Bacillus subtilis, which has been reported to be an inhibitor of the intracellular subtilisin Isp1 from the same organism. The structure of IPI is a variant of the all-beta, immunoglobulin (Ig) fold. It is possible that IPI is important for protein-protein interactions, of which inhibition of Isp1 is one. The intracellular nature of ISP is questioned, because an alternative ATG codon in theipi gene would produce a protein with an N-terminal extension containing a signal peptide. It is possible that alternative initiation exists, producing either an intracellular inhibitor or a secreted form that may be associated with the cell surface.  Homologues of the IPI protein from other species are multi-domain proteins, containing signal peptides and domains also associated with the bacterial cell-surface. The cysteine peptidase inhibitors chagasin and amoebiasin also have Ig-like folds, but their topology differs significantly from that of IPI, and they share no recent common ancestor. A model of IPI docked to Isp1 shows similarities to other subtilisin:inhibitor complexes, particularly where the inhibitor interacts with the peptidase active site.We report the crystal structure solution of the Intracellular Protease Inhibitor (IPI) protein from Bacillus subtilis, accounting for more than 80% of the intracellular azocasein- or azocollagen-degrading activity1. It is a subtilisin homologue and a member of peptidase family S8. It is unclear what the physiological role of Isp1 is; it has been shown not to be important during sporulation where the majority of intracellular proteolysis occurs, even though intracellular proteolysis was correspondingly low when a defective mutant of Isp1 was created2. Potential physiological substrates that have been identified include elongation factor Tu  and ClpC, a negative regulator of genetic competence (with one cleavage occurring in a disordered region and another in a coiled-coil region)3. The peptidase is synthesized as a precursor with an N-terminal, 18-residue propeptide that blocks the active site. Release of the propeptide is probably by Isp1 itself, inter-molecularly4. The structures of both the precursor and the mature peptidase have been solved and show that a proline residue (Pro8) introduces a kink in the backbone that shifts the scissile bond in the propeptide away from the catalytic serine. Once the propeptide is released, the peptidase active site forms correctly4.The Isp1 peptidase  is the major intracellular peptidase inet al.5 described an inhibitor of Isp1 fromBacillus subtilis known as Intracellular Protease Inhibitor (IPI). This inhibitor was effective against a number of subtilisin homologues, using casein as a substrate following pre-incubation of peptidase and inhibitor, but was ineffective against cysteine, aspartic and metallopeptidases. AKi for inhibition of the Isp1 peptidase was estimated to be in the order of 10-9 M6. The gene for the peptidase has been cloned7, and the deduced protein sequence showed, at the time, no similarity to any other protein. There are now 149 homologues in the Pfam familyPF12690 (BsuPI). The family was included in theMEROPS database (as I228), but subsequently removed, because it was not clear whether the proteins in the family were inhibitors or competing substrates. In particular, the inhibition of intracellular peptidases Isp2 and Isp3 was “repressed” in the presence of the substrate casein6, which suggests the activity of competing substrates.The activity of a powerful endopeptidase within the cell must be controlled in order to prevent unwanted proteolysis of cellular proteins, and in 1986 Nishino9; calpastatin, which inhibits the intracellular peptidase calpain10; chagasin from the zooflagellateLeishmania (family I42), which also inhibits cysteine peptidases11; three intracellular coagulation inhibitors from the horseshoe crabTachypleus that are serpins from family I412; and pinA from family I24, which is an inhibitor of the ATP-dependent serine endopeptidase Lon, but is of unknown structure13. Despite the inhibitors being intracellular, the known target peptidases are all extracellular, with the exceptions of calpain and endopeptidase Lon. Given the paucity of known intracellular peptidase inhibitors, it would not be a surprise if the fold of IPI were different from any known inhibitor structures, especially the secreted inhibitors which are stabilized by disulfide bridges, because intracellular proteins lack disulfides.Structural similarity to other known peptidase inhibitors might argue for reinstatement of the family in the MEROPS database. However, most peptidase inhibitors are secreted and intracellular peptidase inhibitors are rare. The only examples are cystatins A and B from family I25, which inhibit cysteine peptidases14). This inhibitory mechanism is known as the standard or the Laskowski mechanism15. The residues that form the reactive bond will vary from inhibitor to inhibitor, according to the specificity of the peptidase that is inhibited. The chymotrypsin-like specificity of the Isp1 peptidase implies that the P1 residue in the reactive bond of the intracellular inhibitor should be a hydrophobic residue.Most serine peptidase inhibitors act as if they were super-substrates, binding so tightly to the active site that they are either not cleaved, or if cleavage occurs then the fragments are not released from the peptidase. There is a bond, known as the reactive bond, which occupies the peptidase active site with residues either side occupying the S1 and S1′ binding pockets . Protein production and crystallization of IPI was carried out by standard JCSG protocols17. The crystal was obtained using the vapor diffusion method in a sitting drop format where sitting drops composed of 100 nl protein solution mixed with 100 nl crystallization solution were equilibrated against a 50 μl reservoir at 293 K. The crystallization reagent consisted of 48.5% polyethylene glycol 600, 0.1M CHES pH 9.7. Data were collected at wavelengths corresponding to the inflection and high energy remote of a selenium MAD  experiment at 100 K using a MARCCD 325 detector (Rayonix) at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 9_2. Data processing were carried out using XDS18 and the statistics are presented in19 and autoSHARP20, and refinement was carried out using REFMAC521. The structure was validated using the JCSG Quality Control server (http://smb.slac.stanford.edu/jcsg/QC). Atomic coordinates and experimental structure factors to 2.6 Å resolution (PDB code: 3ISY) have been deposited in the Protein Data Bank (www.pdb.org22).The American Type Culture Collection (ATCC) provided the genomic DNA used to clonehttp://consurf.tau.ac.il/;23). The results were visualised with PyMOL (http://www.pymol.org/), which was also used for structural figures.Sequence conservation among homologues of IPI was mapped onto the crystal structure using ConSurf , around 50% identical to IspA, as the sole template. The high degree of sequence identity with the target and the small number of insertions and deletions between target and template  assured a high quality model. Additionally, the insertions and deletions lie distant from the catalytic site which was the main region of interest.A homology model ofhttp://vakser.bioinformatics.ku.edu/resources/gramm/grammx/;25), ZDOCK  and ClusPro 2.0 .The new structure of IPI was docked to the model of IspA using three different webservers, GRAMM-X  is 3.2 Å3 Da-1 and the estimated solvent content is 60.9%. The Ramachandran plot produced byMolProbity29 shows that 98.0% of the residues are in favoured regions, with no outliers. Two residues, Lys 86 and Glu 87, are flagged as having unusual Ramachandran plot properties by the PDB validation report although not with Molprobity. Electron density for them strongly supports their modelled conformations.The crystal structure of an intracellular proteinase inhibitor  fromThis is the first structure in a family of peptidase inhibitors derived from Bacillus subtilis. The structure has an unusual Ig-like fold.Click here for additional data file.16 (The structure of the IPI protein is similar to the immunoglobulin (Ig) fold. This common fold consists of a beta-sandwich formed of seven strands in two sheets with a Greek-key topology and proteins with this fold are involved in a variety of functions, including cell-cell recognition, cell-surface receptors, muscle structure and the immune system. The limits of the B-strands seen in the structure coincide well with those determined by NMR16 . At the http://ekhidna.biocenter.helsinki.fi/dali_server/30) the protein with the closest structural similarity is the RbmA protein fromVibrio cholerae  2.9 Å, residues 38–152), which is one of the three major protein components of the biofilm matrix important for cell-to-cell contacts31. The A chain from coagulation factor XIII32 is also structurally similar, with z-scores in the range of 9.4–9.9 depending on the source species. The structural similarity with IPI is over residues 518–629 for human factor XIII, which is a domain from the transglutaminase, C-terminal Ig-like domain family (PF00927). Factor XIII is a transglutaminase important for stabilizing fibrin clots by cross-linking chains with isopeptide bonds32. These similarities probably reflect only the all-beta nature of the structures rather than any common physiological functions.From a comparison using the Dali website  are similar but distant . The structures of IPI and amoebasin (PDB:3M86) are also similar .The folds of chagasin and amoebiasin (both members of inhibitor family I42) have also been described as Ig-likeB. subtilis was not available, a homology model was created. The ~50% sequence identity shared with available templates assures a high quality model: at this level of similarity an RMSD error of around 1 Å is expected35. Since it is often difficult to pick out the true solution from the multitude of poses typically resulting from a docking experiment, we applied three cross-checks to improve confidence in our results. First, we required that the interface of the inhibitor with protease involve the conspicuously conserved wedge structure  is predicted to be intracellular.IPI homologues are found in both bacteria and archaea, but no homologues have been detected in eukaryotes. All bacterial homologues are from Gram-positive bacteria (Firmicutes), with the exception of homologues from the Gemmatimonadetes bacteriumHalobacteriaceae, which includes high salt tolerant species. The archaeans with completely sequenced genomes that possess an IPI homologue also possess at least one subtilisin homologue, which in halophilic archaeon DL31 andHaloterrigena turkmenica include proteins predicted to be intracellular. In none of the species with completely sequenced genomes is the gene for the IPI homologue in close proximity to a gene for a subtilisin homologue, and so they are not part of the same operon or regulon. In total, there are 33 species of bacteria and archaea that possess both an IPI homologue and a subtilisin homologue that is predicted to be intracellular . There are 43 species from which an IPI homologue is known, but an intracellular subtilisin homologue is unknown, but of these 43 species only four have had their genomes completely sequenced .All of the 30 archaean homologues are from species of the familyBacillus subtilis protein. The presence of a signal peptide shows that many members of the family are secreted rather than intracellular proteins. Even among the proteins that contain only the IPI-like domain, a large number also possess signal peptides, including homologues fromBacillus species. One homologue from aGeobacillus species is predicted to be a type II membrane protein with an N-terminal transmembrane domain. The longest protein to include an IPI-like domain is the 585-residue Chte0880 protein fromClostridium thermocellum, which has three repeats each composed of an S-layer homology (SLH) domain followed by an IPI-like domain. The presence of SLH domains indicates that the protein binds to the proteoglycan of the cell wall.It is relatively common for inhibitors to occur as multiple domains within the same protein.5. Later, when the gene was sequenced7, no signal peptide was detected in the coding sequence. Now that the full genome forBacillus subtilis strain 168 has been sequenced39, it is clear that a possible alternative initiating methionine exists for theipi gene and the coding sequence could be extended at the N-terminus, adding an extra in-frame 33 residues (MKRLLVMLLPVLLLIGCGKDEQTEPDKEVSGG). The predicted protein sequences of IPI fromB. subtilis strains BSn540 and QB92841 include this N-terminal extension. When the extended sequence was submitted to the SignalP  a signal peptide was predicted with cleavage at the Thr23-Glu bond. The N-terminal sequence determined by Nishinoet al. (1986)5 was Glu34, with the assumption that Met33 was the initiating methionine. Signal peptidase 1 is unlikely to cleave the Met33-Glu bond, because of the hydrophilic nature of the region Glu24-Gly32. This may mean that alternative initiation exists for this protein; a secreted form starting at Met1 and an intracellular form starting at Met33. It is worth adding that even if a majority of IPI is in fact secreted, a protease inhibitor function is still physiologically plausible since most subtilisins are extracellular.IPI itself was initially characterized as intracellular. It was purified by lysing cells with lysozyme and its N-terminal sequence determined by Edman degradation6. Binding to an intracellular subtilisin may therefore not represent the primary physiological function of IPI. However, there are similarities between the IPI protein and chagasin, and not only in terms of structure. Chagasin inhibition of cysteine peptidases is tight but reversible33. If IPI inhibition of subtilisins were also reversible, then the repression seen with casein would be explained. IPI inhibition would not be via the classical Laskowski mechanism, in which a reactive site bondpermanently occupies the peptidase active site, but access to the active site would instead bereversibly physically blocked.The nearest structural neighbours of IPI, including the RbmA protein and coagulation factor XIII, are involved in protein-protein interactions. This may indicate that rather than functioning as an inhibitor, IPI is binding the peptidase in some non-specific way. Such an interaction might explain the peculiar results obtained with the substrate casein, which apparently “repressed” inhibition of the peptidase38. It is intriguing that IPI may exemplify a second intracellular peptidase inhibitor family, after the chagasin and amoebiasin group, with an Ig-type fold. However, structural similarity between chagasin and IPI is weak and the two groups clearly share no recent common ancestor. We hope the resolution of the structure encourages others to further characterize IPI and proteins bearing IPI-like domains to further probe their functions.Without further kinetics studies it is not possible to state categorically that IPI definitely is a peptidase inhibitor. However, the tertiary structure and docking predictions illustrate how it could feasibly inhibit its cognate enzyme. That hypothetical mode bears strong similarities to those seen for other well-characterised, unrelated serine peptidase inhibitor families such as the Kazal and Kunitz groups in agreement with a substantial literature on convergent evolution in peptidase inhibition I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Looks good.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.  B. subtilis, a family of protease inhibitor proteins with homologues across many different species. Although being assigned to the general IG-like fold, the structure shows a distinct topology, hence could also be regarded as a novel fold and a significant contribution to the protease inhibitor field. Additional studies are needed to understand the functional aspects of this new protein and structural scaffold.This article reports the first crystal structure of the IPI protein fromI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thorough discussion of the inhibition mechanism and the protein's possible biological role(s) make this manuscript of interest to a wide audience.  Minor changes to improve clarity include delaying (or removing) discussions of ISP in the Abstract because ISP is not the central subject of the manuscript, expanding comments on the NMR structure and possibly citing similarities beyond the common identification of beta strands, and fixing Figure 1A where greater separation between the stereo images is needed.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Recent studies have shown the importance of nonlymphoid chemokines thymic stromal lymphopoietin and IL-27, so studying responses in situ is critical. Most sources of allergens have 1 or 2 dominant allergens, and for house-dust mite, it has been shown that people have a predictable responsiveness to high-, mid-and poor-IgE-binding proteins regardless of the total size of their response. This allergen hierarchy can be used to design improved allergen preparations and to investigate how antiallergen responses are regulated.This overview describes the nature of the immune responses induced by the inhalation of allergens. There is a dichotomy in that B cells have multiple mechanisms that limit the amount of immunoglobulin E (IgE) antibody production, whereas T-cell responses are large even in nonallergic subjects. With the possible exception of responses to cat allergen, however, T cells from nonallergic subjects have limited effector function of helping IgG antibody, and in house-dust mite allergy, this declines with age. Regulation by interleukin 10 (IL-10)-producing cells and CD25 Vh genes to the 0 gene in class switching is intrinsically weak compared with the switching for gamma subclasses, [+ cells, and a post-IgE-switching phase, in which IgE cells differentiate swiftly into plasma cells. This leaves a deficiency in IgE-bearing memory cells that would maintain long-lived responses. Studies on the immunoglobulin heavy chain repertoire expressed by antiallergen IgE antibodies show that there are no restrictions with respect to the macroelements of repertoire diversification such as the selection of Vh gene families or VDJ recombinations, [Vh genes. Selection from continued exposure to low doses of antigen or limitations of antigen binding imposed by the unique structure of IgE could account for this. For the last possibility, it has been proposed that steric hindrance of antigen binding might occur because IgE has a bent structure that places the Vh region in close proximity to the C-terminal of the constant region [A hallmark of immediate hypersensitivity is the ability of mast cells and basophils armed with small amounts of immunoglobulin E (IgE) antibody to induce powerful inflammatory reactions. Immunoglobulin E is indeed produced in lower amounts than the more common IgG isotypes. The serum immunoglobulin levels, which vary with atopic status, are in the region of 5 to 500 ng/mL compared with 0.5 and 10 mg/mL of IgG4 and IgG1, respectively. There are few IgE-producing cells, and IgE has a short half-life. Recent studies show that the IgE antibody titers in humans correlate very well with the presence of IgE producing early plasma cells in the blood, [classes,  and RNA classes, . At the t region .Consistent with tight control mechanisms, it has been repeatedly demonstrated that antiallergen antibodies constitute a large portion of the IgE immunoglobulin -9. The sThe engagement of IgE with the high-affinity FcεR1 in the absence of allergen increases the expression of the receptor on the surface of basophils, mast cells, and dendritic cells, so that the IgE levels, and thus the type of allergen-inducing sensitization, can directly affect the allergic response. It was thought that this occurred by inducing the synthesis of FcεR1, but it now seems to occur via a membrane-stabilizing process .The development of allergic symptoms is related to the titers of antiallergen IgE antibody, but even children with the highest titers only have a 60% probability of disease as shown for wheeze in asthma, and although the probability declined with reduced titers, it was still elevated at the low level of 4.4 ng/mL . The cla+ population[+ and CD45RA+ compartments of nonallergic subjects [+ cells [In contrast to IgE antibody, the T-cell responses to allergens are large. The precursor frequencies of T cells responding to allergen extracts have been reported for pollen and HDM opulation,25 compa[+ cells .Although most of the T-cell responses in allergy have been studied with extracts, the use of purified allergens is more informative because the stimulating specificities and the concentration used is known and can be accounted for. The T-cell responses to the dominant allergens from grass pollen, ,32 birch+ cells have been shown to be the predominant source of Th2 cytokines in PBMCs[+ cells although in in vitro cultures, provided that they were supplemented with IL-2 [CD4 in PBMCs and to p in PBMCs. The Derith IL-2 . In a laith IL-2 .+ CD4+ cells and the secretions of the corresponding chemokines TARC and MDC [Studies on cytokine production in situ have shown that the Th2 polarization is best found in the lungs and not the peripheral blood . Thymic  and MDC . Endobro and MDC ,60. The  and MDC ,62. Pati and MDC .+ T cells from atopic dermatitis patients with IgE antibody to the cat allergen Fel d 1 used tetramer binding to show a similar result, with most of the T cells being within the central memory compartment [The chemokine receptor bias of allergen-responsive T cells to CCR4 has also been detected in grass pollen allergy ,63. The partment . Their fThere are many observations that show that patients undergoing immunotherapy produce more IL-10 and TGF-β,  but it iEvidence that IL-10 might regulate the responses of cells from healthy individuals has been obtained by experiments where the addition of anti-IL-10 receptor antibodies to cultures of their PBMCs enhanced proliferative responses to Der p 1 . This ob+CD25+ T-regulatory cells have been demonstrated on the proliferation responses of PBMCs cultured with cat and pollen allergens. There was, however, no convincing difference in the activity of cells from allergic and nonallergic subjects[+ cells, it was possible to detect a suppressive activity on T-cell proliferation for the cells from allergic, but not nonallergic, subjects [+ population itself. The Th2 cytokine responses were not affected by the presence of CD25+ cells. Studies of atopic dermatitis showed that HDM extract stimulated more of the regulatory cell transcription factor FOXP3 from PBMCs from HDM-allergic subjects than PBMCs from nonallergic subjects, [+ CD25+ cells [+CD25+ high or bright cells are considered to have high regulatory activity, with few or no effector T cells. However, the CD4+CD25+ high subgroup has been shown to contain a mixture of both regulatory and effector T cells, and the percentage of CD4+CD25 high T cells correlated negatively with the suppressive capacity of CD4+CD25+ T cells [Suppressive effects of CD4 subjects,79 or th subjects did showsubjects . Inspectubjects,  and that5+ cells . Studies5+ cells . CD4+CD2 T cells . There i T cells . Althoug T cells .Haemophilus influenzae P6 protein antigen using the same technique and other antibacterial responses [There is good agreement that IgG antibodies to grass,  mite, 1,70,86 raesponses ,90. Theyesponses . Cockroaesponses . Grass pesponses .Fel d 1 has been reported to induce IgG antibodies in most people exposed to cats possiblyMost of the IgE binding to the common sources of allergen is directed to a small number of dominant allergens. Bet v 1 is a very dominant allergen binding 90% of the IgE antibodies to birch pollen in sera from people in Scandinavia . Ole e 1To examine the relative binding of the nonmajor mite allergens to the known major Der p 1 and Der p 2 allergens, a panel of 9 allergens was prepared including allergens known to absorb out the IgE binding to nearly all the allergens detectable by IgE immunoblotting of 2-dimensional electrophoresed extracts . The all+ T-regulatory cells have been proposed as important modifiers of the responses of nonallergic subjects. It is however important to note that the evidence for this is limited, and most studies show that peripheral blood cells from allergic subjects produce more or similar amounts of IL-10 to the cells from nonallergic subjects. This, however, could be the result of an interplay between cytokines in the in vitro culture systems. At least some people exposed to cats make high IgG4 responses to Fel d 1 without the production of IgE antibody, and this could be the result of a regulatory function. It is however possible that the frequency at which this occurs is lower than previously suspected. For HDM allergy, it has been shown that the IgE and IgG responses induced by the different allergens are hierarchical, with the proportion of the response directed to each allergen being similar for all individuals. This not only has use for designing improved allergen preparations, but also can be used as an investigative tool for determining how the responses are controlled.The IgE antibodies induced by allergens are produced in low quantity, and because several features of the B-cell response seem to contribute to this, the system seems tightly regulated. In contrast, the precursor frequency and the amount of cytokine produced by T cells responding to allergens are high and of a similar magnitude to those found for antimicrobial responses. They tend to be higher for allergic compared with nonallergic people, but even the responses of nonsensitized people are similar to those induced by vaccine antigens. The IgG antibody titers are also higher in allergic people, at least for responses to HDM, pollen, and cockroach and mouse allergens. The titers to pollen are small, but the magnitude of the responses to HDM allergens in children are similar to antimicrobial responses and include the IgG1 isotype found in antimicrobial immunity and the highly Th2-dependent IgG4 isotype. Only about 70% of children however produce IgG, and the responses decline with age. It was also found to be severely curtailed in children with intermittent asthma experiencing acute exacerbations. The mechanisms that regulate the IgG responses and perhaps the direct influence of the presence of IgG could therefore be important for disease. The lack of any antibody in nonallergic subjects indicates an inefficient helper T cell function, although allergic and nonallergic subjects have good cytokine responses, as shown by IFN-γ release. The IFN-γ responses of both allergic and nonallergic people are similar and of a similar size to those found in microbial antigens. It is becoming clear that chemokine responses by nonlymphoid cells, especially TSLP and possibly IL-27, are critical regulators of the allergic response, so that responses studied in extended in vitro cultures are not necessarily very informative. Regulatory responses by IL-10-producing cells and CD25Supported by the National Health and Medical Research Council of Australia.


ABCB5 is a member of the ABC protein superfamily, which includes the transporters ABCB1, ABCC1 and ABCG2 responsible for causing drug resistance in cancer patients and also several other transporters that have been linked to human disease. The ABCB5 full transporter (ABCB5.ts) is expressed in human testis and its functional significance is presently unknown. Another variant of this transporter, ABCB5 beta posses a “half-transporter-like” structure and is expressed in melanoma stem cells, normal melanocytes, and other types of pigment cells. ABCB5 beta has important clinical implications, as it may be involved with multidrug resistance in melanoma stem cells, allowing these stem cells to survive chemotherapeutic regimes.We constructed and examined in detail topological structures of the human ABCB5 protein and determined in-silico the cSNPs (coding single nucleotide polymorphisms) that may affect its function. Evolutionary analysis of ABCB5 indicated that ABCB5, ABCB1, ABCB4, and ABCB11 share a common ancestor, which began duplicating early in the evolutionary history of chordates. This suggests that ABCB5 has evolved as a full transporter throughout its evolutionary history.From our in-silco analysis of cSNPs we found that a large number of non-synonymous cSNPs map to important functional regions of the protein suggesting that these SNPs if present in human populations may play a role in diseases associated with ABCB5. From phylogenetic analyses, we have shown that ABCB5 evolved as a full transporter throughout its evolutionary history with an absence of any major shifts in selection between the various lineages suggesting that the function of ABCB5 has been maintained during mammalian evolution. This finding would suggest that ABCB5 beta may have evolved to play a specific role in human pigment cells and/or melanoma cells where it is predominantly expressed. ABCB5 is a member of the ATP-binding cassette (ABC) superfamily of transporters that function in the ATP-dependent transport of structurally diverse molecules. A number of transporters in this family are implicated in multidrug resistance and are recognized causes for the failure of cancer chemotherapy These transporters represent the largest family of transmembrane proteins and are classified into seven families (A–G) in humans, based on the sequence and organization of the nucleotide-binding domain + phenotype where it might mediate doxorubicin resistance. ABCB5 is also indicated to be a metastatic melanoma progression marker, and it is suggested that this transporter has a role in the regulation of progenitor cell fusion The human genome contains 48 ABC genes; 16 of these have known functions, while 14 have been associated with human disease states Among the substrates of this rather elusive transporter are doxorubicin and rhodamine 123 We have tried to bridge this gap by carrying out exhaustive in silico phylogenetic and bioinformatics studies, which indicate that this transporter evolved as a full, rather than a half-transporter, which implies that it functions as a full transporter in certain human tissues or developmental stages. The sequence and topological analysis of the half-transporter in humans shows a unique predicted topology in which the beta form of the transporter has two NBDs (nucleotide binding domains) instead of one, and the first NBD lacks the Walker A region, indicating that this NBD may not be completely functional. A number of single nucleotide polymorphisms  have also been detected in/near this gene. Ten of these are non-synonymous (amino-acid changing) cSNPs, which may affect function. Non-synonymous cSNPs were present in key regions of the gene, including TMDs (transmembrane domains), NBDs, extracellular loops, and the intracellular loops. SNPs in ABC transporters have been reported to play an important role in patients' response to medication and also to play a pivotal factor in risk for disease We determined the topology of the testis-specific full transporter and the beta form because they are most likely to have transporter functions. We constructed models using HMMTOP and displayed them using the program TOPO2. In the full length protein HMMTOP predicted the presence of the conventional 12 TMs with the 2 NBDs, which represents the accepted model for most full transporters . For ABCTraditionally transporter topology has been determined using hydrophobicity plots which determine the locations of the hydrophobic and hydrophilic regions of the transporter (based on the properties of the amino acids) which correspond to the TM regions, extracellular/cytoplasmic regions respectively. Hydrophobicity plots for the full transporter were constructed with TMHMM which predicted 8 TM helices (data not shown). Hydrophobicity plots were also constructed for the beta form with TMHMM. The beta form was predicted to have 5 TM helices and the NBD was predicted to be on the extracellular surface, however when we aligned the ABCB5 beta sequence to human Pgp we found that the topology predicted that the transporter would have 6TM helices instead of 5 (data not shown). It is most likely that the structure predicted by HMMTOP is the closest prediction since it conforms to the basic structure of most ABC transporters, however, the likelihood that this transporter may have a unique structure cannot be completely ruled out.The Motif Scan program was applied for predicting functional motifs within the human full-length ABCB5 amino acid sequence. The analysis revealed the presence of 2 ABC_transporter_2 motifs at amino acids 386–622 and 1015–1253; and 2 ABC_ membrane motifs at amino acids 48–339 and 692–968 . The betThe coiled–coil structure is a highly prevalent protein structural motif In ABCB5.ts 3 coiled coils in the N-terminus were also detected using the program Coils, spanning the amino acids 259-278, 490-505 and 621-636 giving rise to the speculation that possibly the smaller variant form of ABCB5 could be interacting with the coiled-coiled domains of the full transporter and may act in a dominant negative manner. Potential coiled-coil domains were also detected in the N-terminus of the B-family transporters ABCB1, ABCB4 and ABCB11(data not shown) which are the three transporters most closely related to ABCB5 suggesting that these domains are conserved in the B-family members closely related to ABCB5.ABCB5 full transporter gene is located on chromosome 7p21, spans 28 exons and 141.8kb of genomic DNA. This gene encodes 11 different isoforms as depicted in ENSEMBL .With the aid of SIFT analysis we found that a number of the substitutions were not tolerated including: F155V, R260K, Q262K, I369V, D370G, G810V, P996Q, C1054W, K1056E, E1124K and D1187G.Drosophila, C. elegans, and yeast. ABCB11 orthologs are present in non-mammals such as Xenopus, birds, and reptiles, while the duplication event that led to ABCB1 and ABCB4 occurred after the split of mammals from reptiles and before the most recent common ancestor of extant mammals, i.e., sometime between 310 million years ago Xenopus, which suggests the ABCB5 gene may have been lost in some lineages such as birds and reptiles. An analysis of the selection pressure operating on each site reveals that a large proportion of sites in each gene are under strong purifying selection, indicating strong functional constraint  substitutions per nonsynonymous site (Ka) to the number of synonymous (non-amino acid changing) substitutions per synonymous site (Ks). This can be used as an indicator of selective pressure acting on that particular gene. Under neutral conditions Ka/Ks is equal to 1, i.e., mutations occur randomly and the probability of a nonsynonymous mutation becoming fixed in the population is equal to that of a synonymous mutation. A Ka/Ks ratio that is significantly less than 1 indicates that nonsynonymous mutations are less likely to become fixed in a population. This is known as purifying selection as potentially deleterious mutations are purged from the population in order to maintain the sequence of a functionally important site or region within the coding gene.The REL analysis of DataMonkey found that 628 of the 1,464 codons in the full-length alignment of ABCB5 were highly significant for purifying selection. All of the transmembrane domains and NBDs display low Ka/Ks values, indicating that these domains have been under functional constraint. The predicted first NBD of ABCB5 shows comparable levels of purifying selection to the NBDs of the other ABCB genes, revealing that it has been functionally constrained throughout mammalian evolution, and confirming that ABCB5 has evolved as a full transporter. A comparison between the PAML, DataMonkey, and Selecton results gave no consensus of any sites adjudged to be under strong adaptive selection with high probabilities. However, 2 regions, a cytoplasmic region at the start of the gene and a portion of sites at the end of the first NBD, displayed a number of residues with a Ka/Ks value >1, although these were not found to be significantly greater than 1.The GABranch analysis of DataMonkey did not suggest that any specific lineages were undergoing a shift to adaptive evolution. The mean Ka/Ks of each branch was estimated to be under 0.5, indicating that ABCB5 has been undergoing strong to moderate purifying selection along each lineage. The probability that any of these branches was undergoing adaptive evolution was estimated at less than 5%. The only exception was the branch leading to the 6 Laurasiatherian taxa , which was estimated to have a Ka/Ks value of 0.75; the probability that this lineage was undergoing positive selection was adjudged to be 33%. The absence of any major shifts in selection between the various lineages would also suggest that the function of ABCB5 has been maintained throughout mammalian evolution.ABCB5 gene returned a pair of residues with a posterior probability of 0.95 of co-evolving. These residues, 444 and 1196 on the human full-length transcript, are in the predicted first and second NBDs, respectively, and thus may be important for interactions between these two domains.The Spidermonkey analysis of the full-length We have shown in this study that according to topological structure, ABCB5.ts (the full transporter) could be a functional form of ABCB5 and demonstrates the conventional 2-TMD and 2-NBD arrangement generally found in full transporters. Each of these TMDs was determined to contain 6 transmembrane helices using HMMTOP software. We also found 2 potential N-glycosylation sites located in the extracellular loops, which may be important for the trafficking of the protein to the plasma membrane. On the other hand, the ABCB5 beta form demonstrates a rather unconventional structure consisting of 2 potential NBDs and 1 potential TMD, suggesting that: (i) this transporter might have a unique catalytic cycle; (ii) some mechanism of post-translational modification may function to potentially edit the beta form into a more conventional structure to give rise to the functional form of the transporter; or finally (iii) this protein may have some function in addition to transport.Ten non-synonymous cSNPs were found on ABCB5 beta. Of these, one SNP, rs6461515, maps to the potential substrate site (of the protein), suggesting that it may play a role in substrate specificity. We also found this SNP to be slightly more prevalent in South American populations when compared to other populations in the Human Genome Diversity Panel (unpublished data from our laboratory). The rest of these SNPs were in diverse regions of the transporter, including the NBDs. rs58795451, which is in the D-loop of the protein, was determined to be the most deleterious substitution, which agrees well with the fact that the D-loop plays an important role in communication between the NBDs of ABC transporters. The ABCB5.ts (full transporter) has 23 non-synonymous cSNPs, which map to different functional regions of the putative transporter. rs76179099, which maps to the first nucleotide binding domain of the protein, would cause a stop codon to be inserted in this position and may cause the premature translational termination of this ABC protein.From phylogenetic and evolutionary analyses, we have shown that ABCB5 evolved as a full transporter for most of its evolutionary history. There is an absence of any major shifts in selection between the various lineages, which would suggest that the function of ABCB5 has been maintained throughout mammalian evolution. The full transporter form shows similar patterns of site-specific selection when compared to the other B-family full transporters with high levels of purifying selection in both TMDs and NBDs. This indicates that these motifs have retained functional significance throughout mammalian evolution. The full length predicted form in humans also retains these motifs, suggesting that this could be the functional form with respect to normal physiology. However, this form of the transporter has extremely restricted expression in humans (it has only been documented in the testis) nd NBD was located in the cytoplasmic region (www.enzim.hu/hmmtop/). This program operates based on the hypothesis that the topology and localization of the transmembrane segments is determined, not by the specific amino acid composition of the various parts of the protein, but by the amino acid distributions in various regions of the structure www.sacs.ucsf.edu/TOPO2/). TMHMM v2 program (www.cbs.dtu.dk/services/TMHMM/) was used to compute the hydrophobicity plots using the hidden Markov model http://hits.isb-sib.ch/cgi-bin/PFSCAN), which is primarily a data retrieval and analysis system that can match protein sequences to precomputed patterns and profiles from Pfam and Prosite The HMMTOP program was used to predict the topological structure of ABCB5, using the constraint that the 2www.isrec.isb-sib.ch/webmarcoil/webmarcoilC1.html) www.ch.embnet.org/software/COILS_form.html) were used to predict coiled–coil domains. COILS is a program that compares a particular sequence to a database of known parallel two-stranded coiled–coils and computes a similarity score. On comparing this score to the distribution of scores in globular and coiled–coil proteins, the program then calculates the probability that the sequence will adopt a coiled–coil conformation Marcoil (http://bioinf.cs.ucl.ac.uk/psipred/) was used to verify that the regions were actually coiled–coils The Psipred server (www.pantherdb.org) and SIFT (http://sift.jcvi.org). Panther estimates the likelihood of a particular nonsynonymous (amino acid-changing) coding SNP to cause a functional impact on the protein. It calculates the subPSEC (substitution position-specific evolutionary conservation) score based on an alignment of evolutionarily related proteins. Then it scores the likelihood of a single amino acid at a particular position (amino acid PSEC/aaPSEC) or the likelihood of the transition of one amino acid to another . When aaPSEC  = 0, this is the evolutionarily most common allele , whereas more negative values of aaPSEC indicate that the allele is less likely to be observed across evolution (inferred to be less likely to conserve function). The subPSEC score is the difference between the aaPSEC scores for the two alleles. The algorithm takes the absolute value in order to make the scores symmetric, and then multiplies by –1 to adhere to the substitution matrix convention that more negative scores correspond to more severe substitutions. When subPSEC  = 0, the substitution is interpreted as functionally neutral, whereas more negative values of subPSEC predict more deleterious substitutions. The cutoff subPSEC < –3 indicates a deleterious substitution The predicted functional significance of the amino acid substitutions was calculated using Panther software (ABCB gene for each species were downloaded from the Ensembl database (http://www.ensembl.org). If more than one transcript was available, the longest transcript was chosen. Alignments were constructed on the amino acid sequences with the online MAFFT server , using the G-INS-i setting http://www.bork.embl.de/pal2nal/) http://www.datamonkey.org/) http://selecton.tau.ac.il/) rd model that added a category for positively selected sites. In DataMonkey, the Random Effects Likelihood (REL) method was used to estimate the Ka/Ks value for each codon.Amino acid and nucleotide sequences of each In Selecton, the mechanistic-empirical combined (MEC) model was used, which takes into account differences between amino-acid replacement probabilities, under the assumption that more radical changes in amino acid type represent a larger evolutionary step. The GABranch algorithm in DataMonkey was used to measure changes in selective pressure acting along different lineages. The Spidermonkey analysis of DataMonkey was used to infer sites with conditional evolutionary dependencies indicating pairs of sites that may have a functional or structural importance.


They also have a potential role in the osteolysis associated with bone metastases. They are formidable partners in tumor metastasis. © 2010 American Society for Bone and Mineral Research.The growth and metastasis of solid tumors not only depends on their ability to escape from immune surveillance but also hinges on their ability to invade the vasculature system as well as to induce the formation of new blood vessels. Gr-1 Metastasis is the cause of cancer mortality. The growth and metastasis of solid tumors not only depend on their ability to escape from immune surveillance but also hinge on their ability to invade the vasculature system as well as to induce the formation of new blood vessels. Thus tumor blood vessels are promising therapeutic target in the treatment of neoplastic diseases. The approval of bevacizumab, a humanized monoclonal antibody that recognizes and blocks vascular endothelial growth factor A (VEGFA), by the US Food and Drud Administration as a first-line therapy for metastatic colorectal cancer validates the ideas that VEGF is a key mediator of tumor angiogenesis and that blocking angiogenesis is an effective strategy to treat human cancer.–3The microenvironment of solid tumors contains regions of poor oxygenation and high acidity. Growing evidence from clinical and experimental studies points to a fundamental role for hypoxia in metastatic progression. Prolonged hypoxia increases genomic instability, genomic heterogeneity, and epigenetic alteration and thus may act as a selective pressure for tumor cell variants.+CD11b+ cells into the breast carcinoma tumor microenvironment with deletion of TGF-β signaling(One of the responses of the tumor host to hypoxic conditions is an inflammation reaction. This is regulated through hypoxia-inducible factor 1α (HIF-1α) and NF-κB–mediated chemokine and cytokine secretion, resultinsignaling Fig. 1)+CD11b+ c+CD11b+ cells or MDSCs are significantly overproduced in the bone marrow and spleens of tumor-bearing mice. There are high numbers of these cells in peripheral blood of tumor hosts, including cancer patients. MDSCs express CD11b, a marker for myeloid cells of the macrophage lineage, and a marker for granulocytes, Gr-1; thus they are called Gr-1+CD11b+ cells. MDSCs have been demonstrated to be immune suppressive since the 1980s. They inhibit natural killer (NK), B, and T cells through the production of arginase and reactive oxygen species; they also inhibit functional maturation of dendritic cells and promote type II macrophage development and thus represent one mechanism of tumor escape from immune system control and compromise the efficacy of cancer immunotherapy. and low-density polymorphonuclear cells (immature neutrophils). Both populations suppress antigen-specific T-cell responses, but through distinct effector molecules and signaling pathways. Recently+CD11b+ immature myeloid cells are a major source for high levels of TGF-β in the tumor host.(+ cytotoxic T lymphocytes (CTLs) such as perforin and granzymes, cytotoxins that act through the granule exocytosis pathway., which suppressed apoptosis of tumor cells. and by directly incorporating into tumor endothelium. Furtherm locally. These st+CD11b+ to tumor metastasis particularly interesting: (1) These cells are overproduced in tumor hosts that include cancer patients with a variety of tumors. (2) MDSCs are composed of immature myeloid cells at the early stages of differentiation. They are different from terminally differentiated tumor-associated macrophages (TAMs), identified as Mac-1 (CD11b) and F4/80+, which have been shown to promote tumor progression and metastasis through elevated CSF-1 production and enhanced epidermal growth factor (EGF) signaling in cancer cells.–expressing cells activate nuclear IKKα and inhibit the transcription of tumor metastasis suppressor Maspin, thereby promoting tumor metastasis. TGF-β1 nn cancer. Furthermn cancer. In additn cancer.+) are shown to be recruited to colon cancers with deletion of Smad4 and promote tumor invasion.Fig. 1). invasion. Indeed, invasion. In TGF-βinvasion.+ myeloid cells.(In the distant premetastatic lung, TGF-β is one of the factors produced by tumor cells responsible for the production of the chemoattractants S100A8 and S100A9, which attract Mac1id cells. Through id cells. In additid cells.Hematopoietic progenitors, or MDSCs, play an important role in the progress of metastasis at any site and certainly in the bone marrow. Therefore, they are likely important in the progression of multiple myeloma and other tumors that frequently metastasize to bone, including breast cancer, prostate cancer, and lung cancer. Since MDSCs have a cell surface phenotype that overlaps with that of progenitors in the osteoclast lineage, this suggests that they also may have a capacity to develop into osteoclasts at this site. Our group has examined their role in the progression of the osteolytic bone disease associated with multiple myeloma. Myeloma bone disease is characterized by osteoclast activity adjacent to myeloma deposits. Nothing is known of the origin of osteoclasts in either patients or preclinical murine models of myeloma. We do not know where they come from or what their precursors are. However, it is clearly likely to be an issue of importance. The molecular signals that pass between osteoclast precursors and myeloma cells in the bone microenvironment are likely critical to progression of the disease, raising the possibility that myeloma cells may influence MDSC differentiation and/or function.+ mice bearing myeloma. Following coinoculation of these lacZ+ MDSCs with myeloma cells into recipient mice, cells positive for both lacZ and TRACP were observed on the bone surface, demonstrating the MDSCs have the capacity to differentiate into osteoclasts in vivo.(We have studied this in the 5T model of myeloma. The 5T model of myeloma was described originally by Radl and colleagues.,38 They  in vivo.,40 What  in vivo.. Future +GR-1+Ly-C6−, was demonstrated in the long bones of a syngeneic mouse model of breast cancer bone metastasis.(Myeloma serves as a potential paradigm for similar events occurring in solid tumors that metastasize to bone. In these situations, it is possible that the molecular mediators may be different, but the general principles may remain the same. In support of this, an increase in the proportion of granulocytic myeloid-derived suppressor cells, defined as CD11btastasis.If these cells are so important to the skeletal complications associated with cancer, then one question is whether depletion of MDSCs affects tumor progression. There are potential mechanisms by which this could be achieved, for example, by liposome-mediated clodronate or other biosphonates, and it would be interesting to see the efficacy of these approaches when used in this manner. Bisphosphonates are in clinical use for the treatment of myeloma bone disease, and their effects on mature osteoclasts are well studied. However, little is known of their effects, if any, on osteoclast precursors such as MDSCs. Melani and colleagues demonstrated that zoledronate decreased the expansion of MDSCs associated with tumor-bearing mice. This wasMDSCs are a heterogeneous population of cells that are enhanced in tumor-bearing hosts and appear to play a number of roles in immune suppression and metastasis. It is exciting to speculate that these cells may have a distinct role in bone metastases, related not only to their immune suppressive properties but also to their potential to contribute to osteoclastogenesis and the subsequent destructive osteolytic bone disease. Future studies to elucidate their specific role in osteolytic bone disease not only will increase our understanding of the pathophysiology of cancer-induced bone disease but also may reveal novel therapeutic targets for the treatment of this destructive characteristic feature of bone metastases.


These findings add to growing indications of richly diverse roles of adapters in p97-mediated signaling functions.In the ubiquitin-proteasome system, a subset of ubiquitylated proteins requires the AAA+ ATPase p97  for extraction from membranes or protein complexes before delivery to the proteasome for degradation. Diverse ubiquitin adapters are known to link p97 to its client proteins, but two recent papers on the adapter protein UBXD7, including one by Bandau http://www.biomedcentral.com/1741-7007/10/36See research article:  The ubiquitin-proteasome system mediates degradation of misfolded or damaged proteins to ensure cellular protein homeostasis, and selectively removes regulatory proteins in critical signaling pathways. The key step is the posttranslational modification of the substrate with the small protein ubiquitin and extension to ubiquitin chains that serve as a signal for degradation by the proteasome. The ubiquitylation reaction is mediated by a cascade of enzymes, comprising the E1 ubiquitin-activating enzyme, an E2 conjugating enzyme and an E3 ligase that functions in the attachment of the ubiquitin to the substrate  . Althouget al.  establiset al. .Importantly, while various p97-adapter complexes associate with different ubiquitin ligases, the UBXD7 adapter  stands out because it is the only adapter to have been implicated in direct binding to CRLs. UBXD7 has now been shown, in parallel studies by the Alexandru and Deshaies groups , to bindβ-TrCP ligase and reduces substrate ubiquitylation [What might be the functional significance of UBXD7 binding to neddylated CRLs? In the case of the Cul3 substrate Rpb1 in yeast, the UBXD7 orthologue Ubx5  is required together with p97 for efficient degradation . This sutylation . MoreoveAlexandru and colleagues  propose The new insights into UBXD7 function add to accumulating evidence that p97-adapter complexes are more multifaceted and their function is more complex than in a simple linear model, where the adapters' sole role is to link the ubiquitylated client to p97 ATPase Figure . One indThis cofactor diversity also entails complexity in ubiquitin binding. Cofactors including UBXD8, like UBXD7, themselves contain ubiquitin-binding UBA domains that work in addition to the UBDs in the p97-Ufd1-Npl4 core complex that they associate with. Conversely, there are core complexes with adapters that do not contain apparent UBDs: p97-p37 and p97-UBXD1 are such complexes [An important class of cofactors with clear functional implications are the downstream processing factors that include a growing number of deubiquitylating enzymes . They caFurther important implications for functional versatility can be drawn from the recognition that UBXD7 binds not only ubiquitin but also the ubiquitin-like modifier NEDD8. This raises the possibility that adapters may interact also with other ubiquitin-like modifiers, thus increasing the substrate spectrum of p97 and even further establishing it as a decoding and integration hub in far reaching signaling networks. Examples are the binding of the p47 adapter orthologue Shp1 to the ubiquitin-like modifier Atg8 during autophagy, or the association of p97 and Ufd1 with the SUMO system in yeast . Lastly,p97 is now established as a key element of ubiquitin-regulated processes. Clearly, ubiquitin adapters link p97 to ubiquitylated clients and thus position its function downstream of ubiquitination. However, it is becoming obvious that there is more to p97 complexes than mere substrate binding and extraction and that they constitute signal integration hubs. Much work lies ahead to understand the individual functions of the adapters, how p97 complexes modulate and coordinate events upstream and downstream of p97-mediated extraction and how this is embedded in complex signaling networks.


Statement of Problem: Studies reported that nearly 60% of endodontic failures have been attributed to inadequate obturation of the root canal system. Thus, complete obturation of the root canal system and proper apical seal are essential elements in the long-term success of root canal treatment.Purpose: This study aimed to compare the apical seal of lateral condensation technique in the room and in body- simulated temperature.Materials and Method: In this experimental study, 70 extracted, single- rooted, human premolar teeth were instrumented and divided up into four groups. All tooth’s canals were obturated by lateral condensation technique except the teeth in the positive control group. Group 1and 2, each with 30 teeth, were obturated in the room and intracanal temperature respectively. The other two groups were positive and negative control group each with 5 teeth. All groups except negative control were covered by two layers of nail polish. Then linear dye penetration was evaluated with a stereomicroscope. Data was analyzed with student-t test and also Kolmogorov- Smirnov Goodness- of- Fit test to make sure of data. Results: Results showed that dye penetration in group one (obturation in room temperature) was 0.6mm more than group 2 (obturation in simulated-body temperature) although this was not statistically significant (p> 0.05).Conclusion: Under the condition of this invitro study, apical sealing ability was better in the body-simulated temperature than the room temperature, although it was not statistically significant. The complete obturation of root canal systems, and the fluid tight seal is the main goal of endodontic treatment to obtain a complete seal along the entire length of root canal systems . It has Lateral condensation technique of Gutta-percha is the most popular method and this technique is still trained in most dental schools -11. AlthTo achieve a better 3- dimensional filling of spaces, different warm gutta percha have been developed that employ the gutta percha thermoplasticity. The use of heated gutta percha improves the homogeneity of the filling mass and adaptation to dentinal wall , 13. Many evaluations of sealing ability of different obturation techniques  have been carried out , 14-16. This study evaluates the sealing ability of lateral condensation technique in two different temperatures, room temperature and intracanal (body simulated) temperature. In this experimental study a very fine sensor, connecting to a digital thermometer , was designed to measure intracanal temperature. This device is operated with alternate current and a 9 volt battery and shows the temperature between 6-500˚c . For evaluation of internal root canal temperature, multiple patients, requisite for root canal therapy were chosen. After cleaning and shaping, each canal was dried with sterile paper points and the disinfected probe was introduced into the canal 3-4 mm beneath the orifice. The temperature in different canals ranged between 33-35˚c. Samples were seventy extracted human mandibular premolars. They all had a single and a straight canal with complete apices. Teeth with different anomalies in crowns or roots with severe curvature, crazes or visible fractures were removed from the study. Radiographs were taken bucco- lingually and mesio- distally to identify whether the samples had a direct and single canal. The crowns were removed so that the remaining lengths of the roots were about 14-20mm. Soft tissues and calculus were removed from the root surfaces mechanically with hand curettes. All roots were immersed in 0.01% sodium hypochlorite concentration for 24 hours. The working length was recorded with ≠10 or ≠15 k file . The file was introduced into the canal till its tip came out in the apical foramen, and working length was established 0.5mm shorter that the apex. 1 to F2 protaper NiTi instrument . Irrigation with 2ml of 2.5% sodium hypochlorite was performed between the applications of each two files. After preparation teeth were divided into four groups randomly. Two experimental groups (30 teeth in each group). The two experimental groups were obturated with cold lateral condensation technique in two different temperatures with ZOE sealer . All root canals were prepared with SGroup 1oc): After root canal preparation, canals were dried with sterile paper points. Master gutta-percha was selected for each root canal. The tug back was confirmed in the working length and the proper spreader was selected. ZOE sealer  was prepared according to the factory instructions. Accessory cones were used until they could not be introduced into the canal with more than 2-3mm of their length. Gutta- percha was removed up to 2 mm apical to the orifice with a flame- heated excavator and were cervically sealed with Coltosol . Specimens were radiographed to confirm the competence of root canal obturation and were kept between two wet gauzes.Obturation in room temperature : For simulating intracanal temperature two- layer glass cavity (60×40×40cm) was designed with automatic thermostat and completely humidified (100%) atmosphere . To confTeeth in group#2 were transferred into the cavity but gutta- percha, sealer and paper points were kept out of the cavity. After root canal preparation, teeth were transferred into the glass box. Canals were dried with sterile paper points. Master gutta-percha was selected for each root canal. The tug back was confirmed in working length and the proper spreader was chosen. ZOE sealer  was prepared according to the factory instructions in the room temperature. Accessory cones were used until they could not be inserted into the canal more than 2-3mm of their length. Gutta -percha was removed up to 2 mm apical to the orifice with a flame- heated excavator and cervically sealed with Coltosol . Radiographs were taken to check and confirm the obturation quality.3- Positive ControlThe similar preparation method was used for this group. Teeth were dried with sterile paper points and one gutta- percha without any sealer were inserted into each canal freely. Coltosol  was used to seal 2-3 mm coronal part of each canal. After taking radiographs, all specimens were preserved at room temperature and in humidified atmosphere. All specimens in the negative control group were prepared and obturated as the group one and sealed with 2-3 mm of Coltosol in the coronal part. After canal obturation, all the samples were evaluated regarding the quality of obturation by taking radiographs. Experimental groups and the positive control group were put in 100% humidity and 37ºc for 3 days in the incubator. The apical part of negative control group was coated with a layer of sticky wax in order to prevent dye penetration. Total root surfaces were covered by two layers of nail polish. Following obturation root surfaces of experimental groups and positive control group were coated with two layers of nail polish up to the apical 2mm. Then all the teeth were put in room temperature for 72 hours in pelican ink  in the vertical position so that 3mm of the crown was out of ink. The roots were rinsed with tap water for 1 minute and then the nail polish was completely removed with acetone and cotton wool balls. The root surfaces were then evaluated by Zeiss stereomicroscope  at × 6 magnification for fracture. In order to evaluate dye penetration in the teeth, teeth were cleared by the Tagger method . Maximum linear dye penetration was observed under a stereomicroscope  at× 6 magnification with an accuracy of 0.25 mm and by three different observers. For each tooth maximum linear dye penetration was recorded and measured by a special ruler with 0.1 mm accuracy. Finally collected data was compared using student T-test and also Kolmogorov- Smirnov Goodness of fit test to make sure of the data. The negative control group demonstrated no dye penetration while the positive control showed dye penetration along the entire root length. The mean linear dye leakages for the two groups are shown in p> 0.05) although there is a difference between written results. Mean dye leakage of group one with 29 specimens (one tooth was omitted during the procedure) was 1.67 mm and this was 1.09 mm for group 2. The difference was nearly 0.6 mm   .We do also Wilcoxon test in order to evaluate inter observer agreements in data . Three oApical sealing ability is one of the important criteria of a three-dimensional obturation of the root canal system. Microleakage is a good indicator to assess the sealing ability of root end filling materials. A variety of techniques have been developed to evaluate the microleakage including fluid filtration , cross sWe used dye penetration technique using a stereo microscope to evaluate the micro leakage of specimens like many other studies , 31-37.Temperature is an important element that can cause different changes in the gutta percha characteristics. It seems that high temperature makes gutta-percha softer and affects its sealing ability. This study was designed to compare the sealing ability of gutta- percha in the room and body- simulated temperature. In this study, the samples filled in the body-simulated temperature showed less microleakage although not significant. Many of studies in this field are evaluating the sealing ability of the different techniques with and without sealers in room temperature -5, 38-40Most studies have found that injection- molded thermoplasticized techniques yield apical seals better or similar to the lateral condensation techniques , 41-42. Sadeghi et al found no significant difference in the density and sealing ability of lateral compaction using two different spreaders and vertical compaction using a BeeFill device [40]. Dadresanfar et al, in 2010, compared the sealing ability of lateral condensation technique and BeeFill system and found that the dye leakage in the BeeFill system was less than lateral condensation technique, although this wasn’t statistically significant .Another study in 2010, showed the better sealing ability of thermoplasticized gutta percha compaction in comparison with the lateral condensation technique, although it was not statistically significant either [4].The results of these studies are in accordance with the results of our study. However, these and other studies have not evaluated the sealing ability of gutta-percha in body simulated temperature .This study showed better penetration of spreader in the group two (35˚c) and subsequently, more accessory cones were placed in each tooth in this group. Results showed that dye penetration, in group 2, was nearly 0.6 mm lesser than group1; although this was not statistically significant. The absence of any significant statistical difference can be explained in this way that this 10-12˚c difference in temperature could not affect the sealing ability of gutta percha in the lateral condensation technique. Thus it seems that the results of the previous studies, regarding the lateral condensation technique, can be extended to invivo situations. Evidently, more studies are needed to evaluate different kinds of obturation techniques in body-simulated temperature. For a better appraisal, other studies can be designed, with more samples and different techniques, to consider these results more reliable.Under the condition of this invitro study, apical sealing ability was better in the body-simulated temperature compared to the room temperature, even though it was not statistically significant.


Join2move. The intervention incorporates core principles of the behaviour graded activity theory (BGA). The aim of this study was to investigate the preliminary effectiveness, feasibility and acceptability of Join2move in patients with knee and/or hip OA.A large proportion of patients with knee and/or hip osteoarthritis (OA) do not meet the recommended levels of physical activity (PA). Therefore, we developed a web-based intervention that provides a tailored PA program for patients with knee and/or hip OA, entitled Join2move. Besides the pilot study we performed two usability tests to determine the feasibility and acceptability of Join2move. In the first usability test, software experts evaluated the website from a list of usability concepts. In the second test, users were asked to verbalize thoughts during the execution of multiple tasks.A non-randomized pilot study was performed among patients with knee and/or hip OA. Primary outcomes were PA (SQUASH Questionnaire), physical function (HOOS and KOOS questionnaires) and self-perceived effect . Baseline, 6 and 12 week follow-up data were collected via online questionnaires. To assess feasibility and acceptability, program usage (modules completed) and user satisfaction (SUS questionnaire) were measured as secondary outcomes. Participants from the pilot study were invited to be interviewed. The interviews focused on users’ experiences with Join2move. In particular, performing exercise at one's own pace without time or travel restrictions was cited as convenient. However, some minor flaws were observed. Users perceived some difficulties in completing the entire introduction module and rated the inability to edit and undo actions as annoying.Twenty OA patients with knee and/or hip OA between 50 and 80 years of age participated in the pilot study. After six weeks, pain scores increased from 5.3 to 6.6 (p=0.04). After 12 weeks this difference disappeared (p=0.5). Overall, users were enthusiastic about This paper outlines the preliminary effectiveness, feasibility and acceptability of a web-based PA intervention. Preliminary results from the pilot study revealed that PA scores increased, although differences were not statistically significant. Interviews and usability tests suggest that the intervention is feasible and acceptable in promoting PA in patients with knee and/or hip OA. The intervention was easy to use and the satisfaction with the program was high.NTR2483The Netherlands National Trial Register. Trial number:  Osteoarthritis (OA) in the knee and hip is a degenerative joint disorder with a high prevalence that increases with age. The disease is associated with pain, functional disability and impaired quality of life ,2. OA isSince general practitioners (GP) are considered the first and main point of contact for people with OA, the general practice is ideally situated to promote PA. In practice, however, a GP’s ability to encourage physical exercise is limited by time constraints and lack of standard protocols ,14. In pJoin2move. Over the course of one year, we used an iterative design methodology to test, analyse and refine the Join2move program. As part of the iterative development process, this paper focuses on the preliminary effectiveness and the usability of Join2move.The World Wide Web provides an alternative medium for reaching outside care patients. In Europe 61% and in North America 79% of the population have internet access . AlthougJoin2move intervention is a fully-automated web-based intervention which contains automatic functions (automatic text messaging and automatic e-mails) without human support. Participants are initially presented with the homepage http://www.artroseinbeweging.nl. The password-secured PA program is available 24/7 from the homepage and is provided without charge. In keeping with the BGA treatment, the Join2move intervention is a self-paced nine week PA program in which patients’ favourite recreational activity is gradually increased in a time-contingent way. In the first week of the program, users select a central activity , perform a 3-day self-test and determine a short term goal for the next eight weeks. Based on test performances and a short term goal, eight tailored weekly modules are automatically generated. Every week, new weekly assignments and evaluation forms (pain and performance) are posted on the password-secured website. If a scheduled weekly module is missed, users can choose to repeat the module, adapt the difficulty or continue with the next module. Since personal messages are updated on a weekly basis, users are encouraged to log in once a week. Automatic e-mails are generated if participants do not visit the website regularly. A description of the intervention is provided in Table Development was based on a systematic review  and a prJoin2move program. Consequently, our research question was:“ What is the preliminary effectiveness , feasibility and acceptability of Join2move in patients with knee and/or hip OA?” “Feasibility” concerns whether we are capable of carrying out Join2move in a larger study. “Acceptability” is whether participants support or reject Join2move.Extensive exploration is needed in order to examine the potential of the Join2move intervention. A second purpose was to determine program use and user satisfaction with the Join2move intervention. This pilot study, which aimed to provide a basis for a large Randomized Controlled Trial (RCT), was part of a research protocol which has been approved by the ethics committee of the VU University Medical Center Amsterdam .This pilot study used a non-randomized design. Our primary focus was to determine the preliminary effectiveness of the Patients with self-reported knee and/or hip OA were recruited through advertisements in Dutch newspapers and online health-related websites. Eligibility criteria were 1) age 50–80; 2) self-reported OA in knee and/or hip; and 3) no physical therapy and/or treatment from a medical specialist for OA in the last six months. Potential participants were excluded if they 1) had no internet access at home, 2) were unable to understand the Dutch language and 3) had contra-indications (loss of consciousness and cardiovascular disease) for PA without medical supervision. To verify self-reported diagnosis, we performed clinical tests to assess the presence of knee and/or hip OA. Assessments were performed by a physiotherapist after the study period, according to the American College of Rheumatology (ACR) ,27.Interested patients who met the inclusion criteria were sent an invitation letter requesting informed consent. Once written informed consent was obtained, participants were invited to fill out a baseline questionnaire. After the baseline assessment, participants were assigned to the intervention. We conducted two online posttests at 6 and 12 weeks after baseline.Join2move intervention, primary outcome measures in this study were PA, physical function and self-perceived effect. Secondary outcomes were OA symptoms, sport and recreation and quality of life. The first primary outcome, self-reported PA, was measured by the Short Questionnaire to Assess Health-enhancing PA (SQUASH) [To assess the potential effectiveness of the (SQUASH) . Pain sc(SQUASH) ,30 and t(SQUASH) ,32. The To assess the feasibility and acceptability of the intervention, program usage and user satisfaction were measured as secondary outcomes. Program usage was measured by the number of weekly modules completed. Once a participant read the weekly assignments and filled out the evaluation form, the module was defined as completed. Adequate exposure to the program was achieved if users interacted at least 75% with the program content. This cut-off point was determined by the research team on the basis of previous research . User saJoin2move intervention, viz.,1) heuristic evaluation, and 2) the Thinking Aloud approach. For the heuristic evaluation, four software experts from Netherlands Institute for Health Services Research (NIVEL) were invited to participate. With respect to the Thinking Aloud approach, five patients between the ages of 50–80 years with self-reported knee and/or hip OA were recruited via the Dutch Arthritis Foundation. The sample size for the Thinking Aloud approach was based on previous research by Nielsen [Two qualitative tests were performed to determine the usability of the  Nielsen . The autDoes the system behave consistently?”, “Does the site use words that older adults know?”, “Is the program perceived as helpful?”(see Appendix 1 for the full list of heuristics). Software experts individually evaluated the website, based on the list of heuristics. Subsequent discussion yielded a list of usability issues. The second instrument, the Thinking Aloud approach [The first usability test, the heuristic evaluation, was performed by means of a set of usability criteria created by Jakob Nielsen  and Danaapproach , was useOf the 47 registered patients, fifteen 32%) did not meet the inclusion criteria. Reasons for exclusion were: no OA symptoms (n=3); receiving treatment from a physical therapist for OA (n=2); OA in other joints than knee or hip (n=7); and not meeting the age criteria of 50–80 years (n=3). Furthermore, seven (15%) participants did not return the informed consent document and five (11%) participants withdrew after returning informed consent. A total of twenty (42%) participants were finally included. Sixteen (80%) participants agreed to be interviewed. According to the ACR criteria, thirteen of the sixteen participants (81%) had clinical knee and/or hip OA, and three participants (19%) had no OA. Participants’ demographic characteristics are shown in Table % did notPA results at baseline, six weeks and twelve weeks are given in Table Join2move. As one user commented “When I skipped my weekly PA exercise due to other commitments, I had no opportunity to repeat that exercise. That was frustrating”. The results from the SUS among 15 participants revealed an average score of 73 points (SD 15) on a 100-point scale questionnaire. According to the study of Bangor et al. [The website was easy to use” and nearly all patients disagreed with the statement “I think I would need technical support to be able to use the program”.The majority of participants  selected walking as the central activity. Other selected activities were floor exercises , cycling , domestic tasks , gardening , and rowing . A total of twenty participants commenced the intervention with the program introduction. Login-file analyses revealed that 100% (n=20) of the users completed the introduction module. Overall, 55% (n=11) of the participants completed at least 75% of the program (≥7 week assignments). 70% (n=14) achieved 60% program exposure and 30% (n=6) were exposed to at least 30% of the intervention. The exposure percentage declined over time. The most listed reasons for skipping a weekly PA were other commitments or of lack of time. Adverse events, such as extreme pain or injuries, were not reported during the program. The 16 interviews revealed that performing the activities in one's own time and at one's own pace was regarded as convenient. In general, participants perceived the website as an additional motivation to perform PA. However, the interviews also revealed an important usability issue. It became clear that patients were dissatisfied with the rigid character of r et al. , this scExperts in heuristic evaluation rated the rigid character of the intervention as a disadvantage. This was in accordance with results from the interviews. Results of the Thinking Aloud test are given in Table Based on the results of the interviews and the heuristic evaluation, we changed the program's time contingent structure (i.e. fixed time periods) into a more flexible format. In the most recent version, options have been included which give users the choice of repeating modules and adapting the difficulty of the modules. The usability errors from the Thinking Aloud approach had more to do with the design of the website and the location of several buttons. These relatively minor problems were also addressed.Join2move is a plausible, feasible and acceptable program for patients with knee and/or hip OA. Although effectiveness was not proved due to the lack of power, results do indicate that Join2move has the potential to increase PA levels in patients with knee and/or hip OA. Participants reported higher levels of PA, particularly (and as expected) involving moderate activities like walking and cycling (200 minutes). In line with other research [Results from this study indicate that research , walkingresearch . In the research ,43.Providing an intervention does not automatically mean that patients will use it, particularly when it is self-directed, with minimum personal contact. Since the success of web-based interventions requires active participation, non-usage attrition has been pointed out as a common concern in the field of web-based education. In line with other studies, ,22,44, tWith respect to usability, the involvement of end-users was extremely valuable for identifying usability issues and system flaws. Along the way, we incorporated greater flexibility into the program. The implemented changes resulted in a less rigid version with more options tailored to the performance of the individual user.Join2move intervention. Typically, these patients are disproportionately less educated and have a lower income. Particularly with regard to these under-served populations, GPs should refer sedentary OA patients more frequently to a physical therapist or other health care provider. Further, it will be important to translate Join2move for other self-help platforms, such as videos, brochures and self-help books. A final limitation is that we only performed one Thinking Aloud test to detect and resolve usability issues. Unfortunately, we did not retest the redesigned intervention. In order to optimize usability for the implementation phase, a repetition of this procedure is advised.The findings from this study need to be interpreted in light of the study's limitations. The small sample size, single group design and lack of long-term assessments limit conclusions of causality, long-term effects and generalizability. Furthermore, the potential presence of the so-called Hawthorne effect may have contributed to an overestimation of PA scores. This implies that observed PA changes may be partly the result of study participation. Besides the Hawthorne effect, self-reported PA measures may also contribute to an over-estimation of PA levels in this study. This may be a consequence of recall error, perceived social desirability and other biases. To obtain the best results, a combination of validated questionnaires with objective measures would be preferable in future studies. Another limitation concerns outside-care patients who lack computer skills or internet access. These groups are mostly excluded from web-based interventions. Unfortunately, this disadvantage applies also to the Join2move has the potential to contribute to meeting this need. The intervention is unique, since this is the first web-based PA intervention focusing on outside-care patients with OA. Moreover, while most web-based PA interventions have additional human contact, the Join2move intervention is fully computerized. Given the fully automatic character, the program has the potential to reach large populations while placing a minimal burden on our scarce health resources. This paper illustrates how involving end-users and experts can contribute successfully to the development of a web-based self-help intervention. The results suggest that the intervention is feasible and acceptable in promoting PA among patients with knee and/or hip OA. The intervention was easy to use and satisfaction with the program was high. This suggests that the intervention is acceptable for patients with knee and hip OA. Preliminary results from the pilot study revealed that PA scores increased, although differences were not statistically significant. A randomized controlled trial is needed to determine the effectiveness of the Join2move program.Strong evidence indicates that regular PA is important in the management of OA. To date, however, many patients with knee and/or hip OA remain sedentary. Unfortunately, the vast majority of these patients do not receive any help in the promotion of PA. Low-cost, effective and accessible PA interventions are needed. Although our results are not conclusive, this study suggests that Usability items used for the heuristic evaluationInteraction1) Are the links to websites consistent throughout the website?2) Do buttons and links show that they have been clicked?3) Does the ‘back’ button appear on the browser toolbar on every page?4) Are error pages descriptive, and did they provide a solution to the user?5) Does the system inform users what is going on through appropriate feedback within a reasonable time frame?6) Does the system behave consistently?7) Does the system eliminate error-prone conditions and present users with confirmation options before they commit to the action?Information and architecture8) Is the path for any given task a reasonable length (2–5 clicks)?Visual design9) Is the default font size 12-point or larger? If not, is there an obvious way on the page to increase the font size? If not, does changing the font size in the browser enlarge all of the text?10) Are text and interaction elements a different colour from the background? Are clickable items highlighted differently from other non-clickable highlighted items?Information design11) Has the amount of text been minimized; is only necessary information presented?12) Is the content written in the active voice, directed to “you”?13) Does the site use words that most older adults know? Are instructions written in plain language?14) Is a relevant help button provided? Does the system provide documentation about the website?Persuasive principles15) Can users relate to and feel familiar with the context, images and figures that appear in the program?16) Does the system contain the knowledge to be learned?17) Is the program easy to use and are the tasks easy to perform with a small number of steps and keystrokes?18) Can users learn about how they solved the tasks on previous occasions when the system was used?19) Are users aware that the moderator can observe and see the results?20) Do users get rewards or praise when a task is performed correctly21) Is the program perceived as helpful?22) Does the program act as a coach?The authors declare that they have no competing interests.DB was responsible for day to day management of the project, developed the web-based intervention, collected all data, analysed the data and wrote the paper. CV had the idea for the study and managed the project. JD and DdB contributed to the intellectual content of the manuscript. All authors read and approved the final document.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6947/13/61/prepub