. The Indiana University Genome Informatics Laboratory houses wFleaBase, along with related genome databases FlyBase  and euGeDaphnia's) to be matched by sequence similarity, then annotated with reference gene information. When Daphnia or other new organism sequences are matched to this data, it suggests their gene function and provides starting points for experiments into their ecological and evolutionary genetic significance.To start wFleaBase, we copied a genome database/web server template from Argos infrastructure, including Chado database and genome informatics tools, loaded the database with a first set of sequences, and produced BLAST comparisons of these against 10 other eukaryote genomes from the euGenes project. The euGenes project provides a standard summary of gene and genome information from eukaryotic organisms, and includes over 200,000 named genes and their functions, with 900,000 genome features. This eukaryote genome collection allows new genomes (like Daphnia sequences from other public databases (PB), mitochondrial sequences from molecular systematic studies of the genus (MT) and amplicons of microsatellite DNA markers (MS) are also categorized. To date, wFleaBase contains 14,451 records, including EST (WFes0000001-WFes0012408), GSS (WFgs0000001-WFgs0001495) and MS (WFms0000001-WFms0000548) sequences. Each sequence header provides a short description on the type of sequence, the species and strain from which the sequence was obtained, the library identification code for the cloned fragment with synonyms and contact name. Full contact information is provided elsewhere [Daphnia sequences to the corresponding author, for processing and archiving the data into wFleaBase. Submissions undergo quality assurance checks for vector contamination and correct taxonomy before they enter the database.Sequences are assigned unique and stable accession numbers upon entry into the Chado database, which are organized into seven divisions according to whether they are derived from genome survey sequences (GSS), expressed sequence tags (EST) or high-throughput genomic (HG) and cDNA (HC) projects. D. pulex. Beginning at the welcome page, the user can navigate via the hyperlink located at the top menu towards the Blast page of wFleaBase  to perform sequence-similarity searches on the archived data using the BLAST family of programs. The user enters a nucleotide sequence, whose gene function is well characterized and evolutionarily conserved, with a goal to find the homologous gene in Daphnia. For example, a Drosophila melanogaster mRNA sequence obtained from GenBank (NM 165564) or from FlyBase (FBgn0003082) for the gene photorepair is used to query all Daphnia sequences using the default settings of the tblastx program. Alternatively, the user can select to query species-specific GSS or EST databases. This search retrieves record WFgs0000440, which is a 917 nucleotide sequence with a best match score of 83 bits and an E-value of 5e-45. Using this information, the user can then download the Daphnia sequence onto their personal computers as a text file, design primers using their own software to probe the arrayed Daphnia cosmid library by the Polymerase Chain Reaction (PCR), identify bacterial clones containing the gene, and characterize the entire locus by sequencing. Indeed, this specific exercise identifies at least three cosmids  containing a likely homologue to photorepair from Drosophila [As an information and gene discovery system, wFleaBase focuses on providing efficient tools for searching and retrieving records of interest. Its current features are best highlighted by co-navigating the web pages along with a user interested in locating ecologically relevant genes, for example, genes that confer resistance to elevated levels of ultra-violet radiation encountered by closely related species to osophila . by following the "Genomics" hyperlinks located at the top and side menus. At present, Daphnia EST and GSS sequences are each compared to the protein coding genes and to genomic sequences in euGenes. Many options exist for sorting the BLAST tables. The user can specify what BLAST result columns to show, and can sort these columns based on the ascending or descending order of their entries. The tables can also include BLAST results against all organisms within euGenes or the tables can be filtered to include results from comparisons against a single taxon. For example, the same user, now looking to find a Daphnia homologue to genes known to confer salt-resistance to species inhabiting saline environments, begins by searching for names or euGenes accession numbers of functionally related genes within the Blast tables using the wFleaBase search function located in the top menu of the Blast tables. If the user chooses to search for "ATPα", which is a sodium/potassium-exchanging ATPase shown to be under positive selection in brine shrimp populations adapted to ultra-saline waters [Daphnia sequences via hyperlinks located in the first column of the search results, or further uncover the extent of evolutionary conservation for this gene by examining the euGene Reports, also via hyperlinks located in the last column. Alternatively, if the user chooses to use the FlyBase accession number for this gene (FBgn0002921) to retrieve Daphnia homologues using the search function, the same 11 records are obtained.Returning to the welcome page, the user can instead choose to explore tables containing data extracted from automated BLAST searches against the euGenes database, which includes annotated genome sequences from 10 eukaryotic model organisms. Although this option for gene searching is more tedious, it does allow users to focus precisely on the data currently available in wFleaBase. Four tables of BLAST results are offered at e waters , 11 EST Daphnia genes of ecological interest is limited by the levels of sequence and functional conservation among characterized genes in other model organisms. Work is underway by the DGC to create the required tools for identifying ecologically relevant genes by positional mapping using microsatellite markers. wFleaBase presently archives 528 microsatellite markers [D. pulex and D. magna, wFleaBase integrates a suite of computational programs that (i) identifies microsatellites from raw DNA sequencer trace files, (ii) designs optimal primers for amplifying the markers and (iii) indexes the amplicon, microsatellite motifs and primer information into the Microsat database [Daphnia genome sequencing project.Although effective, the candidate gene approach to finding  markers . Yet, todatabase . The MicDaphnia Medline references and of Daphnia web documents. LuceGene [wFleaBase uses LuceGene to support rapid search and retrieval of the sequence database, of Blast table entries, of LuceGene , based oLuceGene  search sDaphnia into a functional genomics research model, with the added advantage of interpreting observations in the context of natural ecological challenges. wFleaBase is currently designed to facilitate gene discovery for immediate use in research projects. However, the functionality of this service will grow in equal pace with the rapid accumulation of genomics data. Within the next year, this site will host the full genome sequence for D. pulex – a collaborative project involving the U.S. Department of Energy Joint Genome Institute, the Environmental Protection Agency and the DGC. Genetic maps for D. pulex and D. magna are also under construction. Therefore, wFleaBase will soon be enhanced by implementing the CMap module of GMOD and allow users to compare the Daphnia genetic and physical maps emerging from current research and to choose the most appropriate set of markers for quantitative trait locus (QTL) mapping projects. Simultaneously, wFleaBase will also assemble cDNA sequences from another large DGC sequencing effort aimed to document most of the Daphnia transcriptome. Daphnia gene reports will be created in accordance to standards set by current model organism databases [Daphnia genomic information to other model species will be reinforced, allowing greater opportunities to apply the candidate gene approach for identifying genes of ecological importance.The wFleaBase project is young. Its future success is linked to the DGC and its goal to develop atabases . Gbrowse using web browsers and at  by using internet file transfer protocols.wFleaBase is publicly available and can be accessed at JC contributed data, web documentation and aided in the overall design and functionality of this database. VS contributed programming for both the database and Blast searches, and contributed to its design and development. DG contributed the generic database framework design, the overall web structure, the euGenes data and built GMOD database and Blast tools. All authors read and approved the final manuscript.


The CBFA2T3 locus located on the human chromosome region 16q24.3 is frequently deleted in breast tumors. CBFA2T3 gene expression levels are aberrant in breast tumor cell lines and the CBFA2T3B isoform is a potential tumor suppressor gene. In the absence of identified mutations to further support a role for this gene in tumorigenesis, we explored whether the CBFA2T3B promoter region is aberrantly methylated and whether this correlates with expression.Aberrant hypo and hypermethylation of the CBFA2T3B promoter was detected in breast tumor cell lines and primary breast tumor samples relative to methylation index interquartile ranges in normal breast counterpart and normal whole blood samples. A statistically significant inverse correlation between aberrant CBFA2T3B promoter methylation and gene expression was established.CBFA2T3B is a potential breast tumor suppressor gene affected by aberrant promoter methylation and gene expression. The methylation levels were quantitated using a second-round real-time methylation-specific PCR assay. The detection of both hypo and hypermethylation is a technicality regarding the methylation methodology. Allelic loss of heterozygosity (LOH) of the human chromosome 16q in several sporadic cancer types, including breast, prostate and ovary cancers, suggests this chromosome arm harbors tumor suppressive loci . The mosCBFA2T3 encodes for a protein that belongs to the eight-twenty-one (ETO) family, which also includes the genes CBFA2T1, CBFA2T2 in mammalian cells and nervy in Drosophila . The mamMutation analysis in breast tumor cell lines and primary breast tumor samples failed to identify any CBFA2T3 sequence aberrations . It was In this study, the methylation status of a high-density CpG island promoter region located five prime to the exon 1B sequence of the CBFA2T3B transcript is described Figure . We explCBFA2T3 encodes two alternative transcripts, CBFA2T3A and CBFA2T3B Figure . It was Based on the idea that this altered expression might develop via aberrant methylation, the promoter activity and methylation status of a high-density CpG island located five prime to the exon 1B sequence of CBFA2T3B was examined Figures  and 1B. To understand this complex promoter methylation, sodium bisulfite sequencing of the hypermethylated breast tumor samples was used to examine the pattern and frequency of methylation with this region. This analysis revealed that specific cytosines appeared more susceptible to methylation compared to others Figure . A charaTo assay CBFA2T3B promoter methylation levels at this Sp1 site, a bisulfite sequencing amplicon spanning this region was initially PCR amplified and column purified from 24 breast tumor cell lines, 55 primary breast tumors, 22 normal breast counterparts and 46 normal whole blood samples. Second-round real-time MSP was performed on the amplicons using internal forward primers to detect for either unmethylated or methylated cytosines at the Sp1 site. Standard curve dilutions of previously prepared internally primed clones representative of either unmethylated or methylated sequence were used to extrapolate methylation levels and normalize for differences in amplification efficiencies. The methylated cytosines were expressed as a fractional ratio of unmethylated cytosines to determine the methylation indices [mi = m/(m + u)]. On plotting the indices, a clear difference between the tumor, normal groups and complex promoter methylation levels was revealed Figure  and 4.2 = 0.63; r = -0.8, P = .0002). Based on the possibility that five prime RNA degradation and secondary structures may have affected the exon 1B complementary DNA (cDNA) synthesis, a correlation between methylation and the total expression is alternatively shown . At a hypermethylated index of around 0.9, approximately 4 mRNA molecules (mlcls) per 104 cells were detected compared to a hypomethylated index of 0.0001 and 120,000 mRNA mlcls per 104 cells. The number of CBFA2T3B promoter mlcls methylated per cell for each breast tumor cell line was also calculated by multiplying the methylation index values by the number of 16q24.3 DNA mlcls per cell as previously determined by FISH  and absolute methylation ratios (u/m). The methylation ratios are simply an alternative way of expressing the methylation data . The calThus, by using this method it was possible to detect CBFA2T3B promoter methylation in all samples. This phenomenon may actually be widespread for genes under the control of methylation as in accordance with the replication model of maintenance methylation -20. ThisBased on the idea that promoter methylation may occur ubiquitously, the concept arises; what is hypo and hypermethylation. In this study, it was demonstrated that hypo and hypermethylation are merely a prediction of levels outside a majority or 'interquartile range' of methylation levels found in the normal samples. For instance, of all the methylation levels detected in the cell lines, only 37.5% of these were predicted as either hypo or hypermethylated relative to the interquartile ranges of normal breast methylation. Moreover, the number of predicted hypo and hypermethylated samples are much lower when compared to the 'full range' of normal methylation ratios as shown in The phenomenon hypomethylation is a reflection of the low methylation levels that can be detected in the 'hypomethylated' samples relative to the methylated and hypermethylated samples. Alternatively, the phenomenon of hypermethylation is readily observed yet indicates that the detection of methylation alone does not simply represent hypermethylation. The CBFA2T3B gene is methylated in all samples and according to MSP in a high-percentage of samples. In effect, several studies which have examined the methylation status of potential tumor suppressors and cancer-related genes often demonstrate methylation in a high-percentage of samples and state hypermethylation based on the detection of methylation. Examples include, ras-effector nore1A (RASSF1A) ,22, straIn biological terms, several scenarios exist as to how these aberrant methylation levels might develop. In the case of hypomethylation, a correlation could be made with severely duplicated chromosome copies of 16q24.3 . A possible scenario here is that the hypomethylation is apparent because of a duplicated copy number and thus, for example, not a direct cause of reduced DNA methyltransferase activity or over-expression per se. In the case of hypermethylation, a correlation could be made with 16q24.3 LOH . An emerging scenario here is that the hypomethylation induces 16q LOH to promote aberrant DNA methyltransferase activity and hypermethylation . AccumulAlthough the nature of such targeted aberrations are unknown, several studies demonstrating that altered maintenance and or de novo DNA methyltransferase activities can induce tumorigenesis, clearly demonstrates that the controlled methylation of CpG islands is crucial for normal cell development -34. AccoOverall, this study lends further support to the idea that CBFA2T3B is aberrantly regulated in breast cancer. Additional clues supporting a role for this gene in tumor suppression may reside within its protein structure. CBFA2T3B contains a characteristic zinc finger myeloid-nervy-DEAF-1 (zf-MYND) domain and nervy homology regions . SeveralRecently, much emphasis has been placed on the development of methylation-based tumor biomarkers for early breast cancer detection to predict disease outcome and strategies for therapy . Interes24 breast tumor cell lines were obtained from the American Type Culture Collection and cultured under recommended conditions. 46 normal whole blood samples, 55 primary breast tumor samples with pathologically classified grade III lesions and 22 adjacent normal breast counterparts samples were obtained with clinical research approval from the Flinders Medical Centre, Department of Surgery. Breast tumor cell line, primary breast tumor and normal breast counterpart genomic DNA was isolated using GenElute for Mammalian Tissues (Sigma). Whole blood genomic DNA was isolated using the QIAamp DNA Blood Kit (Qiagen). Breast tumor cell line and primary breast tumor total RNA was isolated using RNAqueous-4PCR (Ambion). Nucleic acid concentrations were determined using RiboGreen (Molecular Probes). Breast tumor cell line, primary breast tumor and normal tissue (Clontech) total RNA extracts were DNase I treated (Ambion).16 reverse transcribed at 55°C for 2 h using MMLV (Promega) with addition of RNAguard (Promega) and 5% DMSO. CBFA2T3, CYPA, ATP5A and SYK expression levels were assayed by real-time RT-PCR using SYBR Green I (BMA). CBFA2T3 isoform expression levels were assayed using TaqMan probes specific for exons 1A and 1B (GeneWorks). Real-time RT-PCR was performed on a Rotor-Gene 2000 (Corbett Research) using standard 25 μl HotStar Taq conditions (Qiagen) on cDNA equivalent to 100–1,500 ng total RNA. 0.35X final SYBR Green I or 200 nM probe was used for detection. Amplifications were at 95°C for 10 min, 45 cycles at 94°C for 20 s, annealing temperature for 30 s and 72°C for 30 s. Primer sequences and annealing temperatures are shown . mRNA mlcls per cell were calculated at the CT concentration ÷ amount of total RNA (ρg) per reaction multiplied by 4 based on a 4 ρg total RNA per cell estimation. mRNA was shown as raw expression data (n > 4) and or normalized against CYPA or ATP5A. Several other housekeeping and cancer-related gene expression levels were quantitated to ensure the mRNA mlcl per cell estimations were compatible with other methods (data not shown).10–20 μg of total RNA was oligo(dT)re shown . Unknown3 (pH 5.0) (Sigma), overlaid with paraffin oil and deaminated in the dark for 16 h at 55°C. DNA was desalted using Qiagen purification columns, eluted in 500 μl water and desulfonated with 55 μl of 3 M NaOH for 20 min at 37°C. DNA was neutralized and precipitated with 800 μl of 10 M ammonium acetate (pH 7.0), 20 μl linear acrylamide and 5 ml cold 100% EtOH. Modified DNA was pelleted, resuspended in 40 μl 1 mM Tris-Cl (pH 8.0) and the concentrate stored at -80°C.10–20 μg of genomic DNA was digested overnight at 37°C with restriction enzymes XbaI, XhoI, HindIII and EcoRI and cleaned using nucleotide purification columns . The digested DNA was pooled and bisulfite modified . 10 μg oFour 100–200 bp regions spanning approximately 1-kb of the CBFA2T3B promoter region were amplified from ≥ 300 ng of bisulfite modified DNA under standard HotStar Taq conditions using primers specific for either unmethylated or methylated cytosines. Hypermethylated breast tumor samples were used for bisulfite sequencing to generate a CBFA2T3B promoter region methylation map. Four 300–400 bp regions spanning the promoter were amplified under standard conditions as described above using primers simultaneous for both unmethylated and methylated cytosines. The amplicons were sub-cloned into pGEM (Promega) and 5 to 10 clones sequenced using BigDye (Applied Biosystems). Primer sequences and annealing temperatures are shown .5 cells per T75 flask were seeded in 10 ml RPMI-1640 supplemented with 10% FCS, 15 mM HEPES, 10 mg/liter PGS and cultured for 48 h at 37°C with 5% CO2. Cells were treated with 50 μm 5-Aza-dC for 120 h and replenished with fresh medium and 5-Aza-dC every 24 h. These concentrations are not inhibitory to cell growth [MDA-MB-231 was treated with the demethylating agent 5-Aza-dC (Sigma). Approximately 2.0 × 10T from standard curves with known mlcls/μl calculated from the dilution mass in μg/μl ÷ M.W. of dsDNA clone sequence (× 6.02 × 1017 mlcls/μmole). CBFA2T3B promoter methylation levels were expressed as methylation indices [mi = m/(m + u)] and ratios (u/m) . The median gene expression index of 0.2  is calculated from the median expression level of 4,500 mRNA mlcls per 104 cells divided by the number of unmethylated 'active' mlcls in the 104 cells, i.e. . This calculation equates to approximately 4–5 mRNA mlcls expressed per 10 cells and suggests that the CBFA2T3B gene is largely transcriptionally inert. The remaining active alleles may be trans-factor dependent for expression. An inverse correlation between promoter methylation and expression levels per population of cells was established . In hypermethylated MDA-MB-231, approximately 17,000 promoter mlcls were methylated  and 4 mRNA mlcls expressed per 104 cells. In hypomethylated BT-483, approximately 5 promoter mlcls were methylated (mi = 0.0002) and 120,000  mRNA mlcls expressed per 104 cells. This elevated expression equates that 12 (± 4) mRNA mlcls per cell are expressed from an estimated four-promoter mlcls per cell (i.e. four-16q24.3 DNA mlcls per cell) or that one 'active' unmethylated CBFA2T3B promoter mlcl per cell transcribes 2–4 mRNA mlcls. From a methylation index of approximately 2% and greater, a large increase in y may lead to a small decrease in x. Under this condition, the relationship may be asymptotic. The regression equation y = 25,801 x-0.58 (r2 = 0.7669) describes the methylation and expression relationship. When plotted as methylation index values the equation was y = 1.05 x-0.61 (r2 = 0.7665) (pdf file).Click here for fileCBFA2T3B promoter methylation levels assayed using second-round real-time MSP The methylation levels in normal whole blood samples, normal breast counterparts, primary breast tumors and breast tumor cell lines were assayed at the Sp1 site shown in Figure 1C and plotted as absolute methylation ratios (u/m). All samples are labeled corresponding in part to Additional file 2. The asterisks indicate the samples examined by bisulfite sequencing. The gray highlights indicate the normal blood and normal breast counterpart 'full' methylation ranges. Basal methylation ratios in normal bloods averaged 60:1 (cumulative mean) unmethylated to methylated CBFA2T3B promoter mlcls. This average coincided with conventional MSP band intensities of 100:1 based on pUC19 DNA/MspI marker concentrations  and the median methylation index of 0.02 (i.e. 2 mlcls in 100 are methylated). Normal blood ratios ranged 20:1 to 160:1 unmethylated to methylated mlcls. Normal breast counterparts were similar to normal bloods averaging 100:1 but with a larger range of 10:1 to 350:1. Relative to the normal samples, breast tumors displayed highly aberrant methylation ratios clearly resolved by second-round real-time MSP. 75% of breast tumor cell lines were aberrantly methylated outside the full range of normal blood basal methylation. 58% were outside the range of normal breast. Half of the aberrations were either hypo or hypermethylated relative to both normal blood and normal breast. Similar to cell lines, 51% of primary breast tumors were aberrantly methylated relative to normal blood. 35% were aberrant relative to normal breast (i.e. 24% were hypermethylated and 11% hypomethylated). Aberrant methylation ratios ranged from 1:10 in hypermethylated MDA-MB-231 to 7,000:1 in hypomethylated BT-483 (pdf file).Click here for filePrimers, probes and annealing temperatures used (pdf file).Click here for file


HFE mutations (C282Y and H63D) and a group heterozygous for the S65C mutation.The present study is an analysis of the frequencies of HLA-A and -B antigens and HLA haplotypes in two groups of individuals homozygous for the two main HFE and HLA alleles were detected using DNA-based and microlymphocytotoxicity techniques respectively.The study population includes: 1123 healthy individuals, 100 homozygous for the C282Y mutation, 138 homozygous for the H63D mutation and 17 heterozygous for the S65C mutation. An expected significant association between C282Y and the HLA-A3/B7 haplotype was found, but other HLA haplotypes carrying the -A3 antigen were found: HLA-A3/B62 and HLA-A3/B44. Also, a significant association between H63D mutation and HLA-A29/B44 haplotype was found, and again other HLA haplotypes carrying the HLA-A29 antigen were also found: HLA-A29/B14 and HLA-A29/B62. In addition, the S65C mutation seems to be associated with a HLA haplotype carrying the HLA-A26 antigen.These findings clearly suggest that HLA-A3/B7 and HLA-A29/B44 are the ancestral haplotypes from which the C282Y and H63D mutations originated, respectively. The frequencies of these mutations in different populations, their geographical distribution, and the degree of the statistical association to the ancestral haplotypes, suggest that the H63D mutation must have occurred earlier than the C282Y mutation. HFE) was cloned and located on the short arm of chromosome 6 (6p21.3) . (6p21.3) .C282Y lies within a Celtic ancestral haplotype which includes the human MHC (HLA) haplotype HLA-A3/B7 . The HLAIn contrast, the H63D substitution is not restricted to European populations: allele frequencies greater than 5% are found in countries bordering the Mediterranean, in the Middle East, and in the Indian subcontinent. H63D seems to be associated with HLA-A29 and HLA-B44 .A few studies have been performed on the distribution of the S65C mutation in Europe and other countries. In 1999, Barton et al  identifiHFE gene in a sample of the Spanish population.The aim of this study is to find the HLA antigens and haplotypes associated with the three main mutations in the HFE genotyping has been previously performed on a medical care basis because of a presumptive diagnosis of hemochromatosis. In addition, 1113 unrelated, apparently healthy subjects were used as controls for the study. In addition, HLA typing was performed in 230 individuals whom HFE genotyping was negative for the three mutations.A total of 100 unrelated individuals homozygous for the C282Y mutation, 138 unrelated individuals homozygous for the H63D mutation and 17 individuals heterozygous for the S65C mutation were selected for this study. These were subjects in whom HLA-class I typing was performed on freshly collected venous blood samples by the standard complement-dependent microlymphocytotoxicity assay using commercially available alloantisera.HFE gene. To amplify exon 4, a second set of oligonucleotides was used: HEMEx4-5' (5'-GGT GTC GGG CCT TGA ACT ACT ACC) and HEMEx4-3' (5'-A CAT ACC CCA GAT CAC AAT GAG G).Genomic DNA from whole blood samples was extracted by standard protocols. Polymerase chain reaction (PCR) with the pair of primers HEMEx2-5' (5'-CTT TGG GCT ACG TGG ATG ACC) and HEMEx2-3' (5'-CTG GCT TGA AAT TCT ACT GGA AAC C) was used to amplify exon 2 of the The following conditions were used for the PCR reactions: five minutes denaturation at 94°C, 40 cycles of 15 seconds denaturation at 95°C, 15 seconds annealing at 57°C and 30 seconds extension at 72°C. PCR products coming from exons 2 and 4 were 101 and 228 base pairs (bp), respectively.Bcl I (exon 2), Hinf I (exon 2) and Rsa I (exon 4) for 3 hours following the protocol recommended by the manufacturer . The H63D mutation destroys the Bcl I site in the 101 bp PCR product, so while normal DNA is cut into two fragments of 38 and 63 bp, the mutated DNA is not cut. The S65C mutation destroys the Hinf I site in the 101 bp PCR product, so while normal DNA is cut into two fragments of 47 and 54 bp, the mutated DNA is not cut. The C282Y mutation creates a new Rsa I site, the 228 bp DNA product digested with this enzyme is cut into two fragments of 145 and 83 bp in the normal allele, while in the mutated DNA three fragments of 145, 29 and 54 bp are generated after digestion. The digested products were size resolved in 10% acrylamide gel and detected by staining with ethidium bromide.Following the PCR amplifications, aliquots (17 μl) of the reaction mixture were digested with the restriction endonucleases HFE mutations and HLA-A and -B alleles and haplotypes was calculated by 2 × 2 comparison tables and p values were corrected according to the number of alleles or haplotypes compared [2 and Fisher's tests. Odds ratios were calculated as previously described [Allele and haplotype frequencies were estimated using Arlequin V2.0 software . The hapcompared  and usinescribed .The allele frequencies of the HLA-A and -B antigens found in the C282Y carriers group, in comparison with the frequencies in the control population are listed in Table The allele frequencies of the HLA-A and -B antigens in the H63D homozygous group in comparison with the frequencies in the control population are listed in Table From the 17 individuals heterozygous for the S65C mutation, 7 (20%) were HLA-A26 versus 4.6% found in the control population . No association with HLA-B has been found.HFE mutations.The significant associations did not change if we used a control group of 230 individuals without HFE mutations and HLA antigens and haplotypes in homozygous probands. The paper by Barton and Acton [Populations of homozygous individuals for C282Y and H63D are optimal groups to study the HLA haplotypes in which these mutations preferentially appear. To our knowledge, Barton  and the nd Acton  presentsThe strong association between the HLA-A3/B7 haplotype and the C282Y mutation indicates that this haplotype is the main one associated with this mutation in the Spanish population. However, other haplotypes are also associated: HLA-A3/B62 and HLA-A3/B44. This finding supports the founder hypothesis of Simon et al : the anccontaminated by the C282Y mutation, and up until now, these haplotypes may be considered as protector haplotypes.In addition, the high frequency of the HLA-A3/B7 haplotype makes other HLA antigens and haplotypes have reduced frequencies in respect to the controls. It is interesting to see that haplotypes with high-frequency in the Spanish population, such as HLA-A30/B18 and HLA-A2/B51 are absent in the C282Y homozygous group  and hemochromatosis described in the Danish population .H63D and HLA-A29-bearing haplotypes follow a pattern of associations similar to that described for C282Y and HLA-A3-bearing haplotypes. This promotes speculation that HLA-A29/B44 is the ancestral haplotype from which the H63D mutation emerged, since other HLA-A29 carrying haplotypes are also statistically associated with the mutation  declare that they have no competing interests.Authors AP and PM conceived the study, contributed to proband characterisation, performed statistical comparisons and edited the manuscript. Authors EM, MJR, MJC, DO, MGB and LG contributed at different times to the characterisation of probands and HLA typing.The pre-publication history for this paper can be accessed here:


Chemokines are involved in many biological activities ranging from leukocyte differentiation to neuronal morphogenesis. Despite numerous reports describing chemokine function, little is known about the molecular changes induced by cytokines.We have isolated and identified by differential display analysis 182 differentially expressed cDNAs from CXCR3-transfected Jurkat T cells following treatment with CXCL12 or CXCL10. These chemokine-modulated genes were further verified using quantitative RT-PCR and Western blot analysis.One hundred and forty-six of the cDNAs were successfully cloned, sequenced, and identified by BLAST. Following removal of redundant and non-informative clones, seventeen mRNAs were found to be differentially expressed post treatment with either chemokine ligand with several representing known genes with established functions. Twenty-one genes were upregulated in these transfected Jurkat cells following both CXCL12 and CXCL10, four genes displayed a discordant response and seven genes were downregulated upon treatment with either chemokine. Identified genes include geminin (GEM), thioredoxin (TXN), DEAD/H box polypeptide 1 (DDX1), growth hormone inducible transmembrane protein (GHITM), and transcription elongation regulator 1 (TCERG1). Subsequent analysis of several of these genes using semi-quantitative PCR and western blot analysis confirmed their differential expression post ligand treatment.Together, these results provide insight into chemokine-induced gene activation and identify potentially novel functions for known genes in chemokine biology. CXC and CC chemokines are small soluble proteins expressed and secreted by a number of cell types during the initial host response to injury, allergens, antigens, or invading microorganisms . These lin vitro by IL-3, and selectively inhibited by soluble ephrin-B receptor. dATP. Each primer pair was denatured at 94°C for 30 sec, annealed at 40°C for 2 minutes and extended at 72°C for 30 sec for 40 cycles with a final extension for 10 minutes. [33P]-labeled PCR products were resolved on a 6% denaturing polyacrylamide gel. The autoradiogram was inspected on a light box and differentially expressed bands marked by needle punches. The punched film was carefully oriented on the dried gel and the marked bands excised with a scalpel blade. Glycogen (10 mg/ml), 3 M sodium acetate, and 85% EtOH were added and after overnight storage at -80°C, the DNA was precipitated by centrifugation. Each DNA was subsequently reamplified using the same PCR primer set and conditions except that the dNTP concentration was increased to 20 uM and no isotope was added to the mixture. Reamplified PCR products were resolved on a 1.5% AmpliSize™  agarose gel, stained with SYBR® Gold  and extracted from the gel using a QIAEX II kit (Qiagen). Successfully amplified bands were cloned using the PCR-TRAP® cloning vector system (GenHunter) and ligated into GH-competent cells. Following transformation, only clones that contain an insert are capable of growing on LB-Tet agarose plates. The cloned insert was subsequently checked by colony-PCR using primers flanking the PCR-TRAP® vector and the insert sequenced to identify genes differentially expressed between control and chemokine-treated CXCR3-transfected Jurkat T cells.Total RNA was isolated using the Qiagen RNeasyOne microgram of DNase I-treated total RNA from control, CXCL12-or CXCL10-treated CXCR3-transfected Jurkat T cells was reverse-transcribed with 200 units of SuperScript II reverse transcriptase  in a 20 ul reaction for 50 min at 42°C followed by heat-denaturation at 70°C for 15 minutes. Two microliters of the first strand reaction product were amplified in six duplicate reactions using standard PCR conditions  with sequence specific primers. Individual tubes were removed after 24, 26, 28, 30, 32, and 34 cycles and the concentration of specific PCR product determined using an Agilent 2100 BioAnalyzer and DNA 1000 LabChip . Each RT-PCR was performed twice for each RNA preparation. Two housekeeping control genes, GAPDH and ribosomal protein L32 (RPL32), were utilized as controls with specific primers producing a 599 bp product for GAPDH and a 233 bp product for RPL32.Cells were lysed in modified RIPA cell lysis buffer  with 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM sodium orthovanadate, 5 μg/ml leupeptin, 2 μg/ml aprotonin and one Complete Protease Inhibitor Cocktail tablet  per 50 ml of buffer. Whole cell protein extract was used directly for Western blot analysis. Protein concentration was determined using Bio-Rad protein assay kit (BioRad). Twenty micrograms of total protein from each sample was separated on a 10% Tris-glycine polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane . Membranes were blocked for 1 h at room temperature in PBS containing 5% non-fat dry milk and 0.1% Tween-20. The membranes were then incubated overnight at 4°C in primary antibody  diluted 1:1000 in PBS containing 5% non-fat dry milk and 0.1% Tween-20. The membranes were washed in PBS with 0.1% Tween-20 then incubated for one hour in secondary antibody . The blots were washed and the proteins detected using the ECL Plus Western Blotting Kit  and X-MAT AR Film .7/ml. RPMI (0.6 ml) containing 1% FBS with or without 100 ng/ml SDF-1α was added to the bottom wells of the 24-well plate. Transwell chambers with 5 μm pore filters  were then placed into the wells. Cells (1–3 × 106 in 100 μl) were then added to the chambers. After 2 h, the migrated cells in the bottom wells were transferred to triplicate wells of a 96-well plate in 150 μl volumes. Hoechst fluorescence was measured on a Fluoroskan Ascent FL fluorescence plate reader  at λex = 355 nm, and λem = 460 nm. Results are expressed as migration index calculated by subtracting the fluorescence intensity of media alone and comparing the values to the fluorescence intensity (relative number) of cells migrated into the bottom chamber in media alone, which is normalized to a value of 1. Fluorescence values were within the linear range of a standard dilution curve.Jurkat T cell migration was examined using a fluorescence-based Transwell chemotaxis assays as previously described ,61. CXCR6 cells/ml [CXCR3-transfected Jurkat T cells were loaded with the fluorescent indicator, Fura-2AM , for 30 min, then washed and resuspended in PBS containing calcium and magnesium at 10cells/ml . The ratThe cells were found to be functionally responsive to both CXCL12 and CXCL10. As shown in Fig. In an effort to identify genes, which may be upregulated or downregulated by CXCL12 or CXCL10, we examined Jurkat T cells that that expressed endogenous CXCR4 and that had been transfected with CXCR3 using DDRT-PCR analysis Figure  and 3B. To characterize the sequence identity of DDRT-PCR products, the excised and re-amplified bands were cloned into the PCR-TRAP vector and transformed into GH-competent E. coli. Several colonies were selected to be cultured, plasmids were purified, and the inserts wee subsequently screened by colony-PCR using primers flanking the site of the PCR-TRAP vector. The plasmids containing an insert were sequenced. We successfully sequenced a total of 146 cDNAs. Seventeen mRNAs were differentially expressed post treatment with either CXCL12 or CXCL10, each representing known genes with established functions. Five additional RNAs were also identified defining known genes with unknown functions and nine identified hypothetical or predicted genes of unknown function Table .Sequence analysis indicated that the 401 bp A4.7 cDNA is highly homologous to the gene LOC51053 that is also known as geminin, a cell cycle regulator. The 157 bp A6.7 cDNA fragment is 94% identical to the 501 bp human thioredoxin (TXN) gene. The sequence of the band A11.1 was homologous to the transcription elongation regulator 1 gene (TCERG1) also known as CA150. The 329 bp C7.3 cDNA fragment is 87% identical to the published human gene encoding the growth hormone inducible transmembrane protein (GHITM). The sequence of the band C10.4 was homologous to the interferon-induced transmembrane protein 2 (IFITM2) gene, a member of the 1–8 gene family whose members are strongly induced by both type I  and type II (IFNγ interferons). The 102 bp G1.1 cDNA fragment is 97% identical to the human gene encoding flotillin 1 (FLOT1) that is thought to play a role in vesicular trafficking and signal transduction. Sequence analysis indicated that the 444 bp G9.2 cDNA, as well as several other bands, were highly homologous (>95%) to the gene encoding the iron-storage protein ferritin heavy polypeptide 1 (FTH1). The sequences of the other cDNAs included in Table As a large fraction of DDRT-PCR products have been shown to yield weak or barely detectable signals in the Northern blot analysis, we sought to confirm the identity and differential expression of these bands via RT-PCR analysis. We focused our attention on a subgroup of the seventeen DDRT-PCR products representing known genes with established functions. Gene specific primers were designed to produce unique amplimers between approximately 150 and 500 base pairs in size.RT-PCR analysis was performed on the Agilent BioAnalyzer 2100 System. The advantage of using this system to examine competitive PCR products lies in the accurate absolute and relative quantitation of each amplified product. Small differences in the amount of amplimer product, which cannot be detected using slab gel analysis, are more easily analyzed on this equipment permitting RT-PCR to be used to measure changes in gene expression. RNA from CXCR3 receptor-transfected T cells, both before and post treatment with CXCL12 or CXCL10 was reverse transcribed in bulk and aliquots RT product amplified by PCR using specific sets of primers. Initially, a 599 bp GAPDH amplimer was utilized as a housekeeping gene. However, it was noted that GAPDH significantly up-regulated post treatment of the cells with either CXCL12 or CXCL10. To address this issue, a 233 bp ribosomal protein L32 amplimer was utilized as a housekeeping gene for the subsequent comparison of gene expression levels. In Figures Additional studies were performed using primary human T cells derived from several different donors to verify the differential CXCL12-and CXCL10-induced gene expression observed in Jurkat T cells. However, despite the clear differences in gene expression in Jurkat T cells after chemokine stimulation for 24 hr in culture, primary resting and anti-CD3/CD28 mAb-activated human T cells demonstrated variable and non-reproducible expression of the geminin, thioredoxin, DDX1, GHITM and TCERG1 gene products (data not shown). This variability may have more to do with the activation state of the primary T cells being utilized and the chemokine receptor expression and density on primary T cells versus the CXCR3-transfected Jurkat T cell subclone. More detailed studies examining CXCL12-induced gene expression in primary human T cells at various stages of activation are the focus of current studies. Moreover, studies using CXCL10 on primary human T cells are quite difficult as the expression of CXCR3 on resting human T cells is quite low to non-existent (>5% on CD3+ T cells with MFI between 5–12) and may be selectively expressed on certain cell subsets. CXCR3 studies in normal human T cells would require an activation of T cells using IL-2 or Th1 polarizing stimuli and thus may not be a valid comparison with the Jurkat T cells utilized in these studies.To further confirm the expression of several of these genes, Western blot analysis was subsequently performed on total cell lysates of CXCL12-or gp120 IIIB-treated Jurkat T cells. The results shown in Fig. Studies on the alteration of gene expression following chemokine-receptor ligation and the identification of genes that are differentially expressed can provide relevant and important insight into a variety of biological processes and disease etiologies. To date, numerous approaches, model systems, and techniques have been used to search and identify chemokine-modulated genes -33. In thttp://nciarray.nci.nih.gov/cgi-bin/gipo. Many of the genes identified in this study are associated with detoxification, DNA repair, apoptosis, migration, T cell receptor signaling and interferon signaling. While many of the chemokine-induced genes observed in our study are not found on the arrays used by Suzuki et al. [In the present study, we have identified 31 cellular genes that were either up-or down-regulated in CXCR3-transfected Jurkat T cells following treatment with either CXCL12α or CXCL10. Suzuki and colleagues  have reci et al. , we did Although not previously linked to chemokine receptor-ligand signaling, expression of several of these genes such as interferon-induced transmembrane protein 2 (IFITM2) and growth hormone inducible transmembrane protein (GHITM) are recognized to be a part of the interferon signaling system that includes Janus kinases and their downstream target STAT proteins ,40. IFITThe transcriptional cofactor, CA150, whose gene is now designated transcription elongation regulator 1 (TCERG1) is capable of repressing transcription from many viral and cellular promoters whose initiation is dependent upon the presence of a TATA box . CA150 rGeminin (LOC51053) is a 25-kDa protein that inhibits DNA replication and is degraded during the mitotic phase of the cell cycle . This prFlotillin-1 (FLOT1), also known as Reggie-2, encodes a caveolae-associated, integral membrane protein . CaveolaMovement of CCR5 and CXCR4 molecules into lipid rafts is important in the maintenance of receptor conformation and through this mechanism rafts modulate the binding and function of receptors ,62. FlotThioredoxin (TXN) is a 12-kDa oxidoreductase enzyme containing a dithiol-disulfide active site. It possesses a variety of biological functions including the ability to modulate the DNA binding activity of the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) as well as the activity of several other transcription factors including general transcription factor IIIC, NF-κB, and AP-1 ,65. ReacFerritin is a highly conserved iron-binding protein that in its cytosolic form is composed of 2 subunits, ferritin H and ferritin L, each encoded by a distinct gene. Depending on many factors including tissue type, redox status, and the inflammatory state of a given cell or tissue, the ratio of H to L subunits varies greatly . SeveralIn the current report, we do not reveal a specific physiological role for the gene expression changes that have been observed by DDRT-PCR post CXCL12 or CXCL10 signaling. As noted above, many of the more fully characterized genes have multiple interactions utilizing a number of distinct signaling pathways  that are frequently utilized by other cytokine family members. A more detailed understanding of the various genes differentially induced by chemokines via ligation of their cell surface receptors or during the chemotactic process should provide some insight into the process of cell migration and activation and may identify novel targets for therapeutic intervention.None declaredJEN, RJS, LS, DB, VDD, EMS and DDT performed the experiments. JEN and DDT prepared the figures and wrote the paper. DDT also supervised the work and edited the manuscript. All authors have read and approved the final manuscript.differential display, DD; growth hormone inducible transmembrane protein, GHITM; GEM, geminin; transcription elongation regulator 1, TCERG1; thioredoxin, TXN; DEAD/H box polypeptide 1, DDX1


Achilles tendinopathy is considered to be one of the most common overuse injuries in elite and recreational athletes and the recommended treatment varies. One factor that has been stressed in the literature is the lack of standardized outcome measures that can be used in all countries. One such standardized outcome measure is the Victorian Institute of Sports Assessment – Achilles (VISA-A) questionnaire, which is designed to evaluate the clinical severity for patients with Achilles tendinopathy. The purpose of this study was to cross-culturally adapt the VISA-A questionnaire to Swedish, and to perform reliability, validity and structure evaluations.Cross-cultural adaptation was performed in several steps including translations, synthesis of translations, back translations, expert committee review and pre-testing. The final Swedish version, the VISA-A Swedish version (VISA-A-S) was tested for reliability on healthy individuals (n = 15), and patients (n = 22). Tests for internal consistency, validity and structure were performed on 51 patients.r = 0.89, ICC = 0.89) and healthy individuals . The internal consistency was 0.77 . The mean [95% confidence interval] VISA-A-S score in the 51 patients (50 [44–56]) was significantly lower than in the healthy individuals (96 [94–99]). The VISA-A-S score correlated significantly (Spearman's r = -0.68) with another tendon grading system. Criterion validity was considered good when comparing the scores of the Swedish version with the English version in both healthy individuals and patients. The factor analysis gave the factors pain/symptoms and physical activityThe VISA-A-S had good reliability for patients (pain/symptoms and physical activity, and can be used in both research and the clinical setting.The VISA-A-S questionnaire is a reliable and valid instrument and comparable to the original version. It measures two factors:  Achilles tendinopathy is a common overuse injury, especially among athletes involved in activities that include running and jumping -4. SeverDespite the high incidence of Achilles tendon disorders there have been few randomized treatment studies in patients with Achilles tendinopathy ,11-16. IRobinson et al.  developeTherefore, the purpose of this study was to translate and culturally adapt the English VISA-A questionnaire to Swedish, to perform reliability and validity evaluations of the Swedish version, and to analyze the factor structure of the questionnaire.To establish good face validity and content validity, the translation and cultural adaptation of the VISA-A questionnaire into Swedish was performed in several steps . The EngThe panel of experts consolidated the various versions into one pre-final version of the VISA-A questionnaire – Swedish version (VISA-A-S). The pre-VISA-A-S was pilot tested on five patients and five healthy individuals. After pilot testing question 1 was made clearer by entering the minutes in the boxes.The final version of the VISA-A-S  with the results of the two patient groups, the non-surgical group (n = 45) and surgical group (n = 14), in the original article by Robinson et al. (2001). The results of the healthy individuals in our study were also compared with the results from the healthy individuals in the original study.The structure of the VISA-A-S questionnaire was evaluated with a factor analysis.All data were analysed by SPSS 11.5 for Windows. Descriptive data are reported as mean, standard deviation and 95% confidence interval.r, as performed for the VISA-A English version [Test-retest data was analysed by Pearson's  version . Inter-CU test. For comparison of the VISA-A-S with the VISA-A, a two sample t-test was used since only means and standard deviations, and no raw data, were available from the results in the original study [Comparison of VISA-A-S with Stanish et al. (2000) tendon grading system was performed by calculating the Spearman's rank correlation coefficient for non-parametric data. VISA-A-S scores for the healthy group and the patient group were compared using the Mann Whitney al study . The levA principal axis factoring with varimax rotation, eigenvalue over 1.0, was applied for evaluation of the structure of the questionnaire.r > 0.88, ICC > 0.88) and for patients . There was no significant difference between the scores on test days 1 and 2. When analyzing each question separately . The patients with Achilles tendinopathy had a significantly lower score (p < 0.0001) compared with the healthy individuals. The mean VISA-A-S score for patients in the present study was significantly (p < 0.01) lower than the mean VISA-A score for the non-surgical group in the original article by Robinson et al. (2001). When comparing the VISA-A-S score for patients in the present study with the VISA-A score of the surgical group in the original study [The VISA-A-S score correlated significantly with the tendon grading system by Stanish et al. (2000)  and physical activity (questions 7 and 8).The factor analysis revealed two factors of importance : A widely-used clinical outcome measure for patients with Achilles tendinopathy would help comparisons between treatments in various clinics and research studies. The VISA-A questionnaire is an easily self-administered questionnaire. Since research is performed in various countries it is important to properly translate, culturally adapt and evaluate instruments like a questionnaire in order to be able to compare the results .face validity and content validity. The test-retest reliability and the internal consistency were considered good. A significant and strong correlation between the VISA-A-S and the tendon grading system by Stanish et al. (2000) indicates good construct validity. The comparison of the results of the patients and healthy individuals in the present study with the results of the non-surgical group, the surgical group and the healthy individuals, as reported in the original article by Robinson et al. (2001), indicates good criterion validity.This study demonstrates that the Swedish version of the VISA-A questionnaire has good reliability and validity. With careful translation and cultural adaptation, we established good pain/symptoms (questions 1–6) and physical activity (questions 7 and 8), strongly confirming that the questionnaire is valid for evaluating the patient's symptoms and its effect on physical activity. The factor analysis and an internal consistency of 0.77 as measured by Cronbach's alpha indicate that no question should be excluded.The factor analysis gave the two factors: We did not include a separate group of pre-surgical patients as in the original study by Robinsson et al. (2001). This is because the advances in the non-surgical rehabilitation of patients with Achilles tendinopathy during recent years have resulted in markedly reduced number of patients awaiting surgery. The patients in the present study can therefore be viewed as representing patients from both groups  of patients used in the original study . This woThe one week duration between the two tests in the test-retest reliability part was somewhat long. This duration was chosen because this is the usual time that lapses between a patient's first and second visit with the physical therapist. This may explain why two of the questions differed significantly between test-days. During this week the patients have met with their physical therapist, which may have caused the patients to change their view on their symptoms and physical ability.Good criterion validity indicates that the Swedish version and the English version of the VISA-A questionnaire evaluate the same aspects of clinical severity in patients with Achilles tendinopathy. It can, thus, be expected that similar scores in the two versions indicate the same index of severity in patients with Achilles tendinopathy.The 11 physical therapy clinics throughout Sweden, which participated in this study, all reported that the questionnaire was easily administered, and required a minimum of communication between the physical therapist and the patient. The physical therapists perceived the questionnaire as a good clinical tool and useful when treating patients with Achilles tendinopathy.A review of the literature in regards to treatment of Achilles tendinopathy yielded only a few randomized treatment trials ,11-16. Tpain/symptoms and physical activity, reinforces that the VISA-A questionnaire can be used as an index of clinical severity. The present study culturally adapts and validates the VISA-A-S questionnaire  declare that they have no competing interests.KGS conceived of the study, participated in its design, performed data acquisition, analyzed and interpreted the data and drafted the manuscriptRT conceived the study, participated in its design, interpreted the data and helped to draft the manuscript.JK participated in the study design, interpreted the data and helped to draft the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:VISA-A-S questionnaire The Swedish version of the VISA-A questionnaire in MICROSOFT WORD format.Click here for file