Introduction. Reduced kidney function, approximated by elevated cystatin C, is associated with diastolic dysfunction, heart failure, and cardiovascular mortality; however, the precise mechanism(s) that account for these relationships remains unclear. Understanding the relationship between cystatin C and subclinical left ventricular (LV) remodeling, across ethnically diverse populations, may help explain the mechanisms underlying the association of kidney dysfunction with heart failure and cardiovascular mortality. Methods. Measures of cystatin C and LV parameters were obtained from the multi-ethnic study of atherosclerosis (MESA) cohort at baseline . LV parameters; LV end-diastolic (LVEDV) and end-systolic volumes (LVESV), LV mass (LVM), concentricity (LV mass/LV end-diastolic volume), and LV ejection fraction (LVEF) were measured using magnetic resonance imaging. Nested linear models were used to examine the relationship between higher quartiles of cystatin C and LV parameters, with and without adjustment for demographics, height, and weight, and traditional cardiovascular risk factors. Similar analyses were performed stratified by ethnicity and gender. Results. A fully adjusted model demonstrated a linear relationship between higher quartiles of cystatin C and lower LVEDV, . Associations were also observed between higher quartiles of cystatin C and lower LVESV (P = 0.04) and concentricity (P = 0.0001). In contrast, no association was detected between cystatin C and LVM or LVEF. In analyses stratified by race and gender, the patterns of association between cystatin C quartiles and LV parameters were qualitatively similar to the overall association. Conclusion. Cystatin C levels were inversely associated with LVEDV and LVESV with a disproportionate decrease in LVEDV compared to LVM in a multi-ethnic population. This morphometric pattern of concentric left ventricular remodeling, may in part explain the process by which kidney dysfunction leads to diastolic dysfunction, heart failure and cardiovascular mortality. Elevated cystatin C, a marker of reduced kidney function, is associated with increased cardiovascular mortality  and incin = 2,622), 28% African-American , 22% Hispanic , and 12% Chinese (n = 803). Individuals with a history of physician-diagnosed myocardial infarction, angina, heart failure, stroke, or transient ischemic attack, or who had undergone an invasive procedure for CVD  were excluded from the study at baseline (2000–2002). This study was approved by the Institutional Review Boards of each study site, and written informed consent was obtained from all participants.The Multi-Ethnic Study of Atherosclerosis (MESA) design has been previously described . BrieflyMedical history, anthropometric measurements, and laboratory data for the present study were taken from the first examination of the MESA cohort (July 2000–August 2002). Information about age, sex, ethnicity, and medical history was obtained by questionnaires. Resting blood pressure was measured using Dinamap Monitor PRO 100  automated oscillometric device. Three measurements were obtained at 1-minute intervals with the subject in the seated position with back and arm supported after 5 min of rest with an appropriate-sized cuff, with the cuff at the level of the heart, using a standardized protocol. The average of the second and third measurements was recorded as the resting blood pressure. Hypertension was defined as a systolic blood pressure ≥140 mm Hg, a diastolic blood pressure ≥90 mm Hg, or currently taking medications for blood pressure control. Smoking use was defined as never, former, and current smokers. Diabetes was defined as a fasting glucose ≥126 mg/dL or use of insulin or hypoglycemic medications. Total cholesterol was measured from blood samples obtained after a 12-hour fast and measured using a standardized kit (Roche Diagnostics). Cystatin C was measured from frozen sera at a central laboratory  using a BNII nephelometer  and a particle-enhanced immunonephelometric assay  . The anaThe MESA MRI protocol has been described in detail elsewhere . BrieflyDifferences in baseline characteristics were compared across quartiles using analysis of variance (ANOVA) for continuous variables and Chi-square tests for categorical variables. The LV parameters of interest, LVEDV, LVESV, LVM, LVEF, and concentricity (LVM/LVEDV), were compared across cystatin C quartiles using ANOVA. Multivariate linear regression analyses in nested models were performed to evaluate the association of cystatin C quartiles, with the following outcomes: LVEDV, LVESV, LVM, LVEF, and concentricity (LVM/LVEDV). After univariate analysis (Model 1), the following covariates were considered as potential confounders: age, gender, race/ethnicity, height, and weight (Model 2); Model 2 covariates plus traditional cardiovascular risk factors: history of diabetes, antidiabetic medications, hypertension, systolic blood pressure, total cholesterol, antilipid medications, and smoking (Model 3). Similar linear analyses in nested models were performed stratified by ethnicity and gender. Interaction terms using (cystatin C quartile × race) and (cystatin C quartile × gender) were introduced in the model. In a separate analysis, where serum creatinine or creatinine-based estimated glomerular filtration rate (eGFR-CR) was added in the model to assess the association of cystatin C quartiles with LV parameters, collinearity diagnostics such as variance inflation factor was performed to avoid unstable parameter estimates. Sensitivity analysis between log transformed cystatin C and LV parameters was also performed. All statistical analyses were performed using JMP Version 8 .Of 6,814 MESA participants, 1,786 did not have MRI measures of LV structure and function, and 58 had no serum cystatin C measures, leaving 4,970 participants who were included at baseline for analysis. Cystatin C levels ranged from 0.36 to 7.59 mg/L with a mean value of 0.89 mg/L and a median of 0.86 mg/L (interquartile range: 0.76 to 0.98 mg/L). For ease of comparability with previous studies, cystatin C was divided into quartiles: quartile 1 (≤0.76 mg/L), quartile 2 (>0.76 and ≤0.86 mg/L), quartile 3 (>0.86 and ≤0.98 mg/L), and quartile 4 (>0.98 mg/L). Participants with higher concentrations of cystatin C were more likely to be older, to have diabetes and hypertension, and to use antihypertensive medications. Those with higher cystatin C levels were less likely to be Chinese or black and had higher body mass indices. As expected, increasing quartiles of cystatin C were associated with higher levels of creatinine and lower creatinine-based estimated glomerular filtration rates .P values for trend between higher cystatin C quartiles and MRI measures of LV remodeling are demonstrated in The unadjusted associations and P = 0.0001, LVESV P = 0.04, and LVM/LVEDV P = 0.0001) remained highly significant. The results of collinearity diagnostics revealed a variance inflation factor (VIF) of 1.14 for both creatinine and cystatin C, with creatinine and cystatin C in the model, and a VIF of 1.37 and 1.47, for creatinine and cystatin C, respectively, in the full model.  In a separate analysis after further adjustment for serum creatinine, the association between cystatin C quartiles and LV remodeling (LVEDV P = 0.0004), LVESV (P = 0.03), and LVM/LVEDV (P = 0.0001) remained highly significant. The results of collinearity diagnostics revealed a variance inflation factor (VIF) of 1.33 for both eGFR-CR and cystatin C, with only eGFR-CR and cystatin C in the model, and a VIF of 1.55 and 1.66, for eGFR-CR and cystatin C, respectively, in the full model. The associations between log transformed cystatin C and LV parameters were similar, and hence data was not presented.In another separate analysis, after further adjustment for creatinine-estimated GFR (eGFR-CR) instead of creatinine in the full model, associations between cystatin C quartiles and LV parameters: LVEDV  resulting in spurious results due to residual confounding. Some studies suggest that corticosteroids  and thyrCystatin C levels are inversely associated with LV end diastolic and systolic volumes and directly associated with concentricity independent of traditional cardiovascular risk factors including hypertension in a multi-ethnic population. This morphometric pattern of concentric LV remodeling may in part explain previously observed associations between cystatin C and diastolic dysfunction, heart failure, and cardiovascular mortality.


Moreover, phosphorylation of Hsp90α by PKCγ decreases the binding affinity of Hsp90α towards ATP and co-chaperones such as Cdc37 (cell-division cycle 37), thereby decreasing its chaperone activity. Further investigation demonstrated that the reciprocal regulation of Hsp90α and PKCγ plays a critical role in cancer cells, and that simultaneous inhibition of PKCγ and Hsp90α synergistically prevents cell migration and promotes apoptosis in cancer cells.It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications. However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process. The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery. First, we prove that PKCγ (protein kinase Cγ) is a client protein of Hsp90α, and, that by interacting with PKCγ, Hsp90α prevents PKCγ degradation and facilitates its cytosol-to-membrane translocation and activation. A threonine residue set, Thr The present study is the first example to show that a client directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery. Hsp90 (heat-shock protein 90) is one of the most conserved heat-shock proteins and plays an essential role in protection from heat shock . The funThe chaperone function of Hsp90 is modulated at three major levels, its intrinsic ATPase activity, the association with distinct conformation-specific co-chaperones and post-translational modifications . Althoug597) of Hsp90α affects its ATPase activity and N-terminal dimerization, leading to a decrease in its chaperone activity  plus 5–10% dried non-fat skimmed milk for 30 min at room temperature (20–25°C). The membrane was incubated with the indicated primary antibodies in PBST (PBS with 0.1% Tween 20) and 1% dried non-fat skimmed milk for at least 2 h at room temperature or overnight at 4°C, washed three times with TBST for 5 min each time at room temperature, and then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies for 60 min at room temperature. Following five washes with TBST, immunoreactive bands were detected by ECL (Pierce).in vitro phosphorylation of Hsp90α by PKC was performed in PKC reaction buffer . Recombinant Hsp90α was incubated with PKC with or without ATP at 30°C for 30–60 min, and then the sample was mixed with reducing SDS/PAGE loading buffer for further analysis.The The proteins used for these assays were ectopically expressed in HeLa cells and then immunopurified with anti-Myc antibodies. Proteins were first washed three times with cell lysis buffer and eluted with buffer containing 20 mM glycine (pH 2.2). The eluted samples were immediately neutralized with Tris buffer to a pH of 7.5.2+ and 100 mM NaCl (pH 7.5)] were incubated with high-affinity ATP–agarose (Innova) in a cold room for 1 h and then washed three times with the same buffer. The pelleted resin was mixed with reducing SDS/PAGE loading buffer for immunoblotting.For the ATP-binding assay, proteins in Tris buffer .HeLa cells were homogenized on ice in buffer A . Nuclei were pelleted by centrifugation at 1500 To determine whether PKCγ is chaperoned by Hsp90α, first, we confirmed the previously reported physical interaction between Hsp90α and PKCγ. HeLa cells were transfected with a plasmid expressing HA-tagged PKCγ. After whole cells were lysed and immunoprecipitated with a monoclonal anti-Hsp90α antibody, the immunoprecipitates were then analysed by Western blotting with an anti-HA antibody. We found that PKCγ was co-immunoprecipitated with Hsp90α A. MoreovNext we mapped the region of Hsp90α that mediates its interaction with PKCγ. Studies have reported that a conserved motif in the C-terminal domain of PKC mediates its interaction with Hsp90 , whereas514 because it is a major phosphorylation site in PKCγ and, moreover, its phosphorylation status is an indicator of PKCγ kinase activation [PKCγ belongs to the conventional subfamily of PKC, which needs to be transferred from the cytosol to the plasma membrane for activation . After ctivation .514, were strikingly reduced compared with the controls . Our data showed that the mRNA level of PKCγ was unchanged suggesting that the transcription level of PKCγ was not directly influenced by 17-AAG treatment. We then assayed the protein level and Thr514 phosphorylation status of PKCγ by Western blotting after 17-AAG treatment. We found the protein level of PKCγ and its phosphorylation at Thr514 in whole HeLa cell lysates were decreased in a time- and dose-dependent manner compared with the controls , indicating that they form a complex. Hsp70 and Cdc37 did not interact directly with each other, but both of them could interact with Hsp90α and PKCγ , which suggests that only one of the two is the key co-chaperone for the chaperoning process of PKCγ. To test this hypothesis, we knocked down the expression of Hsp70 and Cdc37, individually and simultaneously, by siRNA. As shown in Supplementary Figure S2(C), Cdc37 appears to play a more important role than Hsp70 in stabilizing PKCγ and mediating PKCγ activation. As expected, knocking down the expression of Hsp70 and Cdc37 simultaneously removed completely the protective effect of Hsp90 on the stability and activity of PKCγ. We found that overexpression of Cdc37 in cells with reduced expression of Hsp70 could partially rescue the stabilization and activation of PKCγ, but overexpression of Hsp70 in cells with reduced expression of Cdc37 did not . In addition, the binding affinity between Hsp90α and PKCγ was more significantly decreased in Cdc37-knocked-down cells compared with Hsp70-knocked-down cells . Collectively, these results show that co-chaperone Cdc37 plays a more important role than Hsp70 in recruiting PKCγ to the Hsp90α chaperone machine.We next investigated whether the co-chaperone Cdc37 is involved in the Hsp90α chaperoning of PKCγ. The Hsp90-dependent chaperone cycle requires sequential association and dissociation of various co-chaperones to effectively chaperone and release clients . We focuin vitro (in vivo (http://www.biochemj.org/bj/457/bj4570171add.htm) dependent manners. Having proved that Hsp90α is phosphorylated predominantly on threonine residues, we then showed that threonine phosphorylation of Hsp90α was reduced by knocking down the levels of PKCγ in HeLa cells . We found that both serine and threonine residues of Hsp90α were phosphorylated by PKCγ in vitro A; howeve as potential phosphorylation candidates . To confirm that these sites were phosphorylated by PKCγ, we constructed three Hsp90α non-phospho-mutants , and incubated them with PKCγ in vitro. We found that threonine residue phosphorylation in these mutants was attenuated, although the effect in the T603A mutant was not as strong as in the T115A or T425A mutants . We proposed that the phosphorylation of Hsp90α by PKCγ can regulate the disassociation of PKCγ from its Hsp90α chaperone machinery. To prove this hypothesis, we immunoprecipitated WT and threonine residue-phosphorylated Hsp90α from HeLa cells transfected with control vector or overexpressing Myc–PKCγ and incubated these two forms of Hsp90α with the HA–PKCγ protein. As shown in Since the interaction of Hsp90α and PKCγ regulates the behaviour of both proteins in living cells, we tested whether Hsp90α also regulates the activity of PKCγ in colon carcinoma cells in which PKCγ is overexpressed. We constructed a kinase-inactive form of PKCγ, K380A-PKCγ, and a constitutively active form, A25E-PKCγ . In a ceThe present study is the first to investigate the client-protein-modulating Hsp90 chaperone machinery, which not only reveals a novel regulatory mechanism of Hsp90, but also opens up a new direction of chaperone research. Hsp90 is a chaperone for many kinases which play pivotal roles in signal transduction and cellular homoeostasis, and is itself phosphorylated at multiple sites by different kinases. The results of the present study provide new insights to these processes by showing that Hsp90α is directly regulated by a kinase client and how, in turn, the activity of the kinase client is modulated by the Hsp90α chaperone machinery.The Farmer and the Viper. Prior to interacting with its chaperones, newly synthesized PKCγ is like a frozen viper, unstable and inactive. PKCγ then meets the ‘farmer’, Hsp90α, which interacts with PKCγ with the help of the co-chaperone Cdc37. By this interaction, Hsp90α ‘thaws the frozen viper’ and mediates the stabilization and the cytosol-to-membrane translocation of PKCγ. As the ‘revived viper’, PKCγ translocates to the membrane and is further activated by stimulation from secondary messengers, but first, in order to perform its cellular function, PKCγ must disassociate from the Hsp90α chaperone machinery. Activated PKCγ ‘bites the farmer’ by phosphorylating Hsp90α at a specific threonine residue set (Thr115/Thr425/Thr603), which releases it from the Hsp90α chaperone machinery. The overexpressed and activated PKCγ then promotes cancer cell migration and decreases cancer cell apoptosis. However, the phosphorylated Hsp90α (‘bit farmer’) loses all or part of its chaperone activity  physically interact with Hsp90 and (ii) inhibition of Hsp90's chaperone function must result in reduced client protein activity. Although it was reported that binding of conventional PKC to the Hsp90α–Cdc37 complex is necessary for its maturation and activation , in the in vivo, it phosphorylates both serine and threonine residues in vitro , we propose that PKCγ evolved to be phosphorylated at the minimal number of sites to allow the matured PKCγ to ‘escape’ from Hsp90α, such that its release and cellular activities would occur in a controlled manner.We propose a model whereby phosphorylation of the threonine residue set causes a large conformational change affecting all three Hsp90α domains, such that a cascade reaction occurs causing the loss of its chaperone function. In support of such a model, our data shows that phosphorylation of the Throrylated C. Whereaorylated B and 6C.orylated C in comp115/Thr425/Thr603 threonine residue set serves as a ‘phosphorylation switch’ for Hsp90α: when one or two threonine residues of the threonine set are ‘turned on’ (phosphorylated), Hsp90α loses part of its binding affinity toward PKCγ and when all of the three threonine residues are ‘turned on’, Hsp90α ‘opens’ its doors so that it completely loses its binding affinity toward PKCγ; however, when the threonine set is ‘turned off’ , Hsp90α ‘closes’ its doors and traps PKCγ, that is, Hsp90α has the strongest binding affinity toward PKCγ. In summation, an unphosphorylated threonine set binds PKCγ, whereas a fully phosphorylated form releases PKCγ.The Hsp90 chaperone cycle is known to proceed through association and disassociation of both co-chaperones and client proteins . On the S. cerevisiae) [Hsp90 has been reported to be phosphorylated by known client kinases in mammalian cells or in other species such as Src , Sch9 (Sevisiae)  etc. AltPKCγ is expressed mainly in neuronal tissues, where it plays roles in neuron development and neuropathic signal transduction. The expression of PKCγ has also been shown to be elevated in some cancer cells, especially colon carcinoma . HoweverTaken together, the present study demonstrates a novel regulatory mechanism of the interaction between Hsp90α and its kinase client PKCγ, which provides insights to the regulation of Hsp90α chaperone function by its clients and provides clues to possible therapeutic intervention in PKCγ-elevated cancers.


Portunus pelagicus) allergens in comparison with those of black tiger prawn (Penaeus monodon) by testing reactivity with shellfish-allergic subjects' serum IgE. Cooked extracts of both species showed markedly increased IgE reactivity by ELISA and immunoblotting, and clinical relevance of IgE reactivity was confirmed by basophil activation tests. Inhibition IgE ELISA and immunoblotting demonstrated cross-reactivity between the crab and prawn extracts, predominantly due to tropomyosin, but crab-specific IgE-reactivity was also observed. The major blue swimmer crab allergen tropomyosin, Por p 1, was cloned and sequenced, showing strong homology with tropomyosin of other crustacean species but also sequence variation within known and predicted linear IgE epitopes. These findings will advance more reliable diagnosis and management of potentially severe food allergy due to crustaceans.Shellfish allergy is a major cause of food-induced anaphylaxis, but the allergens are not well characterized. This study examined the effects of heating on blue swimmer crab ( Shellfish play an important role in human nutrition and health, but can provoke serious IgE-mediated adverse reactions in susceptible individuals. Shellfish are a major cause of food-induced anaphylaxis Charybdis feriatus), was published in the International Union of Immunological Societies (IUIS) allergen database . Furthermore, there is little information on shellfish from the southern hemisphere or Asia-Pacific region. Patients frequently report clinical reactions to more than one shellfish species, but whether this is a result of multiple sensitivities or from IgE cross-reactivity between allergens of different shellfish species is unknown The shellfish group includes crustaceans  and molluscs . In the few studies characterizing shellfish allergens to date, including our own, one of the most frequently recognized (major) allergens of species in both shellfish phyla is the abundant muscle protein tropomyosin (TM) Adding complexity, there are reports of altered stability and allergenicity of food proteins after processing Portunus pelagicus), and in particular the identification of the major allergen Por p 1. Evidence of cross-species IgE reactivity with another commonly consumed species, the black tiger prawn (Penaeus monodon), was sought and the effect of heating on allergens of both species and their cross-reactivity was assessed. Clinically relevant IgE reactivity to the shellfish extracts was assessed by a whole blood basophil activation assay.We report here the characterization of allergenicity of a commonly eaten crustacean species, the blue swimmer crab  and the Monash University Human Ethics Committee (MUHREC CF08/0225).A/L) , seven non-atopic controls  and one atopic non-shellfish-allergic subject . Allergic subjects were identified from the Alfred Hospital Allergy clinic seafood allergy database on the basis of clinical history of allergy to shellfish and positive shrimp-specific IgE  . Of thesPortunus pelagicus) and black tiger prawn (Penaeus monodon) were purchased at Prahran market . For raw crab (RC) and raw prawn (RP) extracts, the outer shell was removed and muscle collected. Finely cut muscle was blended with PBS pH 7.2 and left overnight at 4°C with constant mixing. The crude extract was centrifuged at 13,000 rpm at 4°C for 20 minutes. Supernatant was collected and filter sterilized before storage at −80°C in aliquots. For cooked crab (CC) and cooked prawn (CP) extracts, the outer shell was kept during the heating process (20 minutes immersed in boiling PBS) before removal and extract preparation as above. The protein concentration of each extract was determined using the Bradford assay kit  using bovine gamma globulin as a standard.Fresh blue swimmer crab . Pre-stained standards  were used as molecular weight (MW) markers. Proteins were resolved at 200 V for 35 minutes using an Xcell II mini-cell apparatus (Invitrogen) and the gel was stained with Coomassie brilliant blue.450 nm value for the non-atopic subject sera were used to determine the cut-off for positive IgE binding in shellfish-allergic subjects.Wells of a 96-well EIA/RIA plate  were coated with 100 µL extract , and incubated overnight at 4°C. All of the following incubations were performed for 1 hour unless otherwise stated and the plate was washed 4 times in 0.05% Tween 20/PBS (PBS-T) between steps. Blocking was performed using 5% skim milk powder diluted in PBS-T. Serum, 100 µL diluted 1∶10 in 1% skim milk powder/PBS-T, was added to wells and then incubated for 3 hours at room temperature with shaking (45 rpm). Rabbit anti-human IgE antibody  and goat anti-rabbit IgG-HRP  were added sequentially to wells and plates incubated at room temperature for 1 hour with gentle shaking. Plates were then washed 5 times in PBS-T, followed by 3 washes in PBS. IgE binding was detected using TMB  substrate (Invitrogen). After 5 minutes, the reaction was terminated using 1 M HCl and the absorbance (O.D.) at 450 nm measured by spectrophotometry . Sera from seven non-atopic subjects were screened to determine the extent of non-specific binding. ‘No-antigen’, background values were subtracted from test sera data. Two standard deviations above the mean O.D.450 nm was ∼1 and within the linear phase of the titration curve. Using this dilution, serum samples were incubated with an equal volume of shellfish extract , purified recombinant TM from black tiger prawn  or purified recombinant TM from blue swimmer crab (rPor p 1)  for 1 hour at room temperature and then tested for reactivity with either CC or CP extracts. Percent inhibition was calculated using the following equation: percent inhibition  = 100−[(O.D.450 nm of serum with inhibitor/O.D.450 nm of serum without inhibitor) X100]. For comparison of inhibition by different extracts, the extract (inhibitor) concentration that caused 50% inhibition was calculated from dose-response curves. To assess non-specific inhibition by extracts, serum from a non-shellfish-allergic, atopic (Bahia grass pollen-sensitized) subject was incubated with the above inhibitors, and then tested for IgE reactivity with Bahia grass pollen extract in comparison with untreated serum.For inhibition ELISA experiments, individual subject sera were first titrated (1∶10–1∶1280) for IgE reactivity with CC or CP to determine the concentration at which the O.D.Proteins of each of the four extracts  were separated by SDS-PAGE as above with 3 µg/well loaded into a 10 or 15-well 4–12% Bis-Tris gel (NuPage), or 60 µg protein loaded into a 6 cm 2D well 4–12% Bis-Tris gel (NuPage). Proteins were transferred to a nitrocellulose membrane  using an Xcell II blotting apparatus (Invitrogen) at a constant voltage of 30 V for 1 hour. Successful transfer of protein was evaluated by Coomassie brilliant blue staining of the gel as above. The membrane was blocked using 5% skim milk powder/PBS-T for 1 hour at room temperature with gentle rocking. Subject serum (1∶40 in 5% SMP/PBS-T) was then applied to the membrane using a Miniblotter apparatus . To detect IgE binding, the immunoblot was incubated sequentially with rabbit anti-human IgE antibody  and goat anti-rabbit IgG-HRP conjugated antibody  each for 1 hour at room temperature with gentle horizontal shaking. Following incubation with chemiluminescent peroxidase substrate , proteins were visualized using enhanced chemiluminescence technique For inhibition IgE immunoblot experiments, serum samples  were first incubated with whole crude extracts  or rPen m 1 (1.2 µg/mL) for 1 hour at room temperature and then tested for reactivity with the CC or CP extracts by immunoblotting as described above. In a separate experiment to directly compare rPor p 1 with rPen m 1, serum samples were incubated with rPor p 1 or rPen m 1 at 0.048, 0.24 and 1.2 µg/mL. A ‘no inhibitor’ control was also included. Complete protease inhibitor  was added to the diluent (one tablet in 50 mL of diluent) to prevent non-specific proteolytic degradation of IgE antibodies. Non-specific inhibition was assessed as described for the inhibition IgE ELISA above.Jasus lalandii, Genbank accession number JX860677.1). The primer pair used was BSC-TM (F) 5′-GCCGGATCC-ATGGACGCAATCAAGAAGAAGATGCAG-3′ and BSC-TM (R) 5′-GCGGAATTC-TTAGTAGCCAGACAGTTCG-3′. The PCR included one cycle of denaturation at 94°C for 2 minutes, 35 cycles at 94°C for 30 seconds, annealing at 55°C for 45 seconds and elongation at 72°C for 1 minute, and a final elongation step at 72°C for 7 minutes. The amplified full length open reading frame was cloned into the sequencing vector, pCR 2.1 (Invitrogen) using the BamH1 and EcoR1 restriction sites for cloning, and the open reading frame for TM sequenced  to obtain the construct pCR2.1_TM.A band corresponding to the predominant IgE-reactive 39 kDa protein was excised from the SDS gel for mass spectrometric analysis. The band was de-stained, reduced, alkylated and digested with trypsin as reported previously E.coli cells using heat-shock for 30 seconds, incubation in SOC medium at 37°C for 1 hour and grown overnight on Luria Bertani (LB) agar plates with 100 µg/mL ampicillin  at 37°C. Positive colonies were tested using PCR as described above and selected for protein expression.The open reading frame of TM was cross-cloned from the construct, pCR2.1_TM to the expression vector, pProEXHT-B using restriction sites for BamH1 and EcoR1 and ligation was performed using T4 DNA Ligase . This expression plasmid construct was transformed into chemical-competent NM522 E.coli cells using a French-Pressure Cell, purified using nickel charged metal-chelate affinity chromatography  following the manufacturer's instructions and stored at −80°C until further use. The protein concentration of the purified protein was determined by absorbance at 280 nm using a nanodrop spectrophotometer .For recombinant protein expression, 5 mL of fresh overnight culture from a single colony was used to initiate growth in 1 L LB broth containing 100 µg/mL ampicillin. Recombinant protein expression was induced using 0.6 mM isopropylthio-β-galactoside . After expression, the culture was centrifuged at 3500 g for 10 minutes to obtain the bacterial pellet and subsequently resuspended in extraction buffer . Recombinant blue swimmer crab TM containing the 6xHis tag was extracted from the Shellfish extracts were tested for basophil activation using our established methodology The Wilcoxon matched-pairs signed rank test was used to compare overall serum IgE reactivity between shellfish extracts, and Spearman's correlation test was used to assess correlation between individual specific IgE levels against different extracts or using different assays. Analyses were performed using GraphPad Prism version 5.04 for Windows .Analysis of raw and cooked shellfish extracts by SDS-PAGE and Coomassie brilliant blue staining  revealedQuantitation of serum IgE binding to the shellfish extracts by ELISA  showed tSera from 12 subjects with IgE positivity to RC and/or CC by ELISA, and where sufficient serum was available, were used for immunoblotting to visualize IgE-reactive proteins in the shellfish extracts . Immunobhttp://www.allergen.org/index.php). The most similar TMs were from the American lobster (Homarus americanus) and the black tiger prawn (Penaeus monodon) which both showed 98% sequence identity. Sequence identity with other Portunus species TM, and the only crab TM, Cha f 1, listed within the IUIS allergen database was 92%. There were no amino acid differences between relevant regions in the blue swimmer crab TM and published linear IgE epitopes described for Penaeus aztecus (Pen a 1) Penaeus monodon (Pen m 1) Following analysis of allergenic proteins using IgE immunoblot, the highly IgE-reactive 39 kDa protein of the blue swimmer crab was identified as TM by peptide mass fingerprinting analysis . The bluRecombinant blue swimmer crab TM, rPor p 1, was successfully expressed and purified with approximately 95% purity. Coomassie brilliant blue staining of SDS-PAGE of rPor p 1 under denaturing conditions showed a single band with a MW of approximately 41 kDa . The higTo assess biologically relevant shellfish allergen-specific IgE antibody reactivity, the ability of the different extracts to activate basophils from five shellfish-allergic subjects  was analyzed by flow cytometry. Activated basophils were identified by high IgE expression and up-regulation of surface CD63 . No non-Dose-dependent basophil activation to the crab and prawn extracts was observed, with a range of sensitivities for the subjects, consistent with their different crab- and prawn-specific IgE reactivities by ImmunoCAP and our ELISA and immunoblotting assays . When suInhibition ELISA was used to quantitate the degree of IgE cross-reactivity between the two shellfish species and the effects of cooking. No non-specific inhibition of IgE reactivity by the shellfish extracts, rPen m 1 or rPor p 1 was evident using serum IgE from a Bahia grass pollen-sensitized control subject and Bahia grass pollen extract (data not shown). Using shellfish-allergic subjects, both cooked extracts showed greater inhibition of IgE binding than the corresponding raw extracts . For mosInhibition immunoblotting was used to examine the proteins responsible for cross-reactivity of IgE antibodies between the different extracts, and in particular whether there were potentially unique allergens within the blue swimmer crab. Again, non-specific inhibition by the extracts was excluded by testing for inhibition of the binding of serum IgE from a Bahia grass pollen-allergic control subject to Bahia grass pollen extract (data not shown). Four shellfish-allergic subjects  with IgE reactivity to TM (39 kDa) were selected for this assay. For all subjects, rPen m 1 markedly inhibited IgE binding to the TM band, further confirming the identity of this protein as TM . The CC When the IgE inhibitory reactivity of rPor p 1 and rPen m 1 were compared over a concentration range using two shellfish-allergic patient sera, rPor p 1 showed slightly greater inhibition of IgE reactivity with the TM band in CC than rPen m 1, and rPen m 1 inhibited reactivity with CP TM slightly better than rPor p 1. NeitherShellfish allergy is a serious and increasing health issue, with current limitations in accurate diagnosis and management due to lack of information regarding clinically relevant allergens and IgE cross-reactivity between shellfish species. Crustaceans, especially crabs and prawns, are a common cause of shellfish allergy world-wide. This study examined the IgE-reactive components of a commonly eaten crustacean species, the blue swimmer crab, compared with those of a well characterized species, the black tiger prawn. In particular, the effects of heating on IgE reactivity and cross-reactivity of the crab allergens were investigated. In addition, the major TM allergen of the blue swimmer crab was cloned and sequenced and its IgE reactivity characterized.When shellfish-allergic subject serum IgE reactivity with whole extracts was compared, the raw crab extract showed greater IgE reactivity compared with the raw prawn extract by both ELISA and immunoblotting. Whether this was due to inherent differences between the proteins of the blue swimmer crab and black tiger prawn, or to differences in sensitizing species or route of sensitization is not clear. We and others have shown previously that inhalation during commercial processing can result in sensitization to seafood Portunus pelagicus TM, Por p 1. This revealed strong homology of the blue swimmer crab TM with other crustacean TMs, but regions of amino acid sequence difference at sites of known and predicted linear IgE epitopes support the need for crustacean species-specific diagnostic reagents. The purified rPor p 1 showed high IgE reactivity by direct immunoblotting, but in agreement with sequence differences, IgE inhibition immunoblotting showed differential reactivity of rPor p 1 and rPen m 1. When compared for inhibition of IgE reactivity with TM in shellfish extracts, rPor p 1 could better inhibit reactivity with the cooked blue swimmer crab TM and rPen m 1 was the better inhibitor of reactivity with black tiger prawn TM. Several other allergenic proteins of the blue swimmer crab were recognized at lower frequency. For some subjects, these proteins were recognized in the absence of TM reactivity. Testing of a larger subject cohort is required to assess the clinical importance and cross-reactivity of these proteins and hence define an appropriate panel of defined allergens for refined diagnostic assays.IgE immunoblotting studies were performed to examine individual IgE-reactive proteins. As for other crustacean species, TM is a major allergen of the blue swimmer crab. Over 50% of shellfish-allergic subjects showed IgE-reactivity to proteins corresponding to TM in both raw and cooked crab extracts and TM identity was confirmed by TM-specific mAb reactivity and peptide mass fingerprinting of the highly IgE-reactive 39 kDa protein. We report here for the first time the cloning and full sequence analysis of the In view of potential food matrix-associated effects on the outcome of heating of allergens Cross-reactivity between crustacean species is essential to understand in order for shellfish-allergic subjects to receive the best clinical advice on food avoidance. There are limited studies of crustacean cross-reactivity to date, often with small subject cohorts Cancer pagurus or brown crab, a northern hemisphere species). All subjects with IgE-reactivity to crab or prawn TM by immunoblot had moderate to high levels of allergen-specific IgE (≥2.37 kUA/L) as determined by ImmunoCAP to shrimp and/or crab. These subjects were also more likely to have had severe allergic symptoms, such as angioedema and anaphylaxis, upon contact with shellfish. However, there were some subjects who had a strong ImmunoCAP result to shrimp and/or crab and clinical history of severe reactions but showed low or no IgE reactivity by ELISA or immunoblot. These subjects may have species-specific IgE with limited or no cross-reactivity with the crustacean species in our study Screening of shellfish-allergic subjects by IgE ELISA against raw and cooked extracts gave insight into whether the current diagnostic ImmunoCAP for crab is relevant in a southern hemisphere clinical setting. Although a double-blind placebo-controlled food challenge can confirm diagnosis of food allergy, this procedure has a high risk of anaphylaxis for adults with shellfish allergy who may have other comorbidities, and is not routinely performed in our hospital Previous studies have concluded that specific-IgE to TM is an accurate predictor of shrimp allergy In conclusion, heating causes a marked increase in clinically relevant IgE reactivity of blue swimmer crab extract. In particular, we identified and characterized the blue swimmer crab TM, Por p 1, as a heat-stable major cross-reactive allergen. Other IgE-reactive blue swimmer crab proteins were observed, some corresponding to molecular weights of documented shellfish allergens, but others currently unidentified, some potentially unique to the blue swimmer crab. Our findings will advance reliable diagnosis and management of shellfish allergy.


Estrogen rapidly modulates hippocampal synaptic plasticity by activating selective membrane-associated receptors. Reorganization of the actin cytoskeleton and stimulation of mammalian target of rapamycin (mTOR)-mediated protein synthesis are two major events required for the consolidation of hippocampal long-term potentiation and memory. Estradiol regulates synaptic plasticity by interacting with both processes, but the underlying molecular mechanisms are not yet fully understood. Here, we used acute rat hippocampal slices to analyze the mechanisms underlying rapid changes in mTOR activity and actin polymerization elicited by estradiol. Estradiol-induced mTOR phosphorylation was preceded by rapid and transient activation of both extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) and by phosphatase and tensin homolog (PTEN) degradation. These effects were prevented by calpain and ERK inhibitors. Estradiol-induced mTOR stimulation did not require activation of classical estrogen receptors (ER), as specific ERα and ERβ agonists  failed to mimic this effect, and ER antagonists could not block it. Estradiol rapidly activated both RhoA and p21-activated kinase (PAK). Furthermore, a specific inhibitor of RhoA kinase (ROCK), H1152, and a potent and specific PAK inhibitor, PF-3758309, blocked estradiol-induced cofilin phosphorylation and actin polymerization. ER antagonists also blocked these effects of estrogen. Consistently, both PPT and DPN stimulated PAK and cofilin phosphorylation as well as actin polymerization. Finally, the effects of estradiol on actin polymerization were insensitive to protein synthesis inhibitors, but its stimulation of mTOR activity was impaired by latrunculin A, a drug that disrupts actin filaments. Taken together, our results indicate that estradiol regulates local protein synthesis and cytoskeletal reorganization via different molecular mechanisms and signaling pathways. A large amount of literature indicates that gonadal steroids have a strong impact on various functions and features of the central nervous system, such as the regulation of reproductive behavior and cognition as well as neuroprotection. The hippocampus is one of the main target regions for estrogen actions in brain. In particular, fluctuations of circulating levels of estrogen during the estrous cycle have been shown to modify spine density in area CA1 of hippocampus , a mechaAmong the physiological events participating in memory formation, reorganization of the actin cytoskeleton within dendritic spines is considered to be necessary for transforming a brief stimulus into a long-lasting memory trace , 10. EstCurrent hypothesis for LTP assumes a critical role for activity-induced release of brain derived neurotrophic factor (BDNF) and activation of its receptor, tropomyosin receptor kinase B (TrkB), resulting in stimulation of the mammalian target of rapamycin (mTOR) and local protein synthesis . EstrogeAnimals were treated in accordance with the principles and procedures of the National Institutes of Health Guide for the Care and Use of Laboratory Animals; all protocols were approved by the Institutional Animal Care and Use Committee of Western University of Health Sciences.3 (26), glucose (10), KCl (2.5), NaH2PO4 (1.25), MgSO4 (2), and cut into 400 μm thick transverse slices using a McIlwain tissue chopper. Hippocampal slices were maintained in a recovery chamber with artificial cerebrospinal fluid (aCSF) medium, containing (in millimolar): NaCl (124), KCl (2.5), CaCl2 (2.5), MgSO4 (1.5), NaH2PO4 (1.25), NaHCO3 (24), d-glucose (10), and saturated with 95%O2/5%CO2, for 1 h at 37°C. Hippocampal slices were then transferred into screw-cap microfuge tubes containing 3 ml of freshly oxygenated aCSF medium in the presence of various drugs.Hippocampi were rapidly dissected from 2 to 3 month-old male Sprague-Dawley rats, transferred to oxygenated, ice-cold cutting medium containing (in millimolar): sucrose (220), NaCl (20), NaHCO2PO4 (1.18), NaHCO3 (24.9), MgSO4 (1.18), CaCl2 (2.5), d-glucose (10), HEPES (1), pH 7.4, and equilibrated with 95%O2/5%CO2. Cortices were homogenized in a 7-ml Kontes tissue Dounce homogenizer with 10 strokes. Homogenized tissue was filtered through a 100-μm nylon mesh filter  and the resulting suspension was filtered again through a 5-μm pore size Acrodisc syringe filter with a Supor membrane . The filtrate was then centrifuged at 1000 × g for 15 min at 4°C, washed once, and centrifuged again. The pellet was resuspended in mKrebs and various drugs were directly added to the pre-warmed  synaptoneurosome suspension for the indicated periods of time.Synaptoneurosomes were prepared from 2- to 3-month-old male Sprague-Dawley rats, as previously described , with miH]-pyrazole-1,3,5-triyl)trisphenol (PPT), 10 nM diarylpropionitrile (DPN), and 100 nM (±)-1-quinolin-8-yl]-ethanone (G1)  for 30 or 60 min. We selected these concentrations for estrogen receptors (ERs) specific agonists based on previous reports showing a relatively low selectivity of DPN (about 30-fold) for ERβ over ERα, as compared to the much higher selectivity of PPT (around 1000-fold) for ERα over ERβ (bis(trifluoromethyl)pyrazolopyrimidin-3-yl]phenol , cycloheximide (25 μM) , calpain-inhibitor  , (±)--4--3a,4,5,9b- tetrahydro-8-(1-methylethyl)-3H-cyclopenta[c]quinoline , H1152 , PF-3758309 , rapamycin , and latruncullin A .Slices were incubated with 10 nM 17β-estradiol  at 37°C for the indicated periods of times. Alternatively, slices were incubated with 100 ng/ml BDNF (Millipore), 100 nM 4,4′,4′′- for 30–45 min at room temperature. After three washes, lysates were collected in 200 μl/slice of PBS and fluorescent intensity  was determined using a POLARstar Omega fluorescence polarization microplate reader (BMG Labtech).Actin polymerization was quantified by measurement of “rhodamine-phalloidin fluorescent enhancement,” as previously described , with li2+ lysis buffer  containing a protease inhibitor Cocktail (Thermo Scientific). Protein levels were measured and equalized. Samples were incubated with Rhotekin RBD-agarose beads and gently rocked for 1 h at 4°C. Agarose beads were collected by centrifugation  and washed three times with Mg2+ lysis buffer, resuspended and boiled in Laemmli buffer for 5 min, and then separated by SDS-PAGE. Western blot analysis was performed using mouse anti-RhoA antibody .RhoA activity was determined by pull-down of RhoA-GTP with Rhotekin binding domain (RBD)-linked agarose beads (Millipore), as described previously . RhotekiProtein lysates were prepared at a final concentration of 4–8 μg protein/μl by homogenization of rat hippocampal slices in ice-cold lysis buffer  containing a protease and phosphatase inhibitor cocktail (Thermo Scientific). After sample processing, 25–40 μg of denatured proteins were subjected to 6–15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk dissolved in Tris-buffered saline (TBS) for 30 min at room temperature and probed overnight with different primary antibodies at 4°C. Membranes were then washed three times with TBS containing 0.1% Tween 20 (TBST) for 10 min followed by incubation with Odyssey infrared-conjugated secondary antibodies diluted 1:10000 in Odyssey blocking buffer (LI-COR Biosciences) containing 0.1% Tween 20 and 0.05% SDS for 1 h at room temperature. After 3× 10 min washes with TBST, membranes were scanned using an Odyssey infrared imager (LI-COR Biosciences).All primary antibody solutions were prepared in TBST containing 5% bovine serum albumin at a 1:1000 dilution, except otherwise stated. The following primary antibodies were used: anti-phospho-mTOR (Ser2448) and anti-mTOR , anti-phospho-ERK1/2 (Thr202/Tyr204) and anti-ERK1/2 , anti-phospho-Akt (Ser473 and Thr308) and anti-Akt , anti-phospho-p70S6K , anti-p70S6K , anti-phospho-cofilin  and anti-cofilin , anti-phospho-PAK1/2/3 (Invitrogen) and anti-PAK3 , anti-spectrin , anti-Arc , anti-actin , and anti-PTEN .p < 0.05, **p < 0.01, and ***p < 0.001, as compared to control (DMSO or time = 0 min); and #p < 0.05, ##p < 0.01, and ###p < 0.001, as compared to BDNF or E2 alone; absence of symbol indicates p values >0.05, which were considered as not significant.Statistical comparisons were made by using one-way analyses of variance (ANOVA) followed by Dunnett’s multiple comparison test or two-way ANOVA followed by Bonferroni posttest analysis. Results were generally calculated as means ± standard error of the mean (SEM) from the indicated number of independent experiments and expressed as fold of the indicated control. Statistical significance was always referred to as: *Based on our previous study on the regulation of the mTOR pathway by BDNF , stimulaTo determine the link between estrogen-stimulated calpain activity and mTOR-dependent protein synthesis, we treated hippocampal slices with 10 μM calpain-inhibitor III (CI-III) before estradiol application and then examined mTOR activation. Calpain inhibition completely blocked estradiol-elicited increase in mTOR phosphorylation Figure A. Pre-trRapid calpain activation by estradiol and BDNF in dendritic spines is triggered by ERK-dependent phosphorylation , 28. If To further examine the mechanism by which estradiol activates mTOR, we used specific agonists and antagonists of the two major ER isoforms, ERα and ERβ. Neither the non-specific ER antagonist ICI 182780  nor the specific ERβ antagonist PHTPP (1 μM) affected estradiol-induced mTOR phosphorylation Figure A. ConsisEstrogen has been shown to rapidly stimulate actin cytoskeleton dynamics via the RhoA/ROCK/cofilin pathway . It can Ki values of 14/19 nM, respectively), as compared to PAK2/3   . Thus, wctively) A suggestctively) A. Interectively) B,C, indiWe also tested the effects of ER and TrkB receptor antagonists on estrogen-mediated actin reorganization. Treatment with either ICI or PHTPP blocked the effects of estradiol on PAK and cofilin phosphorylation. In contrast, K252a failed to inhibit estradiol-induced PAK activation, but it prevented the increase in cofilin phosphorylation caused by the hormone Figures A,C. ConsWe next evaluated the relationship between protein synthesis and cytoskeletal reorganization after exposure to estrogen in order to determine whether these two events are independent. To address this question, we determined the effects of protein synthesis inhibitors on estradiol-elicited actin polymerization in hippocampal slices. Neither 25 μM cycloheximide nor 1 μM rapamycin blocked estradiol-induced actin polymerization . Estrogen-mediated stimulation of dendritic protein translation has been shown to be ERK-dependent . HoweverWe then determined whether actin polymerization was necessary for estradiol-mediated mTOR stimulation. Thus, we pre-treated hippocampal slices with Latrunculin A (Lat A), an agent that sequesters actin monomers, thereby preventing filamentous actin (F-actin) assembly. Two concentrations of Lat A were used (0.5 and 5 μM) based on the different sensitivities of F-actin to this drug depending on its subcellular location; low concentrations of Lat A specifically target actin filaments within the dendritic shaft, while higher concentrations are required to affect dendritic spine F-actin . TreatmeIf actin polymerization regulates estradiol-mediated mTOR activation/phosphorylation, it could be due to an activity-dependent event triggered by activation of classical pathways  or be due to a structural requirement linking activated Akt to mTOR. We addressed this question by determining the effects of specific ROCK and PAK inhibitors on estradiol- and BDNF-induced mTOR and Akt activation. Treatment with H1152 did not affect mTOR phosphorylation stimulated by BDNF or estradiol in hippocampal slices. In contrast, 500 nM (but not 50 nM) PF-3758309 completely blocked BDNF- and estradiol-induced mTOR activation Figure , 36. OveIn the present study, we found that estradiol stimulates mTOR in acute hippocampal slices through a signaling pathway involving TrkB receptor activation, ERK phosphorylation, and calpain activity. Specific inhibitors of each of these enzymes abrogated the increase in mTOR phosphorylation elicited by estradiol. These results are consistent with the recently identified mechanism linking BDNF-induced activation of the PI3K/Akt signaling cascade and stimulation of mTOR-dependent protein synthesis through calpain-2-mediated PTEN truncation  see sch. In a prPrevious studies have shown that the stimulation of dendritic protein translation by estradiol involves ERK and Akt activities , 22. WhiRecent studies have focused on the importance of estradiol and BDNF cross-talk signaling in the regulation of synaptic function , 4, 40, The stimulation of the mTOR pathway by estrogen did not require activation of classical ERs, as both the agonists, PPT and DPN, failed to mimic the effect of the hormone on phospho-mTOR levels, and the antagonists, ICI and PHTPP, to inhibit it. Although some studies have shown that estradiol stimulates Akt/mTOR signaling via classical ER activation , 45, theClassical ER activation was required for PAK and cofilin phosphorylation, as well as for actin polymerization elicited by estradiol. Furthermore, both ERα and ERβ agonists stimulated actin signaling and cytoskeletal reorganization. In a previous study , only anThe reorganization of the actin cytoskeleton plays a major role in the structural and functional changes that occur in dendritic spines during LTP , 10. EstInterestingly, actin polymerization/stabilization was required for estradiol-induced stimulation of protein translation signaling, as the depolymerizing agent Lat A suppressed BDNF- and estradiol-induced mTOR and p70S6K phosphorylation. To the best of our knowledge, this is the first study showing that rapid mTOR activation requires F-actin treadmilling. In contrast, protein synthesis was not required for estradiol-induced actin polymerization. Furthermore, we provided evidence that PAK activity was also necessary for mTOR activation, since the PAK specific inhibitor (at the highest concentration used only) but not the ROCK inhibitor blocked Akt and mTOR phosphorylation induced by both BDNF and estradiol. Although some studies have linked PAK1/2 to the Akt signaling pathway independently of its regulatory action on actin filaments –58, our In summary, the results present here shed new light on the molecular mechanisms involved in estrogen modulation of hippocampal synaptic plasticity, and underscore the roles of distinct signaling pathways in mediating its effects on protein synthesis and actin cytoskeleton reorganization.Victor Briz performed all the experiments, prepared the figures, and wrote the manuscript. Michel Baudry directed the research and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fendo.2014.00022/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


The majority of tinnitus cases are related to cochlear dysfunction, leading to altered peripheral input to the central auditory system . These aTo further investigate the processes involved in tinnitus manifestation we used neuroConstruct  to impleAfferent input from the auditory pathway was modeled as trains of spikes. Their frequency depended on the strength of the input whereas their target site depended on the frequency of the input. Additionally, synaptic background noise was introduced to the system as Gaussian noise. Excitatory cells made connections to other excitatory in their 1st, 2nd and 3rd surrounding ring and to inhibitory cells in their 3rd, 4th and 5th surrounding ring with decreasing probabilities . Inhibitory cells made connections with excitatory cells in their 1st, 2nd, 3rd and 4th surrounding ring allso with decreasing probability . This connectivity pattern results in a tonotopic organization of the auditory cortex layer. The simulation also exhibits spontaneous and spike driven firing rates comparable with experimental data [Increasing the background noise and simultaneously decreasing the afferent input to one tonotopic region creates a state that may correspond to the early stage of tinnitus manifestation after cochlear damage. This state also shows an increased basic level of activity in the absence of input and furthermore, a diminished increase of activity during sound presentation. Moreover, a change of the ratio between excitatory and inhibitory weights towards more excitation and less inhibition results in a normal increase of activity during sound presentation. This suggests that the auditory cortex might react upon this diminished ability to detect sound in the damaged frequency region by increasing excitation and decreasing inhibition.The presented simulation constitutes an excellent starting point for a deeper investigation of the early phases in the generation of tinnitus. We are currently extending the simulation to include more structures of the classical pathway and to incorporate synaptic plasticity.


MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling.Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a  Aberrant receptor tyrosine kinase (RTK) activity provides growth and survival signals crucial for the development and progression of many cancers. Treatment of patients with targeted inhibitors of key oncogenic kinase drivers such as sunitinib, erlotinib, gefitinib, and imatinib have demonstrated clinical success MET gene locus are very responsive to c-Met inhibitors such as the highly selective small molecule PF-04217903 (METi). Not unlike the eventual resistance that emerges against other RTK inhibitors, several studies have described development of resistance to c-Met inhibitors via c-Met amplification The c-Met/HGFR receptor tyrosine kinase is a promising therapeutic target as mutations of c-Met  and focal amplifications of the MET gene locus  may indicate an oncogenic dependence on c-Met signaling in vitro screen to select for METi resistant clones of GTL16, a c-Met dependent gastric carcinoma cell line that harbors a high-level focal amplification of the MET gene locus To anticipate potential resistance, we utilized an BRAF is a proto-oncogene that promotes cell growth and proliferation by transducing signals from growth factor receptors as part of the MAP kinase pathway via MEK and ERK. Mutations to this protein such as G469A, E586K, V600E, and K601E can increase BRAF catalytic activity in vitro. Together, these findings suggest that targeted inhibitors can be bypassed at multiple levels and that inhibiting the nodes where the signal converges might be a more robust strategy for therapy.In our model, the resultant SND1-BRAF fusion protein contains a constitutively active BRAF kinase which increases phosphorylation of ERK. Functionally, this fusion protein signals downstream of c-Met and bypasses its inhibition by METi. We demonstrate that a MEK inhibitor or the combination of c-Met and RAF inhibitors suppresses phosphorylation of ERK and reduces the proliferation of the resistant clones GTL16 cells were seeded in a 96-well plate at a density of 20,000 cells per well and treated with 0.5 µM PF-0461903 (METi), a selective c-Met kinase inhibitor . The conCell viability assays were performed using the GTL16 line and GTL16 resistant clones. Briefly, cells were seeded at a density of 4000 cells/well into 96-well plates and allowed to adhere overnight. The following day, cells were treated with either single agent or combination of METi and a Raf inhibitor, PF-04880594 (RAFi)  or a MEKBriefly, readings from the Envision multi-reader were processed using the R package 'drc' (drug response curves) to generate IC50 values The drc package was then used to fit the TGI as a function of the concentration of the compounds. A four-parameter logistic model was used to fit the dose response curves and infer the IC50, slope, and upper and lower limits.GTL16 and resistant clones were grown to approximately 80% confluency and then treated with inhibitor compounds or DMSO vehicle control at the indicated concentrations and time duration. For Western immunoblotting, cells were rinsed with phosphate buffered saline (PBS) and subsequently lysed with cell lysis buffer  supplemented with 2 mM sodium orthovanadate and 2 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were harvested, sonicated briefly, and incubated for 1 hr at 4°C. Lysates were then centrifuged at 14,000 rpm for 10 min at 4°C to pellet cell debris, and supernatant was collected.GTL16 and resistant clones were treated with 2.5 µM METi or vehicle control for 1 hr and lysed with CLB lysis buffer according to the vendor’s directions . Lysates were sent to Zeptosens for analysis. RPPA data from Zeptosens was processed by the vendor and further processed by median centering. Heatmap plots were generated using a custom R script with scaling to a range of −3 to 3 .The protein concentration of cell lysates was quantified using a DC protein assay kit . Forty to seventy-six µg of total protein per lane were loaded in a 4–12% gradient BIS-TRIS SDS-PAGE gel  or Criterion XT gel (Bio-Rad), and was transferred onto nitrocellulose membrane . Membranes were blocked with Phosphoblocker  and incubated overnight with primary antibody diluted in Phosphoblocker. Primary antibodies were used according to manufacturer’s recommendation: phospho BRAF S445 ; BRAF N-term ; BRAF c-TERM C-19 ; phospho c-Met Y1349 ; c-Met ; phospho ERK T202/Y204 ; ERK ; phospho AKT S473 ; AKT ; actin .RNA and DNA were isolated using RNeasy and DNeasy kits  for microarray analysis. Whole genome expression profiling and copy number analysis were performed using Affymetrix HGU133 Plus 2.0 and SNP 6.0 arrays , respectively, per manufacturer’s protocol. HGU133 Plus 2.0 data were GC Robust Multiarray Average normalized using R and the gcrma package from bioconductor.org Affymetrix SNP 6.0 data were processed using the aroma.affymetrix R package according to the methods of H. Bengtsson et al., using the GTL16 as the reference baseline All array data is MIAME compliant and will be publically available from the Gene Expression Omnibus (GEO) under accession GSE27692.BRAF exons 11 and 12 junction [5′-catcaccatgccactttcccttgt-3′] were used to amplify a first-strand cDNA generated using random hexamers. PCR was performed using 1X Titanium Taq PCR buffer, 0.2 mM dNTP, 0.4 µM forward generacer and reverse BRAF primers, and 1X Titanium Taq DNA polymerase . The sequence of cycling was 1 cycle at 95°C for 60 sec; 5 cycles at 95°C for 15 sec, 72°C for 90 sec; 5 cycles at 95°C for 15 sec, 70°C for 90 sec; 25 cycles at 95°C for 15 sec, 68°C for 90 sec, and an additional extension at 68°C for 7 min using Peltier Thermal Cycler 200 . The PCR product was run on a 1.2% agarose gel and a 2.1 Kb amplicon was gel-purified, cloned into pCR®4-TOPO®R cloning vector , and sequenced using M13 forward and reverse primers on a 3730XL capillary sequencer . Sequence data was analyzed using Sequencher .RNA ligase-mediated, rapid amplification of cDNA ends was performed on total RNA from GTL16, GTL16R1, and GTL16R3 clones using a Generacer™ kit . PCR primers specific for 5′ generacer oligo sequence [5′-cgactggagcacgaggacactga -3′] and 3′ primer targeting The METi and RAFi were screened against kinase panels . In these selectivity assays the concentration of ATP was set to the Km value of ATP for each kinase tested to allow for a relative potency comparison between the various kinases. The compounds were tested at 1 µM final concentration. Some of the kinases that showed modest potency in the percent inhibition biochemical assays were further evaluated in a dose response cell based assay. For example METi showed biochemical inhibition of IGF1R but did not inhibit IGF1R kinase activity in the follow-up cell based assay.de novo assembled into contigs using velvet (v1.0.13) http://www.ebi.ac.uk/~zerbino/oases/). Putative fusion transcripts were then aligned by BLAST RNA-Seq libraries were generated and quality assessed as previously described Computational search was performed using publically available CNV (Copy Number Variation) arrays from:http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp).TCGA (http://www.sanger.ac.uk/genetics/CGP/Archive/).Sanger .Broad (http://www.ncbi.nlm.nih.gov/geo/) .GEO (https://cabig.nci.nih.gov/caArray_GSKdata/).GSK  or squamous-like (S) . ResistaTo examine the effect of METi on the activation state of signal transduction pathways, protein lysates from GTL16 and the resistant clones were assayed with Reverse Phase Protein Arrays (RPPA) for select total and phosphorylated proteins. Treatment of GTL16, GTL16R1, and GTL16R3 with 2.5 µM METi for 1 hr inhibited phosphorylation of c-Met residue Y1235 . Total cTo further assess the mechanism of resistance to c-Met inhibition, METi-resistant clones were profiled for gene expression with Affymetrix U133 Plus 2.0 chips and for DNA Copy Number Variation (CNV) using Affymetrix SNP 6.0 arrays. GTL16R1 and GTL16R3 clones have a nearly identical CNV profile to GTL16 except for a 4-fold amplification of ∼356 KB in 7q31 and ∼2 megabases within 7q34. Relative to GTL16, most genes within 7q34 had a corresponding increase in mRNA expression with BRAF expressed more than 32-fold in both GTL16R1 and GTL16R3 .SND1  to be the fusion partner . Here, SND1 happens to be bisected by the small 7q31 amplification observed in the GTL16R1 and GTL16R3 clones. The translocation yields a SND1-BRAF fusion transcript with exon 16 of SND1 fused in-frame to exon 9 of BRAF, resulting in a fusion transcript coding for SND1 amino acids 1-593 coupled to BRAF amino acids 381–766  and 1202–2947 from RefSeq transcript NM_004333.4 (BRAF). Wild type BRAF transcripts were observed in the GTL16R1 and GTL16R3 clones as evidenced by paired reads spanning between exons 8 and 9 (data not shown). GTL16 did not contain any paired reads where one end mapped to SND1 and the other mapped to BRAF. In contrast, GTL16R1 contained 628 chimeric pairs and GTL16R3 contained 702 chimeric pairs  to compare transcript coverage between samples. Relative to GTL16, the RBM of bases 1-2006 of SND1 in GTL16R1 and GTL16R3 is approximately 2-fold higher, while bases 2007–3522 are equivalent . SimilarTo test the functional significance of the BRAF fusion, we treated with METi, RAFi, or a combination of both in cell viability assays. Single agent treatment with RAFi did not alter growth of GTL16, GTL16R1 or GTL16R3 , x-axis.To further confirm that the BRAF fusion protein is indeed signaling through the MAPK pathway, we treated GTL16R1 and GTL16R3 with MEKi as a single agent or in combination with METi. Surprisingly, unlike RAFi, single agent MEKi was very potent and was able to inhibit tumor cell growth with an IC50 value of 1.5 and 4.5 nM for GTL16R1 and GTL16R3, respectively . GTL16 cTo determine if MEKi can synergize with METi, we tested with a sub-optimal dose of MEKi (ranging from 10 nM to 156 pM). Combination with 2.5 nM of MEKi yielded the best synergy as demonstrated by the ΔBLISS plot .To elucidate the mechanism of synergy, we examined modulation of signal transduction pathways by Western immunostaining total cell lysates from GTL16, GTL16R1 and GTL16R3 treated with 200 nM of METi, RAFi, or MEKi as single agents and in combination . PhosphoIn GTL16R1 and GTL16R3, SND1-BRAF was constitutively phosphorylated on BRAF S445 and levels did not respond significantly relative to total SND1-BRAF protein upon treatment with any inhibitor . The SerThe c-Met tyrosine receptor kinase activates two major signal transduction cascades, the RAS-RAF-ERK MAP Kinase pathway and the PI3K/AKT pathway. We examined AKT activity via phosphorylation of S473 on AKT using Western immunoblot. Compared to GTL16, untreated levels of AKT pS473 were approximately two-fold and six-fold lower in GTL16R1 and GTL16R3, respectively . TreatmeWe also investigated ERK phosphorylation levels in response to compound treatments. The basal phosphorylated ERK levels in GTL16R1 and GTL16R3 were approximately 3 fold higher than GTL16. In response to METi, ERK phosphorylation was completely inhibited in GTL16, however, ERK phosphorylation only decreased slightly in GTL16R1 and GTL16R3 .Treatment with RAFi increased ERK phosphorylation two fold in GTL16 as RAF inhibitors can transactivate homo/heterodimers of RAF isoforms to increase MEK signaling Since GTL16R1 and GTL16R3 seemed to shift signaling towards the MAPK pathway and away from the PI3K/AKT pathway, we tested whether the resistance was solely dependent upon the MAPK pathway using MEKi alone. Single agent MEKi was able to completely inhibit ERK phosphorylation in all cells. Consistent with BRAF activation, these data indicate that METi inhibition is bypassed downstream of c-Met and upstream of MEK.MET locus is amplified or mutated in selected tumor types, and these patients are predicted to be responsive to treatment with c-Met inhibitors In some cancers, dysregulated c-Met signaling results in oncogene addiction, a dependence on the pathway to maintain cancer cell growth and survival Here we describe a GTL16 cell line model of METi resistance. We identify an activated SND1-BRAF fusion protein which confers resistance to METi. BRAF promotes cell growth and proliferation by transducing signals from growth factor receptors as part of the MAP kinase pathway via MEK and ERK. Our findings are consistent with a model of resistance where the fusion activated BRAF can bypass upstream c-Met inhibition by hyperactivating the MAPK pathway in a c-Met independent manner. As summarized in Like previously described BRAF fusion proteins, the specific role of the N-terminal partner seems limited. Despite containing an intriguing array of tandem Staphylococcal nuclease homologue domains which function to capture RNA targets, the SND1 portion of the fusion protein likely does not directly contribute to c-Met resistance because use of RAFi was able to restore sensitivity to METi. Rather, we think transcription of the gene fusion is driven by the SND1 promoter and SND1 replaces the N-terminal regulatory region of BRAF which normally regulates BRAF catalytic activity Importantly, the simultaneous combination treatment with both METi and RAFi is required to reduce the phosphorylation of ERK in GTL16R1 and GTL16R3 more than 50% compared to basal pERK levels in GTL16 . The inaThe role of PI3K activation in our particular resistant model seems minor. Lower basal pAKT S473 levels in GTL16R1 and GTL16R3 suggest less dependence upon PI3K signaling. Additionally, while METi treatment suppresses pAKT S473, MEKi treatment dramatically increases pAKT S473, and yet still inhibits cell viability, suggesting that suppressing AKT phosphorylation is not a requirement for blocking cell proliferation . Indeed,de novo SND1-BRAF fusion translocations, or if resistant clones arose from an enrichment of pre-existing cells with these translocations. The GTL16R1 and GTL16R3 are not identical clones despite possessing the same SND1-BRAF fusion protein product. RNA-Seq and exome sequencing data show clone- specific SNPs. Furthermore, the CNV segmentation pattern appears to be distinct in the 7q32–7q34 region. However, because the breakpoints are so similar, it is possible that GTL16R1 and GTL16R3 are subvariants of a common precursor cell that was present in the GTL16 tumor which has since diverged over time creating a subpopulation of SND1-BRAF cells in culture. Additionally, the SND1-BRAF fusion is highly amplified in GTL16R1 and GTL16R3, but is not detected via Next Generation Sequencing in GTL16. At this point we cannot rule out the possibility of a small pre-existing pool of SND1-BRAF expressing clones in GTL16.It is unclear if prolonged exposure of GTL16 cells to METi resulted in The clinical significance of the SND1-BRAF fusion is unknown. A computational search of publically available CNV arrays representing more than 5000 cell lines and primary samples did not identify any co-occurring SND1-BRAF breakpoints consistent with the known fusion. It is likely that selective pressure from prolonged drug treatment is required to generate SND1-BRAF fusions in primary tumors.The acquisition of additional oncogenic drivers has important implications for targeted therapy. The sequential treatment of tumors with targeted agents may allow sequential resistance to develop if inhibitors are applied individually. Conceivably, resistance mechanisms to BRAF inhibition like IGF-1R Figure S1Kinase Selectivity Screen of METi. (A) Kinase selectivity screen (KSS) performed at Upstate Biotechnology. Values in % inhibition of phosphorylation given 1 µM of METi compared to control. (B) KSS performed at University of Dundee . Values in % inhibition of phosphorylation given 1 µM of METi compared to control. (C) Cell based dose response kinase inhibition of indicated kinases by METi.(PDF)Click here for additional data file.Figure S2Kinase Selectivity Screen of RAFi. (A) Kinase selectivity screen (KSS) performed at University of Dundee . Values in % inhibition of phosphorylation given 1 µM of RAFi compared to control. (B) KSS performed at Invitrogen (Selectscreen Service) compared to control. Values in % inhibition of phosphorylation given 1 µM of RAFi. (C) Cell based dose response kinase inhibition of indicated kinases by RAFi.(PDF)Click here for additional data file.Figure S3Phosphorylation fold decreases upon PF-04217903 treatment of GTL16, GTL16R1 and GTL16R3 cells compared to vehicle. Values are taken from RPPA analysis of cells treated with 2.5 µM METi for 1 hr.(PDF)Click here for additional data file.Figure S4Amplification at regions 7q34 and 7q32 form a fusion transcript. (A) CNV data show breakpoints between exons 16–17 of SND1 and 8–9 of BRAF. (B) RNA-Seq paired end reads aligned to the putative fusion transcript show reads that overlap or span the SND/BRAF junction only in clone GTL16R3 but not GTL16.(PDF)Click here for additional data file.Figure S5(A) Table of raw coverage or normalized Reads/Base/Million (RBM) reads of RNA-Seq that align to full length or fusion regions of SND1 or BRAF. Fold is calculated relative to GTL16. Fusion regions are overrepresented in GTL16R1 and GTL16R3 relative to GTL16. (B) Graphic of the raw coverage and RBM at each base position in SND1 and BRAF shows the uneven distribution of coverage and high expression of the fusion transcript.(PDF)Click here for additional data file.


Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites , and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules.Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments.Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in  Ascaris lumbricoides infects around 2 billion people and may influence the pathogenesis, evolution and diagnosis of allergic diseases A. lumbricoides is very allergenic and there is evidence that it may enhance Th2 responses . Based on the strength of the specific IgE response and the frequency of sensitization, cockroach GST (Bla g 5) is considered the most allergenic Dermatophagoides pteronyssinus (known as Der p 8). The prevalence of IgE sensitization to GSTs is higher in allergic patients living in tropical environments Allergic diseases such as asthma are public health problems and, together with other immune mediated diseases, are increasing worldwide Schistosoma ssp., GST have been detected in endemic communities Wuchereria bancrofti and cockroach might boost cross-sensitization A. lumbricoides (GSTA) to study their IgE binding properties and allergenic potential. We found that GSTA has allergenic properties. Serum specific IgE levels to this molecule positively correlate with the IgE levels to mite and cockroach GSTs. In addition, there are at least six GSTA isoforms that are recognized by human IgE, one being the most prominent. Furthermore, natural GSTA (nGSTA) induced type-I hypersensitivity reactions in sensitized subjects.GSTs from helminths also induce IgE antibodies in infected individuals A. lumbricoides extract was separated by Ion-Pair Reverse-Phased micro High Performance Liquid Chromatography (IP-RP-µHPLC) and each fraction was analyzed by mass spectrometry. The component of 23 kDa was detected at 33 minutes of retention time with the mass spectrum of glutathione transferase  as well as the recombinant GST1 from A. suum , but median IgE levels against the nGSTA were significantly higher than those to the rGSTA . In addition, serum IgE levels to nGSTA were comparable to the specific IgE levels to other Ascaris allergens such as the nematode-specific rABA-1  and tropomyosin   .D. pteronyssinus (rDer p 8) and Blomia tropicalis (rBlo t 8 and nBlo t 8). Interestingly, 20 out of 26 asthmatics sensitized to Ascaris GSTs had also detectable specific IgE to at least one of the mite GSTs , nBlo t 8 , and rDer p 8 . Specific IgE levels to Ascaris and mite GSTs showed significant positive correlations . Also, age was inversely related to the levels of IgE to rGSTA as a continuous variable . There was no difference in the specific IgE levels to rGSTA between cases and controls after adjusting by age using linear regression models  compared to controls  in the age group of 14–27 years . There were no differences in the prevalence of positive IgE reactivity to rGSTA between asthmatic patients and non-asthmatic controls . Considering that age have been described as a predictor of the antibody response to parasite GSTs, the study participants were stratified according to age's percentiles in five groups of comparable size. We found that the frequency of positive IgE to rGSTA was significantly greater in children with age 7–13 years  than to individuals in the groups of 14–27 years , 28–37 years , 38–48 years  or 49–70 years  and to the cockroach rBla g 5  were low with IgE levels to rGSTA being significantly higher than to rBla g 5 (p = 0.0001); however, compared to Der p 2 , IgE levels to GSTs were typically of low intensity  and other non-GST allergens such as Bla g 2 and Der p 2. All the asthmatic patients with a positive IgE sensitization to Ascaris were selected for analysis . We found a significant correlation between the IgE levels to rGSTA and rBla g 5  but not between the IgE levels to rGSTA and rDer p 2 . Specific IgE levels to rGSTA  using a LTQ Linear Ion Trap Orbitrap XL™ mass spectrometer. The corresponding chromatogram included several peaks in the range of 25 to 45 min. The largest peak was found at a retention time of 35.75 min . Four in extract . Similar extract .A. suum (UniProt P46436), resulting in 73.3% of sequence coverage (A. suum GST2 (Uniprot P48429). An alternative version of this peptide (LTYFNLR) was also detected with two amino acid substitutions at positions 10(D→N) and 11(I→L) that overlapped the sequence of Gallus gallus GST5 (Uniprot P20137). In addition, the peptide AEPIR with amino acid substitutions at positions 15(G→A), 17(G→P) and 18(A→I) and the peptide KFGLAGK with one amino-acid substitution at position 74(Q→K) were found to match the sequence of the nematode Caenorhabditis elegans GST4 . Twenty-eight peptides matched with the sequence of the GST1 of coverage . In addians GST4 . Detailer spot 4 . Interes mulatta .A. lumbricoides extract using strips with a broader range (pI 3–10) (A. suum GST1 increased towards the most basic isoform (pI 8.0) and revealed the polymorphic peptide DILPVELAK between amino acid positions 120 to 128, which overlapped the sequence of Wuchereria bancrofti π class GST (Q86LL8) (Dirofilaria immitis (UniProt P46426)  , confirm(Q86LL8) . Even th(Q86LL8) . There w P46426) . AltogetA. lumbricoides is well-known as a powerful inducer of Th2 immune responses and IgE synthesis in humans. There are several IgE binding components in the extract of Ascaris and their characterization is essential for better understanding the allergenicity of this nematode, as well as to define an array of cross-reactive and species-specific allergens for component resolved diagnosis of allergy in places where helminthiases are not yet controlled. In the whole extract of A. suum, we have previously observed an IgE-binding component of 23 kDa with sequence similarity to GST A. lumbricoides have allergenic properties since they not only bind specific human IgE antibodies but also induce type-I hypersensitivity reactions in skin of sensitized subjects. Interestingly, the strength of the IgE reactivity to this molecule was greater than to mite and cockroach GSTs which have been previously recognized as allergens The soil-transmitted nematode W. bancrofti and cockroach (Bla g 5) A. lumbricoides had shown to induce cross-reactive antibodies that recognize antigens in the extract of the mite D. farinaeSchistosoma japonicum GST A. lumbricoides is the primary sensitizer. Here, we were not able to detect cross-inhibition of IgE binding between Ascaris and mite GSTs, not even using depletion ELISAs, and a possible explanation may be the strong interference by high titers of competing IgG antibodies (data not shown).We found a strong correlation between IgE levels to GSTA and other invertebrate GSTs . Since tlow reactivity” is a hallmark of the IgE response to GSTA in adults. This was even more evident in asthmatic patients in which the strength of IgE levels to GSTA and cockroach GST (Bla g 5) where much lower compared to other non-GST allergens such as Der p 2 (D. pteronyssinus), we hypothesize that the low strength of IgE reactivity to GSTs is reflecting particular features of the humoral response to these molecules. Our findings are in agreement with previous studies reporting that IgE levels to allergenic GSTs are low The evaluation of a random sample set of asthmatics and controls revealed that at the population level the prevalence of positive IgE sensitization to GSTA was low. In addition, the strength of the IgE response to GSTs from Ascaris, mites and cockroach were consistently low. The same finding in the two sample datasets analyzed strongly support that this “ Der p 2 . Since tSince previous reports have described that human antibody responses to parasite GSTs may change with age A. suum have detected most of the GST expression in the brush border of the parasite's gut and weak to moderate expression in the cytoplasm of longitudinal muscle cells, oviducts and the lateral cords of the hypodermis A. lumbricoides. Intact masses analysis detected four abundant isoforms and mass spectrometry analysis after trypsin digestion on the affinity purified nGSTA showed several amino acid substitutions, mainly located on the N-terminal end. The mass shift pattern of the isoforms compared to the monoisotopic mass of GSTA suggested glycosylations; however experiments to detect sugars found that GSTA is non glycosylated. Although we did not experimentally evaluate other potential post-translational modifications, the results support that GSTA isoforms are due to amino acid substitutions. In agreement with our observations, the draft genome of A. suumA. lumbricoides was striking , the allergenic properties of GSTA should be considered when using helminth GSTs for vaccination; especially because A. lumbricoides is distributed worldwide, commonly found in poly-parasited subjects and different degrees of cross-reactivity between helminth GSTs have been described. However, the fact that natural GST isoforms have differential intensity in IgE binding could be considered for selecting natural or engineered non-allergenic vaccine candidates.The results of this study, beyond its relevance for allergy diagnosis and the understanding of the allergenicity, provide important information to the field of helminth GST research. The finding that A. lumbricoides of potential relevance for asthmatic patients predisposed to recognize invertebrate pan-allergens and co-sensitized to mite and cockroach GSTs. As has been described for other allergenic GSTs, the strength of IgE reactivity to GSTA is low and the prevalence and IgE levels to GSTA are highest during childhood. GSTA has several isoforms with differential IgE recognition.In summary, GSTA is a new allergen from This study was conducted following the ethical principles for medical research stated in the Declaration of Helsinki. The Bioethics Committee of the University of Cartagena approved the study. A full verbal explanation of the investigation was given to each participant and written informed consent was obtained from all subjects or their parents or legal guardians.B. tropicalis and D. pteronyssinus, we tested sera from 26 asthmatic patients and 3 non-asthmatic controls with positive IgE sensitization to Ascaris (Mean age 27±13.3 years). This set of sera plus 7 Ascaris negative sera were used to study the correlation between the IgE levels to purified GSTs from Ascaris and mites . To evaluate the IgE binding capacity of purified GSTs from Ascaris and the house dust mites  n = 36, . The freA. lumbricoides extract was obtained as described previously B. tropicalis (nBlo t 8). The eluates containing the purified GSTs were visualized in denaturing 12% SDS-PAGE gels after Coomassie staining.A. suum (rGSTA) was cloned into the pQE30 vector based on the sequence deposited at GenBank under accession number (X75502.1). The protein was expressed in E. coli M15 and purified as a 6xHis-tagged protein. The recombinant GST of B. tropicalis (rBlo t 8), was amplified from a cDNA library of B. tropicalisD. pteronyssinus GSTmu (rDer p 8) (AY825938) and Der p 2 (ABG76196) were subcloned into the pET45b+ vector and expressed in the Origami BL21 (DE3) bacterial system. Induced cultures were re-suspended in native buffer , incubated with lysozyme  and sonicated. Lysates were incubated with Ni-NTA resin (Invitrogen) for one hour, washed with native buffer plus 20 mM imidazole, and eluted with native buffer plus 250 mM imidazole as a 6xHis-tagged protein. The Ascaris allergens rABA-1 and rAsc l 3 were produced as previously described E coli (Bla g 5) or Pichia pastoris (Bla g 2) as previously described Recombinant GST1 from ρ-nitrophenylphosphate substrate  and the enzymatic reaction stopped after 30 minutes of substrate incubation by adding 3 N NaOH. Absorbance was measured at 405 nm using a Spectrophotometer . All the determinations were done by duplicate and IgE levels above 0.130 OD405 nm were considered positive (mean OD of 7 non-Ascaris sensitized subjects +3 SD). Specific IgE levels to nBlo t 8 (0.5 µg), rBlot 8 (0.75 µg), rDer p8 (0.75 µg), rBla g 5 (0.75 µg), rBla g 2 (0.75 µg) and rDer p 2 (1 µg) were measured by ELISA as aforementioned. Positive and negative control sera were included in each plate.The prevalence of specific IgE antibodies against nGSTA and rGSTA were determined by indirect ELISA. Appropriate antigen concentrations were obtained by titration. Briefly, each well of a 96-well plate was coated with 0.5 µg of nGSTA and 0.75 µg of rGSTA diluted in PBS (pH 7.4), incubated overnight at 4°C, washed with 0.1%-Tween-20/PBS (TPBS) and blocked during 3 hours with 3% bovine serum albumin. Sera samples diluted 1∶5 in blocking buffer were added and incubated overnight, washed and incubated with 100 µl of anti-human IgE Alkaline-Phosphatase Conjugate (Sigma). The assay was developed with The ability of nGSTA and nBlo t 8 to induce mast cell degranulation was evaluated by skin prick tests in eight asthmatic patients with a positive IgE result to the rGSTA [OD >0.130], two parasited children, and two asthmatic patients with a negative IgE result to the rGSTA, who provided consent to perform the procedure . The purA. suum GST, Bla g 5 (O18598), Blo t 8 (ACV04860) and Der p 8 (P46419) structures were done in the SWISS-MODEL server using as templates the experimentally defined structures 2on5H (O. volvulus), 1m0uA (D. melanogaster), 1gsuB  and 1c72d , respectively. Models were refined in Deep-View (energy minimization and rotamer replacements). Their quality was evaluated by several tools, including Ramachandran plots, WHATIF, QMEAN4 index and energy values (GROMOS96 force field). Relative values of accessible solvent area (r-ASA) were determined by ASA-view Homology-based molecular modeling of A. lumbricoides extract was done by high-performance liquid chromatography , using the following conditions: columns: 200×0.1 mm I.D. PS-DVB monolith column; 10×0.2 mm I.D. monolithic PS-DVB trap column; eluent A: water +0.05% trifluoroacetic acid; eluent B: acetonitrile +0.05% trifluoroacetic acid; 3 min trapping with 0.10% aqueous heptafluorobutyric acid (10 µl/min); temperature: 55°C; flow rate: 1 µl/min; gradient: 5–60% B in 60 min; injection of 1.0 µl of 60.5 µg/ml protein extract. The PS-DVB monolith and monolithic PS-DVB trap column were prepared at Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, Salzburg, Austria and are commercially available as ProSwift® reversed-phase monolith columns from Thermo Fisher Scientific, Vienna, Austria. Mass spectrometry analyses were performed using an electrospray ionization – time-of-flight mass spectrometer .The separation of the The separation of nGSTA was carried out using ProSwift® PS-DVB monoliths  with a length of 50 mm and an inner diameter of 1 mm in an HPLC system with low pressure gradient formation  using the following set-up: eluent A: 0.05% aqueous trifluoroacetic acid; eluent B: acetonitrile +0.05% tri-fluoroacetic acid; gradient: 20–50% B in 45 min; flow rate: 60 µl/min; temperature: 60°C; injection volume: 15 µl. The HPLC system was hyphenated to a linear ion trap - Orbitrap mass spectrometer  equipped with an Ion Max electrospray ionization source with a 34-gauge metal needle (both Thermo Fisher Scientific). Electrospray ionization was conducted in positive mode. Data were acquired with a resolution of 100,000 in a mass range of 700–3,500 m/z. For data acquisition and evaluation, the Xcalibur 2.0.7 software (Thermo Fisher Scientific) was used. Mass spectra were deconvoluted with the Xtract software (Thermo Fisher Scientific).The Ascaris extract (200 µg) and the purified nGSTA (25–50 µg) were separated by 2D-SDS-PAGE as previously described A. lumbricoides extract and Coomassie G250-staining were excised and in-gel digested using the ProteoExtract® All-In-One Trypsin Digestion Kit . Besides, four spots resulting of the two-dimensional separation of the nGSTA were excised from the gel and digested as follows: pieces containing the spots of the nGSTA were washed with MilliQ water and 100 mM ammonium bicarbonate buffer (ABC-buffer), then unstained with acetonitrile (ACN) and dehydrated at 30°C with a concentrator . The reduction was performed at 56°C for 30 min with 10 mM dithiothreitol in 100 mM ABC-buffer and the alkylation was done at room temperature for 20 min with 55 mM iodacetamide in 100 mM ABC-buffer. After washing with ABC-buffer and acetonitrile the slices were dried again at 30°C in the concentrator. The digestion was accomplished as prescribed with sequencing grade modified Trypsin . The peptides were extracted from the gel slices with buffer  and concentrated to a final volume of 100 µl.The three visible spots resulting from the two-dimensional separation of the Tryptic peptides were sequenced after separation by ion-pair reversed-phase capillary high-performance liquid chromatography (HPLC) directly coupled to an electrospray ionization-quadrupole-time-of-flight mass spectrometer  as previously described 125I] - labeled α-human IgE . Radiographic signals were visualized by overnight exposure to a phosphorimager screen and subsequent scanning using a Fujifilm BAS-1800 II reader. Specific IgE to the Ascaris extract and HPLC fractions was detected using patient's sera as primary antibody and alkaline phosphatase-labeled anti IgE (αIgE-AP) as secondary antibody .For immunoblotting, proteins were electro-transferred onto nitrocellulose membranes, blocked and washed. After overnight incubation with sera from Ascaris sensitized patients, membranes were washed and bound IgE was detected using [Glycodetection of the Ascaris extracts was performed using the PAS staining, which reveals the aldehyde groups that are exposed when the sugar rings are open. Briefly proteins were electrotransferred to PVDF membranes at 15V during 50 minutes, washed with distilled water, incubated in a solution of 1% periodic acid/3% acetic acid during 15 min; then, the membranes were washed 3 times with distilled water (5 minutes), incubated 15 minutes with the Schiff's reagent (Fuchsin sulfite) in a dark chamber without agitation and washed 3 times with distilled water. Glycosilation signals were visualized on the membranes. For nGSTA and nBlo t 8, the metaperiodate method was used. Briefly, PVDF-electrotransferred proteins were incubated in the dark for 20 minutes in a solution of 10 mM sodium metaperiodate dissolved in 100 mM acetate buffer (pH 5.5). After washed, the membrane was incubated for 60 minutes at RT with 0.125 mM biotin hydrazide (diluted 1∶5000 in 100 mM acetate buffer). Streptavidine-horseradish peroxidase (1∶100.000 diluted in PBS) was added and incubated during 30 minutes. Membranes were exposed to chemiluminiscence reagents  and images were captured with a CCD camera .2 analysis and adjusted by covariates using logistic regression. The comparison of IgE levels to rGSTA between asthmatic patients and controls was estimated using the non-parametric Mann Whitney U test. To evaluate the effect of age on the IgE levels to rGSTA, the study participants were stratified in five groups , , , ,  as defined by the 20th, 40th, 60th and 80th percentile of the age. Linear regressions were used to analyze the effect of age, gender and disease status on the IgE levels to rGSTA. All statistical analyses were two-tailed and the significance was set at p<0.05. Analyses were performed using IBM Statistics SPSS v20 (IBM Corp) and GraphPhad Prism.IgE levels to purified allergens were expressed as the mean OD  of duplicate wells. Non-parametric paired Wilcoxon signed-rank test was used to compare the strength of IgE levels to GSTs. The relationship between IgE levels to GSTs were calculated by Spearman rank correlation. The comparison between the frequency of IgE sensitization between cases and controls was done by χFile S1Descriptive of specific IgE levels to purified Ascaris and mite GSTs (n = 36).(PDF)Click here for additional data file.File S2Multiple sequence analysis between parasitic and non-parasitic GST plus the amino acid composition of the patches with surface-exposed residues between Ascaris GST1 and non-parasitic GSTs from mites  and cockroach (Bla g 5).(PDF)Click here for additional data file.File S3Tryptic peptides coinciding with known GST sequences after LC-MS/MS of purified nGSTA.(DOCX)Click here for additional data file.File S4Peptides identified in four spots containing nGSTA after 2D electrophoresis and LTQ Orbitrap XL mass spectrometry analysis.(XLS)Click here for additional data file.File S5Peptides identified in three spots containing nGSTA after 2D electrophoresis and LC-MS/MS analysis.(PDF)Click here for additional data file.File S6Sequences with homology to GSTs in the draft genome of A. suum.(XLSX)Click here for additional data file.