Blomia tropicalis is a dust mite and an important source of allergens in tropical regions. Up to now, the assays to diagnose atopy to this mite use whole body extract as antigens. However, anti-B. tropicalis IgE antibodies cross-react with Ascaris lumbricoides antigens, hindering the diagnosis of allergy to this mite. In this study, B. tropicalis recombinant allergens were evaluated with the purpose of developing an immunodiagnostic assay for allergy to this mite with greater specificity than those commercially available.B. tropicalis allergens (Blo t 5 and Blo t 21) were cloned into a plasmidial expression vector, expressed in Escherichia coli and purified by affinity chromatography. Sixty-three sera containing anti-B. tropicalis extract (BtE) IgE antibodies were used to investigate IgE reactivity to the recombinant Blot 5 and 21 allergens. Inhibition assays with 20 sera pre-adsorbed with A. lumbricoides extract were performed using rBlo t 5, rBlo t 21, and BtE as antigens. All the assays were carried using indirect ELISA.Two A. lumbricoides extract affected less the reaction with rBlo t 5 and rBlo t 21 than with BtE.Eighty-two point nine percent and 80.0% of the sera with anti-BtE antibodies from 35 children reacted with rBlo t 5 and rBlo t 21, respectively, whereas 92.8% and 89.3% of the 28 sera with anti-BtE antibodies from adult asthma patients reacted with the same allergens, and 96.4% of these sera reacted with a mixture of rBlo t 5 and rBlo t 21. In an inhibition ELISA, the absorption of sera by B. tropicalis antigens. Moreover, the assays using the recombinant allergens had lower IgE cross-reactivity with A. lumbricoides antigens, a fact which would confers higher specificity to serodiagnostic assays than the crude mite extract. However, additional recombinant allergens should be evaluated in order to reach the same sensitivity of the commercially available assays based on mite extract.The rBlo t 5 and rBlo t 21 allergens contain important epitopes recognized by IgE antibodies of individuals allergic to  Blomia tropicalis and Dermatophagoides pteronyssinus are predominant in house dust and are commonly found together  x 100.Inhibition ELISA was performed to investigate the presence of anti-ous work  and are lsewhere . ResultsThe D’Agostino and Pearson test was used for verification of the data normality. The significance of the differences among the different allergens was assessed by the Wilcoxon signed rank test (when the data distribution was found to be non-Gaussian). Differences with a P value equal to or less than 0.05 were considered statistically significant.B. tropicalis recombinant allergens were expressed and purified as full length fusion proteins containing N-terminal His-tags (6 His). Once the fusion proteins were purified, their tags (6 His) were not removed. The achieved yields of rBlo t 5 and rBlo t 21 were 3.1 to 4.6 mg and 2.9 to 4.5 mg per liter of bacterial culture, respectively. Each His-tagged (rBlo t 5 and rBlo t 21) proteins produced an expected band in SDS-PAGE  of IgE antibodies against rBlo t 5 in the children group, and of IgE antibodies in the adult asthmatic patient group as detected in assays using rBlo t 5 or a mixture of rBlot 5 plus rBlo t 21, were significantly higher than in the assay using BtE .Indirect ELISA was used to evaluate the reactivity of IgE antibodies with the fusion His-rBlo t 5 and His-rBlo t 21 fusion proteins. The IgE reactivity profile of the tested sera with the recombinant proteins is summarized in Table B. tropicalis crude extract IgE antibodies (median inhibition of 39.2%) than in the levels of anti-rBlo t 5 (median inhibition of 3.6%) and anti-rBlo t 21 (median inhibition of 0.1%) IgE antibodies, when they were incubated with A. lumbricoides extract at both concentrations . As expected, the anti-B. tropicalis crude extract IgE antibody levels were lower in sera pre-adsorbed with A. lumbricoides extract than in the non-adsorbed sera . Furthermore, there was also statistically significant difference between sera adsorved with 0.3 μg/mL and 3 μg/mL of A. lumbricoides extract . The proportion of total IgE that was reduced by incubation with A. lumbricoides natural extract (median inhibition of 0.1%) was lower than the proportion of anti-B. tropicalis extract IgE antibodies that was reduced by the same treatment  than D. pteronyssinus (39.9%) in bed dust in Salvador, a major city in Northeast of Brazil [B. tropicalis predominates in the mite fauna, it is common to find a high rate of co-sensitization to Blo t 5 and Blo t 21, as highlighted in our study  [The he world -17. ThesBlo t 21 . In the Blo t 21 . Likely,f Brazil . In envividuals) ,19. Althviduals) . FurtherE. coli and native rBlo t 5 have comparable IgE reactivity in terms of percentage of sera with antibodies [B. tropicalis in order to increase its sensitivity. Blo t 7, which has been shown to be as reactive as Blot t 5 with IgE antibodies in the sera from allergic children in a study also carried out in a tropical environment [A. lumbricoides[This study, despite not comparing the reactivity of IgE antibodies to rBlo t 5 and rBlo t 21 with the reactivity to their native counterparts, suggests that recombinant allergens produced as described in this paper can replace natural allergen extracts in the diagnosis of allergies, confirming previously published data showing that rBlo t 5 expressed in tibodies . Howeverironment , may be bricoides, would nThe relationship between house dust mite sensitization and triggering of asthma and other atopic diseases is well documented ,25. GiveB. tropicalis sensitization with helminth infection in tropical and undeveloped regions of the world. It is in fact believed that about 1.5 billion people worldwide are infected with A. lumbricoides[A. lumbricoides, Trichuris trichiura and Toxocara spp (Toxocara canis and Toxocara cati) infections. In fact, most of the sera studied in the present work had anti-Ascaris IgG antibodies, indicating present and/or past A. lumbricoides infection. In this area, an overlap of parasitic endemicity and high prevalence of allergic diseases is observed [A. lumbricoides[A. lumbricoides extract led to varying degrees of inhibition of the binding of IgE to mite allergens. It would also be useful to further demonstrate this cross-reaction by inhibiting the binding of IgE antibodies to A. lumbricoides extract by incubation of the sera with B. tropicalis antigens. As the high levels of IgG antibodies raised in the immune response to this helminth hinders the detection of IgE anti-Ascaris antibodies, a possible way to carry out the experiment would be to use an assay to detect IgE antibodies more sensitive than the one used in the present work.In addition to the problems described above with the use of whole allergenic extracts in the immunodiagnosis of allergic diseases, there is another factor that should be considered by the companies that commercialize these products: the common association of bricoides,29. Salvobserved -32. Therbricoides. The incA. lumbricoides extract was higher for the binding of antibodies to B. tropicalis crude extract than to rBlot 5 and rBlo t 21, although in a few sera there was also a high level of IgE cross-reaction between the helminth extract and the recombinant allergens. Cross-reactivity to both recombinant allergens was observed in the same sera (data not shown). This finding could be explained if the anti-Ascaris antibody response in these sera donors had been more polyclonal than the immune response of the other serum donors, so that antibodies against additional cross-reactive epitopes would be produced. Indeed, it is known that the repertoire of the antigens that are recognized in complex antigenic mixtures may vary greatly in different individuals of the same species [The inhibition by adsorption with  species .A. lumbricoides extract of anti-rBlo t 5 and anti-rBlo t 21 in relation to anti-BtE antibodies cannot be explained by the presence of lower levels of anti-recombinant allergen antibodies, since the amounts of anti-rBlo t 5 IgE antibody activity, on the contrary, were found to be higher than those of anti-BtE IgE antibody activity. Due to this less intense cross-reactivity, assays using a mixture of rBlot 5 and rBlo t 21 as antigen should be more specific than assays using the crude extract.The observed difference in the degree of absorption by IgE antibodies raised by helminths can cross-react with several epitopes of mite antigenic extracts often used in diagnosis of allergic diseases possibly affecting the correct diagnosis. It becomes increasingly relevant, therefore, to obtain more specific allergens to make an accurate diagnosis of allergy and, consequently, improving immunotherapy for mite allergy. However, the price of assays using bacteria-produced recombinant antigens, as compared to those of assays using crude extracts obtained from mite cultures, is a factor that should be taken into consideration.B. tropicalis-sensitized population evaluated in this work. In addition, we showed that the IgE reactive to these allergens had less cross-reactivity with A. lumbricoides extract than anti-B. tropicalis extract IgE, as assessed by an IgE-binding inhibition assay. Thus, rBlo t 5 and rBlo t 21 expressed in E. coli may be potential candidates to be used in a pool of different recombinant allergens for improving serodiagnosis assays of allergy to B. tropicalis. More studies are needed to obtain other recombinant allergens in order to develop a highly sensitive and specific assay for the diagnosis of allergy induced by B. tropicalis allergens.We demonstrated that rBlo t 5 and rBlo t 21 were antigenic for the Blomia tropicalis recombinant antigen; cDNA: Complementary DNA; pRSETA: Plasmidial vector pUC-derived expression vectors that designed for high-level protein expression and purification from cloned genes in Escherichia coli; BtE: B. tropicalis crude extract; ELISA: Enzyme-linked immunosorbent assay; SPT: Skin prick test; CEAR: Centro de Estudos em Alergias Respiratórias; SCAALA: Social Change in Asthma and Allergy in Latin America; mRNA: Messenger RNA; PCR: Polymerase chain reaction; BLAST: Basic Local Alignment Search Tool; IPTG: Isopropyl β-D-1-thiogalactopyranoside; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF: Immobilon-P polyvinylidene difluoride; PBS: Phosphate-buffered saline; PBS/T: Phosphate-buffered saline containing 0.05% Tween; NFM: Non-fat milk; OD: Optical density; CONEP: National Commission on Ethics in Research.rBlo t: All authors report no conflict of interest financial or otherwise, with the findings of this study.KAC: carried out the laboratory assays and wrote the first draft of the manuscript; OPMN: helped in the study design, supervised the molecular biology work and helped in the manuscript revision; FBM: helped with the molecular biology assays; JCMP: helped in the inhibitory assays and revised the manuscript; FABF: helped in the imunmologial assay and revision the manuscript; GL, AAC and MLB provided the asthma patients and children sera and revised the manuscript; MCAS: has performed the statistical analysis; LCPC: helped in the study design and reviewed the manuscript; NMAN: conceived the work, the study design and supervised the immunological assays; CSP: helped in obtaining the Blo t 5 and Blo t 21. All authors read and approved the final manuscript.


Chlamydia trachomatis growth and blocked sphingolipid trafficking from host cell Golgi into chlamydial inclusions. Since the p38-regulated/activated protein kinase (PRAK) is a known target of rottlerin and is activated in Chlamydia trachomatis-infected cells, we investigated the potential role of this kinase in rottlerin-mediated anti-chlamydial activity. However, we found that a PRAK-specific inhibitor failed to inhibit chlamydial growth, suggesting that the kinase activity of PRAK may not be required for chlamydial intracellular replication. This conclusion was supported by the observation that chlamydial organisms replicated equally well in mouse embryonic fibroblast cells with or without PRAK. Moreover, neither the PRAK inhibitor nor PRAK deficiency altered host sphingolipid trafficking into chlamydial inclusions. Finally, rottlerin maintained its anti-chlamydial activity in PRAK-deficient cells. Together, these observations have demonstrated that PRAK is not required for either the rottlerin-mediated anti-chlamydial activity or rottlerin inhibition of sphingolipid trafficking, suggesting that rottlerin may achieve its inhibitory role by targeting other host factors.We previously found that rottlerin, a plant-derived small molecule compound, profoundly inhibited  Chlamydia trachomatis is the number one cause of sexually transmitted bacterial diseases worldwide trachomatis infection causes diseases remains unclear, it is thought that the obligate intracellular growth and cell to cell spreading may significantly contribute to the C. trachomatis pathogenicity. The infectious elementary bodies (EB) of C. trachomatis invade the epithelial cells of urogenital tract via endocytosis. An intracellular EB then differentiates into a non-infectious but metabolically active reticulate body (RB) for biosynthesis and replication. The progeny RBs finally differentiate back into EBs for spreading to new cells. The whole process is restricted within a cytoplasmic vacuole termed inclusion. Thus, nutrients and energy must be transported into the inclusions to support chlamydial growth. For example, sphingomyelin synthesized from fluorochorme-labeled ceramide in the Golgi apparatus was detected in the chlamydial inclusions Urogenital tract infection with C. trachomatis infection, many infected individuals are undiagnosed and untreated due to lack of obvious symptoms. Thus, searching for non-antibiotics topical reagents that can prevent C. trachomatis transmission is necessary. Previously, we reported that rottlerin profoundly inhibited chlamydial intracellular growth, which correlated with blockade of sphingolipid trafficking from the host cell Golgi apparatus into chlamydial inclusions C. trachomatis infection, we tested whether PRAK plays a role in rottlerin’s anti-chlamydial activity in the current study.Despite the serious health problem caused by MAPKPRAK, also known as MAPK-activated protein kinase 5 (MK5), is phosphorylated and activated by p38C. trachomatis L2/LGV-434/Bu organisms used in the current study were propagated, purified, aliquoted and stored as described previously The C. trachomatis for 16 h or various periods of time as indicated in individual experiments and treated with the corresponding inhibitors including rottlerin  and EGCG . At 48 h post infection, cell samples were harvested and subjected to electrophoresis in a SDS polyacrylamide gel. The resolved protein bands were transferred to nitrocellulose membranes for antibody detection. The following primary antibodies were used: rabbit polyclonal antibody (pAb) against HSP27 , rabbit pAb against phosphorylated HSP27 at serine 78 , mouse monoclonal antibody (mAb) MC22 against chlamydial major outer membrane protein  complexed with 0.034% defatted bovine serum albumin (dfBSA) (Sigma) in MEM at 4°C in the dark. Cells were washed 3 times with Hank’s balanced salt solution and back exchanged with MEM/0.34% dfBSA for 1 hour. After wash, cells were immediately visualized by fluorescence microscope.The sphingomyelin accumulation assay was carried out as described previously C. trachomatis L2 organisms, unpublished data) plus a goat anti-rabbit IgG secondary antibody conjugated with Cy2  was used to visualize chlamydial organisms. Hoechst  was used to visualize DNA. In some experiments, the host cell cytoplasm was visualized by labeling the F-actin with Alexa-Fluor 568 Phalloidin .The immunofluorescence assay was carried out as described previously To understand the mechanism of the rotterlin anti-chlamydial activity, we monitored the activity of the p38-regulated/activated protein kinase (PRAK), a known target of rotterlin, in HeLa cells with or without chlamydial infection and in the presence or absence of rotterlin by detecting the phosphorylation of host cell HSP27, a known substrate of PRAK . The phoC. trachomatis-infected cultures with or without treatment with either EGCG, a known inhibitor for PRAK with an IC50 = 1 µM C. trachomatis organisms replicated equally well in both wild type and PRAK-deficient MEF cells (We next tested whether the PRAK activity is required for chlamydial growth. EF cells . Thus, iEF cells .We also examined the role of PRAK in host sphingolipid trafficking into chlamydial inclusions. The PRAK inhibitor EGCG even at 10 µM failed to affect sphingolipid accumulation inside chlamydial inclusions while rottlerin at 1 µM completely blocked sphingolipid trafficking into chlamydial inclusions although neither inhibitors significantly alter the Golgi accumulation of sphingolipids , suggestC. trachomatis infection and rottlerin inhibited both the PRAK activity and chlamydial intracellular growth, we evaluated the role of PRAK in rottlerin anti-chlamydial activity. However, our experimental results have led us to conclude that PRAK is not required for either the rottlerin inhibition of chlamydial growth or sphingolipid uptake. First, we have shown that a PRAK-specific inhibitor fails to inhibit chlamydial growth; Second, chlamydial organisms replicate equally well in mouse embryonic fibroblast cells with or without PRAK; Third, the PRAK inhibitor nor PRAK deficiency can alter host sphingolipid trafficking into chlamydial inclusions; Finally, rottlerin can maintain its anti-chlamydial activity in PRAK-deficient cells. These observations together suggest that rottlerin may achieve anti-chlamydial activity by targeting other host factors.Rottlerin is known to inhibit various host kinases including PRAK. Since PRAK is activated during The question is what host factors might contribute to the rottlerin anti-chlamydial activity. Rottlerin can inhibit host PKCδ


Several different NMDA receptor (NMDAR) antagonists are used to produce pharmacological models for schizophrenia. These antagonists have different molecular mechanisms of action: non-competitive  and competitive . Because competitive antagonism results in more glutamate availability, this may result in glutamate spill-over either onto other receptors of the same synapse or to neighboring synapses. In this study, we modeled the effects of competitive blockade by simultaneously blocking NMDAR and increasing activity of AMPAR (spillover). We looked at alterations of network oscillations and synchronization using our biophysical model of the CA1 region of hippocampus using the NEURON simulator. We then compared the results with data obtained from the CA1 region of hippocampus in rats injected with CPP.Our network consisted of 800 five-compartment pyramidal cells (PYR), 200 one-compartment basket cell interneurons (BAS), and 200 one-compartment oriens lacunosum-moleculare interneurons (OLM) . All celRecordings from CA1 of rat hippocampus under the effect of CPP, showed that there is a reduction in theta and an increase in gamma. Modeling competitive inhibition to glutamate at all synapse locations  resulted in a reduction in the theta power and an increase in gamma power, differing from the noncompetitive effect with ketamine, which reduced both theta and gamma when given across all synapses. However, this result was also seen when the effect of competitive inhibition was restricted to only OLM cell NMDARs. Therefore, our model demonstrates both of these interpretations of CPP action. We will examine further correlates between simulation and experiment to reduce this uncertainty.


We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins. Recent advances in super-resolution imaging have enabled imaging of cellular structures at close to molecular length scales using light microscopy Over-counting of labels in nano-scale resolution imaging techniques is a common but under-appreciated problem. Over-counting can occur, for example, when target proteins are labeled with primary and secondary antibodies or when antibodies are conjugated to multiple fluorophores. It can also occur when the same fluorophore is counted two or more times because it cycles reversibly between activated and dark states. In all of these cases, over-counting can lead to the artifactual appearance of self-clustering over distances that correspond to the effective resolution of the measurement. In this study we first describe a method to quantify the distribution of labeled molecules in images, and we then develop a simple model to predict the magnitude of apparent clustering arising from over-counting. We show how this formalism applies to deliberate over-counting and thereby provides a useful measure of the effective average lateral resolution of a reconstructed super-resolution fluorescence localization image. We use this analytical approach to quantify high resolution images of the high affinity IgE receptor (FcεRI) on the surface of RBL-2H3 mast cells obtained using both stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We also apply the method to an example of IgE-FcεRI complexes that are deliberately clustered on the cell surface by crosslinking with defined trivalent ligands. In this case, the observed clustering contains contributions from the redistributed proteins in addition to the inherent over-counting of multiple labels. Our approach can also be applied to other types of high resolution imaging methods, including transmission electron microscopy (TEM) and has recently been applied to quantify images obtained using photoactivated light microscopy (PALM/fPALM) Pair correlation functions quantify organization in heterogeneous systems and are easily applied to super-resolution localization data. The pair auto-correlation function, If an ensemble of molecules is distributed on a two dimensional surface with centers at positions If we assume a Gaussian-shaped form of the PSF with a standard deviation of ó, the normalized The estimates of apparent clustering due to over-counting that are presented in the first terms of Eqns. 1 and 2 are valid only when over-counting occurs via a random process. More rigorously, this applies when the number of times a given labeled molecule is sampled is well approximated by a Poisson distribution. This is expected to be the case for the majority of high-resolution measurements that are subject to over-counting, such as stochastic blinking of fluorophores in STORM/dSTORM measurements and reversible switching of fluorescent proteins in some PALM/fPALM measurements. This case should also apply when over-counting occurs through conjugation of multiple organic fluorophores to proteins or ligands, or when labeling of proteins with primary and secondary antibodies. As has been documented previously by others, these equations also hold in diffraction limited images in the limit where an ensemble of photons samples the PSF of each observed fluorophore and similar properties of measured correlation functions have been exploited to extract the oligomizeration state of labeled molecules Our estimates of clustering will not be accurate if over-counting is not randomly distributed over all labeled molecules. The first terms of Eqns. 1 and 2 will over-estimate apparent clustering from over-counting for cases where labeled molecules are sampled less frequently than expected from a Poisson distribution. This would occur, for example, when detection of a signal from a labeled molecule decreases the probability that the same labeled molecule will be detected additional times. This occurs in super-resolution fluorescence localization measurements if there is a significant probability of bleaching a fluorophore after it is activated. If, in fact, imaging is conducted in a manner that ensures that all labeled molecules are counted at most once, then measured correlations are due only to clustering of labeled molecules, and over-counting is not a problem. This is the ideal case for PALM/fPALM measurements if every activated fluorophore is irreversibly bleached after being counted, or for EM measurements if a labeling strategy is employed that ensures at most a single gold particle label per target protein. We note that several recent studies have demonstrated that some popular ‘irreversible’ PALM/fPALM probes show reversible blinking under some imaging conditions The first terms of Eqns. 1 and 2 will underestimate the magnitude of apparent clustering when labeled molecules are sampled more frequently than expected from a Poisson distribution. This would occur, for example, when the act of counting a signal from a labeled molecule increases the probability that additional signals will be detected from the same labeled molecule. This condition occurs in super-resolution fluorescence localization measurements if activated probes are counted once for each frame in which they are imaged, including cases when the same signal remains activated in multiple sequential image frames. A rigorous derivation demonstrating how deviations from a Poisson distribution quantitatively alter the magnitude of the over-counting term can be found in Deliberately over-counting probes is useful for isolating the over-counting term in Eqn. 1 and thereby directly measuring the effective average PSF of the measurement. An example of this approach is shown in In an ideal experiment, the range of For cases in which measured correlation functions contain contributions that cannot be attributed to over-counting, such as when We apply this correlation analysis to two types of super-resolution data obtained with labeled IgE specifically bound to the high affinity FcεRI receptor on RBL-2H3 mast cells. −2, which is in good agreement with previous studies Correlation functions derived from images of localized single molecules from cells labeled with Alexa-647 conjugated IgE show significant auto-correlations at short distances and weak correlations that extend to longer distances, as shown in r = 0, and therefore it is not affected by over-counting, even when an experiment is conducted with finite resolution. A detailed derivation of this statement is included in Strong evidence that the large correlations at short radii arise from over-counting labels on single IgE-FcεRI complexes and not from self-clustering of proteins is provided by measurements of cross-correlation functions calculated from two-color images . SimilarMeasured cross-correlation functions lack the large correlations at short distances that dominate auto-correlations functions tabulated from single color images , but theBT. The shape of the measured cross-correlation function is well fit to an exponential and does not appear to drop below The magnitude of measured cross-correlation functions suggests that IgE-FcεRI clustering arises from a thermally driven mechanism, since −2. This surface density is comparable but somewhat lower than that calculated from our fluorescence measurements, but still within expected values This correlation analysis can also be applied to scanning electron microscopy (SEM) images where target proteins are labeled with primary antibodies followed by secondary antibodies conjugated to gold particles as described in Direct evidence that apparent clustering of labeled IgE-FcεRI complexes is dominated by contributions from over-counting is provided by double-label SEM experiments, where distinguishable but functionally identical pools of IgE-FcεRI are labeled with differently sized gold particles . Just asThus, unlike our super-resolution fluorescence localization measurements , we do nOur analysis of both super-resolution fluorescence localization and SEM images yields results that differ from those of several previous studies which report that IgE-FcεRI complexes are tightly pre-clustered into small domains in unstimulated RBL-2H3 cells by electron microscopy Large-scale clustering of IgE-FcεRI is observed when cells are treated with a multivalent antigen that crosslinks multiple surface-bound IgE antibodies. −2, which is significantly lower than our anticipated surface density of IgE-FcεRI complexes and well below our measured gold surface density of 107 golds/µm2. It is likely that the peak at short radius also contains contributions from IgE-FcεRI complexes organized into small oligomers as a result of exposure to crosslinking ligand. In this case, we can interpret the best fit surface density to represent the surface density of small oligomers. If we assume that the actual surface density of IgE-FcεRI is well approximated by the surface density of gold labels, then we would conclude that IgE is organized into tetramers on average. It is also possible that the gold surface density over-estimates (or under-estimates) the IgE-FcεRI surface density and complexes are organized into trimers (or pentamers) on average. Unfortunately, we do not explicitly know the surface density of IgE under this stimulation condition and it is not possible to clearly distinguish small protein clusters from over-counting in single label experiments.Gold particles labeling IgE-FcεRI from cells incubated for 10 min with Y16-DNP show clear extended clusters in reconstructed SEM images , and thi−2) is much lower than the measured surface density of gold particles labeling IgE (148 µm−2), again suggesting the presence of small IgE-FcεRI oligomers on the cell surface. If the surface density of IgE-FcεRI complexes is well approximated by the surface density of gold particles, then we would conclude that receptor complexes are organized primarily as trimers. Unfortunately we cannot draw quantitative conclusions since we do not have independent measurements of receptor surface density under these conditions. Our previous studies showed that Y46-DNP stimulates less cell activation than Y16-DNP, consistent with the lower amount of extended clustering of IgE-FcεRI with the former that is revealed in these images Extended clusters are less apparent in reconstructed images of gold labeled IgE-FcεRI complexes in cells incubated for 10 min with the larger Y46-DNP ligand . Auto-coIn conclusion, we demonstrate that correlation functions provide an analytical tool to quantify heterogeneous distributions of labeled molecules in super-resolution experiments, even in the presence of over-counting that gives rise to the artifactual appearance of short-range clustering. We present an analytical method that predicts the magnitude of correlations arising from over-counting, and we describe a procedure to measure the apparent PSF of an image for cases when signals can be intentionally over-counted. We have validated this analysis methodology by quantifying the lateral distribution of IgE-FcεRI complexes on the surface of unstimulated RBL-2H3 cells imaged using super-resolution fluorescence localization and SEM. We detect weak clustering of IgE-FcεRI complexes when imaged on the ventral cell surface using TIRFM and super-resolution fluorescence localization methods, and these complexes appear randomly distributed when imaged on flat areas of the dorsal surface by SEM. Our interpretations of single-labeled IgE-FcεRI images are confirmed by direct measurements of cross-correlation functions in double label experiments using both imaging methods. We additionally quantify over-counting and long-range clustering in cells that have been stimulated using defined Y-DNP ligands and discuss the advantages and limitations of applying this correlation method to interpret clustered distributions of proteins. These examples emphasize the importance of explicitly considering over-counting when quantifying images of proteins in membranes, where the extent of heterogeneity may be small and subtle.FITC, Alexafluors 647, 532, 488, and rabbit anti-Alexafluor 488 were purchased from Invitrogen . Mouse anti-FITC, 10 nm gold-conjugated anti-rabbit IgG (whole molecule), 10 nm gold-conjugated anti-mouse IgG (whole molecule), 5 nm gold-conjugated anti-rabbit IgG (whole molecule), β-mercaptoethanol, Glucose Oxidase, and Catalase were purchased from Sigma . 5 nm gold-conjugated anti-mouse was purchased from GE Healthcare . A488-IgE, A532-IgE, A647-IgE, and FITC-IgE were prepared by conjugating purified mouse monoclonal anti-2,4-dinitrophenyl (DNP) IgE with Alexafluor 488, Alexafluor 532, Alexafluor 647, or FITC as previously described Rat Basophilic Leukemia (RBL-2H3) cells were cultured as described previously fminfunc function in Matlab. An example image with fits is shown in Single label samples were imaged on an inverted microscope  under through-objective TIRF illumination by a 100 mW 642 nm diode pumped solid state (DPSS) laser . Double label experiments were conducted on an inverted Olympus IX81-ZDC microscope with a cellTIRF module  under through-objective TIRF illumination by either a 75 mW 642 nm DPSS laser  or a 150 mW DPSS 532 laser . In both cases, images were captured with an Andor iXon 897 EM-CCD camera  using custom image acquisition code written in Matlab . To induce A647 or A532 photo-switching, cells were imaged in the presence of an oxygen-scavenging and reducing buffer containing 100 mM Tris, 10 mM NaCl, 10% w/w glucose, 500 µg/mL glucose-oxidase, 40 µg/mL catalase, and 1% β-mercaptoethanol at pH 8. Movies of A647 or A532 photo-switching were acquired at between 5 and 25 frames per second for at least 2500 frames and analyzed by localizing the centers of diffraction limited spots through least squares fitting a two dimensional Gaussian shape using the RBL-2H3 mast cells were grown overnight to ∼50% confluency on 2 mm×2 mm silicon chips at 37°C under standard cell culture conditions Imaging: Mounted samples were imaged with a Schottky field emission Scanning Electron Microscope (LEO 1550) at 20 KeV. The dorsal (top) surfaces of intact, adherent cells were imaged using secondary electron detection (SED) and backscattered detection (BSD) at high magnification. Flat membrane regions were selected for imaging. For imaging 10 nm gold particles, individual micrographs were obtained at 35 K magnification, and typical images cover 2.4 µm2 of the cell surface. For imaging 5 nm gold particles and in double-label experiments with 10 and 5 nm gold particles, micrographs were obtained at 75 K–100 K magnification. Immuno-gold labeled protein distributions for ≥10 different cells and ≥2 individual experiments were obtained for all experimental conditions presented. Gold particle centers were localized by finding the weighted centroid of identified particles using automated image processing software written in Matlab. Correlation functions were tabulated from these binary images of gold centers. Reconstructed images are formed by convolving an image of the particle centers with a Gaussian shape with half-width given by the gold particle radius.I is the unfiltered reconstructed image of localized probes, generated as described above. For SEM measurements, I is a binary image of localized gold particle centers. In either case, the image I is padded with zeros in both directions out to a distance larger than the range of the desired correlation function  to avoid artifacts due to the periodic nature of FFT functions. The normalization factor Pair auto-correlation functions were tabulated in Matlab using Fast Fourier Transforms (FFTs) as follows:1I), while the second image was reconstructed from localized Alexa 532 fluorophores (2I). In SEM measurements, one image was reconstructed from the locations of 5 nm gold particle centers (1I) and the second image was reconstructed from locations of 10 nm gold particle centers (2I).1 and ρ2 are the average surface densities of images 1I and 2I respectively, and Re{} indicates the real part. This computation method of tabulating pair auto and cross-correlations is mathematically identical to brute force averaging methods. Correlation functions were angularly averaged by first converting to polar coordinates using the Matlab command cart2pol, and then binning by radius. Pair cross-correlation functions were computed using two images. In super-resolution fluorescence localization measurements, one image was reconstructed from localized Alexa 647 fluorophores Click here for additional data file.


This work presents the development of bioassays and biosensors for the detection of insecticides widely used in the treatment of olive trees. The systems are based on the covalent immobilisation of acetylcholinesterase on magnetic microbeads using either colorimetry or amperometry as detection technique. The magnetic beads were immobilised on screen-printed electrodes or microtitration plates and tested using standard solutions and real samples. The developed devices showed good analytical performances with limits of detection much lower than the maximum residue limit tolerated by international regulations, as well as a good reproducibility and stability. The graphs obtained by plotting log of residual activity 2.5.Oxidation of the beads: 60 mg of magnetic beads were stirred for 4 h in 1 mL of 0.5 M sulfuric acid, and then washed twice with distilled water.Functionalization with an amine group: 70 μL of 3-aminopropyltriethoxysilane were added to the beads previously poured in 100 mL of ethanol and ultrasonicated during 5 min, the suspension was kept under mechanical stirring overnight and finally washed three times with ethanol and twice with distilled water.Covalent coupling with glutaraldehyde: 30 μL of aminated beads were washed twice with 1 mL of 0.1 M pH 7 buffer. 820 μL of buffer, 100 μL of electric eel AChE (4.41 UI/mL) and 80 μL of a 25% glutaraldehyde solution were added to the beads and stirred during 30 min at room temperature.1 μL of the obtained enzyme-linked beads suspension was placed either on the surface of the working electrode, beforehand fitted with a 4 mm-diameter magnet (amperometric detection), or in each well of the microplate (colorimetric detection).Nickel magnetic beads with a diameter of 200 nm were activated according to the following steps 100, I and I0 being respectively the current after the and before inhibition.The pesticide detection was made in a three-step procedure as follows: first, the initial response of the electrode to 1 mM ATCh was recorded three times, then the electrode was incubated in a solution containing a known concentration of insecticide, and finally the residual response of the electrode was recorded again. Electrodes were thoroughly washed with distilled water between each measurement. The percentage of the inhibition was correlated with the insecticide concentration, the inhibition rate was calculated according to the following formula: I (%) = [. The efficiency of this oxidation step was controlled using reverse-phase HPLC, it was shown that 3 × 10−5 M NBS was sufficient for the total oxidation of a 10−5 M methidathion solution. The effect of NBS on AChE was investigated to ensure that the enzyme is not affected by the oxidizing agent, it was shown that in assays conditions NBS did not inhibit AChE.In this study we have focused on the detection of oxidized forms of each pesticide, which are less stable but more toxic than the normal forms. The oxidized forms of dimethoate and malathion, respectively called omethoate and malaoxon are commercially available, but in the case of methidathion an oxidation step must be carried out using 3.3.1.i were calculated by performing enzyme kinetic measurements using different pesticide concentrations and by varying the incubation time of the enzyme with the pesticide. The residual enzyme activity was determined according to the Ellman's spectrophotometric method as described previously. The inhibition constant ki is proportional to the affinity of the pesticide for the enzyme and its inhibitory power. ki is therefore a fundamental parameter to compare the inhibitory potency of insecticides and assess the sensitivity of the enzymes studied. The constants ki presented in i shows that the acetylcholinesterase of electric eel is highly sensitive to malaoxon and methidathion but very weakly sensitive to omethoate.The inhibition constants k3.2.3.2.1.The time of reaction of acetylthiocholine with immobilized AChE was studied by measuring every 5 min the appearance of the yellow complex at 412 nm. It was shown that the reaction was completed after 30 min in absence of pesticide; this time was therefore used in inhibition measurements.3.2.2.The inhibition effect of malaoxon, methidathion and omethoate on Electric eel AChE was studied using an incubation time of 10 min and a measurement time of 30 min. As expected from the values of inhibition constants , the inhBeside these results, the microplate assay showed a good reproducibility with a stable response, this detection method has the advantage of saving time as it allows analysing 96 samples in a single assay. Furthermore, due to the small volumes used, it allows one to minimize the use of reagents, however this characteristic can be considered also as a disadvantage because of the difficulty to maintain good reproducibility using small volumes.3.3.−8 of oxidized methidathion, the R.S.D of the inhibition determination was 4% (217 ± 10).The operational stability of biosensors based on AChE covalently bound to magnetic beads was tested to ensure that any decrease in the signal was related to enzyme inactivation and not to enzyme leakage. This parameter was estimated by repetitive measurements of the response of a single electrode to 1 mM ATCh, with intermediate rinses with distilled water. In these conditions, the designed sensors showed a very good stability for at least 10 consecutive measurements with a relative standard deviation (R.S.D) in order of 3% (300 ± 10 nA). The reproducibility of the inhibition was estimated by measuring six times the inhibition percentage by 3 × 1010 and IC50 values obtained for each pesticide are summarized in The incubation time between the biosensor and pesticides was also tested. It was observed that an incubation time of 10 min was sufficient to reach the maximum inhibition rate whatever the pesticide concentration used. This incubation time was thus selected and used for all inhibition assays. 3.4.−2 M of omethoate as final concentration and extracted according to the method described in Section 2.7. The inhibition effect of the resulting extracts was measured after dilution in the working buffer, at a final concentration of 10−4 M. In these conditions, the average response of the sensors was 75 ± 7 nA, showing a good reproducibility of the extraction method. The recovery percentage for the three pesticides was calculated using the formula below:The performance of the biosensors was tested using olive oil samples previously spiked with known concentrations of pesticides. In order to measure the efficiency of the extraction procedure, three olive oil samples were spiked to obtain 10i.e., microplate assay (It was found that the recovery percentage for the three pesticides with both methods varies from 40% to 170%, as shown in te assay  or biosete assay .−2 M of omethoate. It was found that the matrix effect was responsible of a 5.5% supplementary loss of the sensor response, which can explain the increase in inhibition ratios observed using real samples. Additional blank assays were performed to verify that the inhibition was not due to pesticides already present in the olive oil, these tests showed very low inhibition rates, lower than 4%. The inhibition effect of olive oil samples containing NBS has also been studied, it was shown that NBS did not affect the measurement.Despite that, a small increase of the inhibition effect was observed whatever the pesticide studied, especially when using the microplate assay. These small discrepancies could be attributed to matrix effects. In order to evaluate these interferences, a blank assay was carried out using an olive oil sample that was previously extracted and then spiked to obtain 104.−10 M. The application on the spiked olive oil samples showed very satisfactory results, comparable with those obtained using standard solutions.In this work we have developed and optimised the immobilisation of acetylcholinesterase on magnetic microbeads by covalent coupling. The modified beads have been used in two configurations, using either microplates or screen-printed electrodes supports. Depending on the configuration, colorimetry or amperometry have been applied as detection methods and the devices have been tested on standard and real samples for the determination of three pesticides commonly used for the treatment of olive trees: omethoate, malaoxon and methidathion. The results showed a good performance of the developed devices with reasonable limits of detection, as well as good reproducibility and stability. These last two criteria were however weaker for the colorimetric method due to the difficulty in handling small volumes in the microplate assay. On the other hand, this configuration had the advantage of being faster and more economical. This study highlighted the low sensitivity of the electric eel acetylcholinesterase for omethoate compared to malaoxon and oxidized methidathion, which lead to detection limits as low as 10


Drosophila Hippo and C.elegans cst-1/2.Mammalian Ste20-like kinases (MSTs) are the mammalian homologue of Drosophila hippo and play critical roles in regulation of cell death, organ size control, proliferation and tumorigenesis. MSTs exert pro-apoptotic function through cleavage, autophosphorylation and in turn phosphorylation of downstream targets, such as Histone H2B and FOXO (Forkhead box O). Previously we reported that protein kinase c-Abl mediates oxidative stress-induced neuronal cell death through phosphorylating MST1 at Y433, which is not conserved among mammalian MST2, Using immunoblotting, in vitro kinase and cell death assay, we demonstrate that c-Abl kinase phosphorylates MST2 at an evolutionarily conserved site, Y81, within the kinase domain. We further show that the phosphorylation of MST2 by c-Abl leads to the disruption of the interaction with Raf-1 proteins and the enhancement of homodimerization of MST2 proteins. It thereby enhances the MST2 activation and induces neuronal cell death.The identification of the c-Abl tyrosine kinase as a novel upstream activator of MST2 suggests that the conserved c-Abl-MST signaling cascade plays an important role in oxidative stress-induced neuronal cell death. Drosophila Hippo and plays an important role in apoptotic cell death MST2 shares the highest degree of homology with the It has been shown that MST2 is regulated by Raf-1 through a direct interaction, which prevents dimerization and phosphorylation of the activation loop of MST2 independent of Raf-1's protein kinase activity The ubiquitously expressed tyrosine kinase c-Abl is activated by DNA damage agents In this study, we demonstrate that MST2 is regulated by c-Abl tyrosine kinase. C-Abl phosphorylates MST2 at Y81, which leads to enhancement of MST2 autophosphorylation as well as its homodimerization. Consistently, we found that c-Abl mediated phosphorylation inhibits the interaction between Raf-1 and MST2. The MST2-Y81F mutant, which is unable to be phosphorylated by c-Abl, confers a lower kinase activity and pro-apoptotic ability compared to that of WT MST2. In mammalian neurons, Rotenone, a specific inhibitor of mitochondrial NADH dehydrogenase in vitro kinase assay followed by immunoblotting with an anti-pan-tyrosine antibody (Drosophila (Hippo), and C. elegans  the c-Abl-induced phosphorylation of MST2 reduces the interaction between Raf-1 and MST2 and enhances MST2's homodimerization, (3) c-Abl-MST2 signaling plays a critical role in neuronal cell death upon Rotenone treatment. Collectively, we have identified a novel upstream regulator of MST2 underlying the oxidative stress-induced cell death.In this study, we have discovered an evolutionarily conserved signaling link between the tyrosine kinase c-Abl and the MST family of kinases that mediates responses to oxidative stress in mammalian cells. Our findings generalize the substrates of c-Abl from MST1 to other family members of the MST proteins. Our major findings are: (1) c-Abl phosphorylates MST2 at the conserved Y81 The elucidation of the c-Abl-induced phosphorylation of MST2 and consequent disruption of its interaction with Raf-1 proteins provides a molecular basis for how c-Abl kinases activate MST2 signaling in the contexts of oxidative stress in mammalian cells. Previous study has demonstrated that Raf-1 kinase binds to MST2 and prevents its dimerization and autophoshorylation of T180, which results in the inhibition of both MST2 activation and proapoptotic activity Drosophila and mammals integrates multiple upstream inputs, enabling dynamic regulation of tissue homeostasis in animal development and physiology, especially the organ size control and cell death Drosophila Abl (d-Abl) function was obtained by analysis of mutant phenotypes in the embryonic somatic muscles and the eye imaginal disc. The expression patterns and mutant phenotypes indicate a role for d-abl in establishing and maintaining cell-cell interactions in the developing embryonic muscle and adult eyes in vitro  is higher than that of MST1 and Hippo (50%) in vitro . Thus, iDrosophila (Hippo), and C. elegans (cst-1/2), which is absent in mammalian MST1 (Drosophila (hippo), and C.elegans (cst-1/2) , ian MST1 . In contcst-1/2) . We alsoTogether with our previous finding GGAATATTCTCCTCAATAC; c-Abl hpRNA#1, GACCAACCTGTTCAGCGCT;c-Abl hpRNA#2, AAGCAGCTCGATGGACCTCCA; MST2 Rescue plasmids were generated by creating three silent base-pair mutations in the WT or mutation sequences. Unless stated otherwise, all transfections were carried out in complete medium with Lipofectamine 2000 (Invitrogen) or Vigofect (Vigorous) according to the manufacturer's protocols.The plasmids used were as follows: pCMV-Myc-c-Abl was a gift from Dr. Cheng Cao (Beijing Institute of Biotechnology). MST2-Y81F and other mutants were generated by site-directed mutagenesis. All mutations were verified by sequencing. Raf-1 were cloned into pEGFP-C2 vector at Eco RI and Kpn I restriction sites from the HeLa cDNA library. Mammalian RNAi constructs were designed as described 2 in DMEM supplemented with 10% fetal bovine serum. DMEM and fetal bovine serum were purchased from Invitrogen. Cerebellar granule neurons were prepared from postnatal day 6 (P6) rat pups Neuro2A and HEK 293T cells in vitro kinase assay or Western blotting analysis. Protein expression was determined by probing Western blots of immunoprecipitates or total cell lysates with the appropriate antibodies as noted in the figure legends.The antibodies used were MST2, c-Abl, phospho-MST1 (Thr183)/MST2 (Thr180), and ERK1/2 ; GST (Santa Cruz Biotechnology); FLAG-M2 (Sigma); phosphor-tyrosine p-Tyr (4G10) (Millipore); GFP and phosphor-FOXO3 (Ser 207) (Invitrogen). Immunoprecipitations and immunoblotting were carried out as described In vitro kinase assays were carried out as described 2, ATP, 1 µg of GST-MST2 or GST-MST2 mutation as substrate. Immunoprecipitated MST2 from cells was incubated with 0.4 µg of GST-FOXO3-FD or Histone H2B in a reaction buffer containing 30 mM Tris (pH 7.4), 20 mM MgCl2, 1 mg/ml BSA, ATP. Kinase reactions were separated by SDS-PAGE gel electrophoresis and analyzed by autoradiography or by immunoblotting with indicated antibody.Immunofluorescence and cell death assay were carried out as described Statistical analysis of the data was performed with one-way ANOVA followed by Fisher's PLSD post hoc test using Origin software (Version 8). Data are presented as the mean ± SEM and the number of experiments is indicated in each figure. *P<0.05 or **P<0.01 denotes statistical significance.Figure S1Mass spectrum analysis of MST2 phosphorylation by c-Abl kinase. (A). Immunoprecipitate complex from In vitro c-Abl kinase assay was performed using GST-Hippo or –MST2 as substrate. Phosphorylation reactions were analyzed by immunoblotting with anti-pan-tyrosine phosphorylation antibody. Both MST2 and Hippo proteins were tyrosine phosphorylated by the recombinant active c-Abl kinase in vitro. (D). Lysates of HEK 293T cells transfected with pCMV vector, Myc-c-Abl WT, or Myc-c-Abl KD (kinase dead form) were immunoprecipitated with anti-Myc tag to purify the kinases and then in vitro c-Abl kinase assay was performed using GST or GST-MST2 as substrate. The result was analyzed by immunoblotting with anti-pan-tyrosine phosphorylation antibody (p-Y). GST was taken as a negative control. MST2 could be tyrosine phosphorylated by c-Abl WT but not kinase dead c-Abl. Asterisks (*) indicated non-specific bands.(TIF)Click here for additional data file.Figure S2C-Abl knockdown does not alter the MST2 protein levels. Neuro2A cells were transfected with c-Abl RNAi or the control vector. 72 hours after transfection, cells were treated with 50 μg/ml Cycloheximide (CHX) for different time periods. Equal amounts of total protein lysates were subjected to immunoblotting with the MST2 or c-Abl antibody.(TIF)Click here for additional data file.Figure S3C-Abl-mediated Y81 phosphorylation is important for the dimerization of MST2. Lysates of HEK 293T cells transfected with GFP-MST2 alone or together with FLAG-MST2 WT or Y81F or Myc-c-Abl expression plasmid were immuno-precipitated with FLAG antibody and analyzed by immunoblotting against GFP antibody.(TIF)Click here for additional data file.Figure S4C-Abl RNAi efficiently knocks down the endogenous c-Abl in CGNs. Immunocytochemical analysis of rat cerebellar granule neurons (CGN) transfected with β-galactosidase expression plasmids together with the c-Abl shRNA or control U6 plasmid (in a ratio of 1∶3). C-Abl RNAi reduced endogenous c-Abl expression in b-galactosidase positive cells.(TIF)Click here for additional data file.Figure S5Representative pictures of cell death in CGNs under rotenone treatment. CGNs transfected with c-Abl RNAi plasmid or control vector (pBabe/U6), MST2 expressing plasmid or its control vector, together with GFP vector were treated with Retenone (120 nM) for 24 hours. Yellow arrowhead stands for the healthy neurons and red arrowhead indicates apoptotic cells.(TIF)Click here for additional data file.Figure S6C-Abl inhibitor STI571 significantly decreases MST2 expression-induced cell death. CGNs transfected with Flag-MST2 plasmid or control vector (pCMV5) together with GFP vector were treated with Retonone only (120–150 nM) or with Rotenone (120–150 nM) and c-Abl inhibitor STI571 (5 μM) for 24 hours. Under Rotenone treatment, MST2 expression increases neuronal death significantly, while the effect could be reversed by STI571 .(TIF)Click here for additional data file.


Reversible posttranslational protein modifications such as phosphorylation of Ser/Thr/Tyr and Met oxidation are critical for both metabolic regulation and cellular signalling. Although these modifications are typically studied individually, herein we describe the potential for cross-talk and hierarchical regulation.The proximity of Met to Ser/Thr/Tyr within the proteome has not previously been addressed. In order to consider the possibility of a generalized interaction, we performed a trans-kingdom sequence analysis of known phosphorylation sites in proteins from bacteria, fungi, plants, and animals. The proportion of phosphorylation sites that include a Met within a 13-residue window centered upon Ser/Thr/Tyr is significantly less than the occurrence of Met in proximity to all Ser/Thr/Tyr residues. Met residues are present at all positions  within the 13-residue window that we have considered. Detailed analysis of sequences from eight disparate plant taxa revealed that many conserved phosphorylation sites have a Met residue in the proximity. Results from GO enrichment analysis indicated that the potential for phosphorylation and Met oxidation crosstalk is most prevalent in kinases and proteins involved in signalling.The large proportion of known phosphorylation sites with Met in the proximity fulfils the necessary condition for cross-talk. Kinases/signalling proteins are enriched for Met around phosphorylation sites. These proteins/sites are likely candidates for cross-talk between oxidative signalling and reversible phosphorylation. In toto, these factors comprise a well-adapted basis for signalling, regulation, targeting, and interaction, all at least potentially in the absence of de novo protein synthesis [The proteome includes the combination of at least three components; genome-encoded proteins, the products of alternative initiation and splicing, and of posttranslational modifications (PTM). This combination is orders of magnitude larger than could be solely genome-encoded . The divynthesis ,8.Saccharomyces cerevisiae has genes for 113 protein kinases while the Homo sapiens has 518 such genes [Arabidopsis thaliana includes genes encoding 1055 protein kinases [Among PTM, reversible phosphorylation has been the most extensively studied -11. Thisch genes . The gen kinases  and 112  kinases . Clearly kinases .Reactive oxygen species (ROS) are an inevitable consequence of aerobic metabolism -24. HighIn addition to Ser/Thr/Tyr phosphorylation and Met oxidation, other common reversible PTMs include Ser/Thr O-glycosylation , Lys/ArgIt was recently suggested that reversible Met oxidation might serve as a rheostat to control proximal Ser/Thr/Tyr phosphorylation ,51, and 3DB [Bacteroides fragilis, Clostridium botulinum, and Ascaris suum.In order to consider our analysis of potential crosstalk in as broad a context as possible we have employed a data-mining strategy. Selected reference proteome or complete proteome sequences were downloaded from Uniprot , NCBI 55, or TAI3DB . Our aimPhosphorylation sites were predicted for complete proteomes using Musite , NetPhosThe proportion of Ser/Thr/Tyr sites that include at least one Met-residue in a window ranging up to 21-residues was scanned. As most known phosphorylation motifs are less than 13-residues in length , a 13-reArabidopsis thaliana, Glycine max, and Oryza sativa proteins that include a Met-residue within the 13-residue phosphorylation-site window. Proteins from these three taxa account for ~90% of known phosphorylation sites in plants. The results were used to identify related sequences in five additional proteomes , using a significance E-value cutoff of 0.00001. The total number of phosphorylation sites with Met-conservation was then calculated among the eight disparate taxa. Additionally, the proportion of hydrophobic residues  was enumerated for each phosphorylation site.In order to evaluate the extent of conservation of phosphorylation sites that contain a Met, and to identify candidate proteins for Phos-Met oxidation crosstalk in plants, a PSI-BLAST search was conducted for A. thaliana or the g:Profiler webserver [S. cerevisiae and H. sapiens. A hypergeometric test (with Bonferroni correction for multiple comparisons) was used to select significantly enriched (p < 0.05) terms [2 ratio of numbers of each significantly enriched GO term in the test set (phosphoproteins that have Met residues in the vicinity of phosphorylation site) to background set  was calculated. All data analyses were performed using Python ver. 2.7. A Z-test was used to calculate the difference between two group proportions; p < 0.05 was considered significant.A gene ontology (GO) term-enrichment analysis was performed for proteins that contain at least one known phosphorylation site including a Met within our specified ± 6-residue window, using the agriGO webserver  for A. tebserver  for S. c5) terms ,65. A loEvaluating the translated proteome from diverse taxa, it is clear that Met belongs to the group of relatively low-abundance amino acids along with Cys, His, Trp, and Tyr Figure . In planThe proportion of Ser/Thr/Tyr sites which include a Met within the window of ± 6 residues is shown in Figure The overall positional distribution of Met in the proteome of eukaryotic organisms is nearly uniform. In Figure E. coli has been reported to have only 32 P-Tyr-containing proteins and two tyrosine-kinases [The patterns are somewhat more complex when comparing the distribution of other members of the hydrophobic amino acid homology group  within the ± 6 residue window used to evaluate Met distribution  as against the background set .We conducted GO analysis of the sequences of all the plant, yeast, and animal proteins that have a Met-containing phosphorylated-site, in order to test for enrichment in functional domains/motifs. The analyses were conducted versus a total P-proteins background Figure . BacteriWe assume that a phosphorylation-site that includes a Met at the same position within the ± 6 residue-window among diverse taxa would have the greatest probability for regulatory crosstalk. We specifically searched proteins from 8 plant taxa in order to evaluate potential candidates. A list of the consensus 13-residue sequences for potentially conserved P-Ser/Thr/Tyr sites which include a Met residue is presented in Additional file The proteins that contain an experimentally verified phosphorylation site with a vicinal Met show significant enrichment  for many GO terms when the Met is conserved  in eight disparate plant taxa  and anaerobes  have a similar proportion of sites with Met  on phosphorylation then it would be predicted that one group would have a significantly different proportion of  phosphorylation sites including a Met residue. At least superficially this is not the case. In the very limited data available to us, aerobes  different molecular species, but if there are two types of PTM there is the potential to generate 59049 (3n) species [While the regulatory importance of protein PTM has been long known, it has only been more recently that roles for multiple and interacting PTM's have been appreciated . Example species . The eno species .A. thaliana is responsive to cGMP [Our initial interest in crosstalk between Ser/Thr/Tyr-phosphorylation and Met oxidation was stimulated by observations that two important metabolic enzymes could be responsive to cellular redox signalling through this mechanism -52. Whil to cGMP .Results from GO analysis indicated that the occurrence of Met near phosphorylation sites could be more prevalent in proteins related to signalling such as kinases and stress related proteins, and could be common to all three taxa - plants, yeast, and animals Figure  and 6. MOur study identified a large number of phosphorylation sites which include a vicinal Met residue. The proteins containing these sites can potentially function as redox-sensors capable of transducing input from ROS signalling to regulation of phosphorylation. These observations should stimulate further research on PTM crosstalk and control of protein function in response to oxidative signalling.The authors declare that they have no competing interests.RSPR participated in design of the study and conducted the computational analyses. JJT and DX participated in design and coordination of the study. JAM conceived the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.S1.pdf. Percentage of Ser/Thr/Tyr sites  with Met within a ± 6 window in different species.Click here for fileS2.pdf. Distribution of other hydrophobic residues  around non-Ser/Thr/Tyr and Ser/Thr/Tyr  sites in plant (A), yeast (B), animal (C), bacterial (D) and archaeal (E) P-proteins.Click here for fileS3.pdf. Consensus sequences (13-mers) for phosphorylation sites with highly conserved Met (≥ 7 Met among eight taxa).Click here for file


Plasmodium knowlesi sporozoite (csp/ssp2) and two blood stage (ama1/msp142) genes, leads to self-limited parasitaemia in 60% of rhesus monkeys and survival from an otherwise lethal infection with P. knowlesi. In the present study, the role of the blood stage antigens in protection was studied in depth, focusing on antibody formation against the blood stage antigens and the functionality thereof.A DNA prime, poxvirus (COPAK) boost vaccination regime with four antigens, i.e. a combination of two P. knowlesi sporozoites. During immunization and challenge, antibody titres against the two blood stage antigens were determined, as well as the in vitro growth inhibition capacity of those antibodies. Antigen reversal experiments were performed to determine the relative contribution of antibodies against each of the two blood stage antigens to the inhibition.Rhesus macaques were immunized with the four-component vaccine and subsequently challenged i.v. with 100 19 antibody titres in vaccinated animals were low, which was reflected in low levels of inhibition by these antibodies as determined by in vitro inhibition assays. Interestingly, after sporozoite challenge antibody titres against blood stage antigens were boosted over 30-fold in both protected and not protected animals. The in vitro inhibition levels increased to high levels . As growth inhibition levels were not significantly different between protected and not protected animals, the ability to control infection appeared cannot be explained by GIA levels. Judged by in vitro antigen reversal growth inhibition assays, over 85% of the inhibitory activity of these antibodies was directed against PkAMA1.After vaccination, PkAMA1 and PkMSP1in vitro and protection, either after vaccination or after challenge.This is the first report that demonstrates that a DNA prime/poxvirus boost vaccination regimen induces low levels of malaria parasite growth inhibitory antibodies, which are boosted to high levels upon challenge. No association could, however, be established between the levels of inhibitory capacity  Plasmodium parasites to anti-malarial (prophylactic) drugs, development of an effective malaria vaccine is generally considered a public health priority . All GIA and reversal GIA results reported are the averages of two independent GIA assays.After incubation of 24 to 26 hours, 30 μL of the resuspended culture was added to 200 μL ice-cold PBS, pH 7.4. After brief centrifugation, the supernatant was removed and pellets were frozen. Parasite lactate dehydrogenase levels were determined in the thawed pellets, as previously described . From th19 antibody levels was assessed by Spearman's rank correlation, the correlation is presented as Spearman's Rho. Two-sided P values less than 0.05 were considered significant.All statistical analyses were performed using R software version 2.8.1 . IgG titres were log-transformed to obtain normality and significance was assessed by t-tests; a correction for unequal variances was applied where necessary. IgG antibody levels are presented as geometric means with 95% confidence intervals. The statistical significance of changes in IgG titres between time points were assessed using a paired t-test and presented as a ratio with 95% confidence intervals. Between group comparisons of GIA titres were performed using Mann-Whitney U test and titres are presented as medians with quartile ranges. Changes in GIA titres between time points were assessed using a paired t-test and data are presented as a difference (in percent points) with the corresponding 95% confidence intervals. The relation between GIA titre and PkAMA1 or PkMSP1P. knowlesi, after receiving three injections with DNA encoding 4 Plasmodium knowlesi antigens (csp/ssp2/ama1/msp142) and a booster with a mixture of 4 COPAK viruses, encoding the same antigens. Monkeys receiving a similar vaccination regimen, but with DNA and poxvirus comprising sporozoite antigens (csp/ssp2) only, or mock vaccine, were not able to control the parasitaemia (not protected). A summary of the outcomes of this study is presented in Table This study is a further analysis of samples obtained during a vaccination and challenge study, published by Weiss and co-workers . In that19/PkAMA1) were determined in serum samples obtained from csp/ssp2 and csp/ssp2/ama1/msp142 animals, both before and after challenge, and run in a single experiment  while the titre for PkAMA1 appeared higher at 2,305 AU/mL .ELISA titres for the two blood stage antigens PkMSP119/PkAMA119 and PkAMA1 antibody titres from protected and non-protected monkeys, respectively   Figure .19 were boosted significantly. For PkAMA1 the antibody level was 34 fold  higher post-challenge compared to pre-challenge. For PkMSP119 the increase in the antibody titre after challenge was 50 fold . Again no significant differences were observed between protected and not protected animals   showed elevated antibody levels against PkMSP1csp/ssp2 vaccine (N = 5) were also included in the analysis . The animals with the highest growth inhibition levels  in the level of inhibition was observed in all Pk4 vaccinated animals . This suggests that there is also no association between GIA levels and protection.GIA levels between protected and not protected animals were not significantly different (t-test), either before (P = 0.07234) or after challenge .Other studies have shown a positive correlation between antibody levels and GIA inhibition (f.e. ). In thiP. knowlesi parasites in vitro, either before or after challenge , also only after challenge. Antibodies were pre-incubated with increasing concentrations of PkAMA1 or PkMSP119, prior to addition to a growth inhibition assay. Growth inhibition could be reversed to over 85% by addition of the PkAMA1 antigen at 100 μg/mL , but were not able to control the infection below 1.5% parasitaemia [ama1 and msp142, showed a significant delay in the appearance of parasites in the blood (>2 days) and 60% of these monkeys could control the infection below 1.5% parasitaemia. This is a strong indication that the immune response to the blood stage antigens was necessary for controlling parasite growth.Rhesus monkeys receiving sitaemia . By cont19 after vaccination were determined and found to be present at low levels, with corresponding low growth inhibition activity . Challenge of the animals initially resulted in high parasite growth rates, for all animals. Obviously, in vivo parasite growth was not or only marginally inhibited. This is supported by the observation that functional antibody levels after vaccination, as determined in GIA, are low.The cellular immune responses measured in the original study  did not 19 and PkAMA1 appear to be higher in protected animals compared to those in not protected animals , four weeks after challenge are lower than the levels reported for PkAMA1/adjuvant immunization studies . In thes19 antibody levels were detected after challenge of naïve monkeys, while PkAMA1 antibodies were not  [19 antibodies, it has been shown that Fc-tail mediated antibody responses may be important for protection in a humanized mouse model [19 antibodies may be growth-inhibitory rather than invasion inhibitory [19 antibodies.Almost complete reversal of the growth inhibition can be achieved by addition of 100 μg/mL PkAMA1 protein in the GIA assay. Titration of PkMSP1n Figure , implyinn (ADCI) ) that wise model . Moreovehibitory . These m42 part of the vaccine was done using PkMSP119 protein. It cannot be excluded that this may have lead to an underestimation of the antibody titres and of the ability to reverse the inhibition in the GIA reversal experiments. However, a protein/adjuvant immunization study with PfMSP142 in humans has shown that the antibody response against PfMSP142 is strongly directed to the PfMSP119 part [It has to be noted that all analyses of the response against the PkMSP1119 part , warrantThe present study could not establish a correlation between the levels of inhibitory antibodies, either before or after challenge, with the ability to control the infection. Previous analysis of these experiments has shown that also no correlation could be established between T-cell mediated immunity (ELIspot antigen-induced IFN-γ production), and protection .P. knowlesi has a multiplication rate of, on average, 10 per 24 hours (one cycle) [The boost in functional antibody levels observed after sporozoite challenge is very interesting, especially in relation with the observed course of parasitaemia in the Pk4x3/COPAK vaccinated animals . Boostine cycle)  (i.e. eae cycle) ), the inImportantly, this study shows that in a vaccination regimen that is not focused on the production of antibodies, these are produced and their levels are significantly boosted after sporozoite challenge. These antibodies may play a direct role in protection alongside the cellular and antibody-mediated cellular immunity induced after vaccination and challenge.P. knowlesi-rhesus macaque challenge model could be instrumental for the eventual elucidation of factors that contribute to protection upon challenge with P. knowlesi.The above studies show that the The authors declare that they have no competing interests.MMaH, BF, ER and WW made substantial contributions to the conception and the design of the study. MMaH did the work in lab, analysis and interpretation of data was done by MMaH, IH, AH, WW, ER and BF. MMaH drafted the manuscript, DN, AT, CK, ER, WW and BF revising it critically. MMaH, ER and BF gave final approval of the version to be published; all authors read and approved the final manuscript.


Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix which mediate inhibition of axonal regeneration after injury to the central nervous system (CNS). Several neuronal receptors for CSPGs have recently been identified; however, the signaling pathways by which CSPGs restrict axonal growth are still largely unknown. In this study, we applied quantitative phosphoproteomics to investigate the global changes in protein phosphorylation induced by CSPGs in primary neurons. In combination with isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labeling, strong cation exchange chromatography (SCX) fractionation, immobilized metal affinity chromatography (IMAC) and LC-MS/MS, we identified and quantified 2214 unique phosphopeptides corresponding to 1118 phosphoproteins, with 118 changing significantly in abundance with CSPG treatment. The proteins that were regulated by CSPGs included key components of synaptic vesicle trafficking, axon guidance mediated by semaphorins, integrin signaling, cadherin signaling and EGF receptor signaling pathways. A significant number of the regulated proteins are cytoskeletal and related proteins that have been implicated in regulating neurite growth. Another highly represented protein category regulated by CSPGs is nucleic acid binding proteins involved in RNA post-transcriptional regulation. Together, by screening the overall phosphoproteome changes induced by CSPGs, this data expand our understanding of CSPG signaling, which provides new insights into development of strategies for overcoming CSPG inhibition and promoting axonal regeneration after CNS injury. Chondroitin sulfate proteoglycans (CSPGs) are a family of extracellular matrix (ECM) molecules that contribute to the failure of axon regeneration following injury to the adult mammalian central nervous system (CNS) CSPGs are comprised of a protein core with one or more covalently attached chondroitin sulfate glycosaminoglycan (CS-GAG) side chains. Much evidence shows that the inhibitory actions of CSPGs depend on the specific sulfation patterns in CS-GAG chains Reversible protein phosphorylation is one of the most important posttranslational modifications for cellular regulation and signal transduction in eukaryotic cells. Protein mass spectrometry has emerged as a key technology for screening protein posttranslational modifications including phosphorylation. It allows simultaneous phosphorylation site mapping and quantitation in a single experiment. The iTRAQ technology enables the comparison of up to eight different samples in one mass spectrometry-based experiment To monitor CSPG-induced regulation of protein phosphorylation, three pairs of cell lysates with or without CSPGs treatment were collected from three independent primary CGN cultures and subjected to iTRAQ-based quantitative phosphoproteomic analysis as described in methods. A work flow of sample processing and analysis is presented in 2(CSPG/Control) ranged from −3.32 to 1.13, with an average log2(CSPG/Control) = 0.018±0.24 (Mean±SD). There were 511 phosphopeptides changed with mean log2(CSPG/Control) values either greater than 0.262 (Mean+1SD) or less than −0.225 (Mean-1SD). Of these 511 phosphopeptides, 118 had p<0.05 in abundance upon exposure to CSPG. A complete list of these 118 phosphopeptides which meet the dual criteria of p<0.05 and 1SD cutoff are shown in Changes in the phosphorylation level of the phosphopeptides were quantified based on the intensity of different iTRAQ reporter ions. Among 2214 phosphopeptides identified, 1988 phosphopeptides were quantified in all three biological replicates; 2064 phosphopeptides were quantified in at least two of the three biological replicates. The complete list of all quantified phosphopeptides is shown in The set of 118 significantly changed phosphopeptides were annotated in the gene ontology format using the PANTHER classification system. The top three categories were cytoskeletal proteins (n = 25), nucleic acid binding proteins (n = 17) and enzyme modulators (n = 12) . Among tp = 7.8 × 10−7), outgrowth of axons (p = 8.7×10−7), disassembly of filaments (p = 9.1 ×10−6), organization of cytoplasm (p = 1.1×10−5) and outgrowth of neurites (p = 1.87×10−7) as the top 5 most significant functions annotated. Another represented category was the RNA post-transcriptional modification group that is involved in splicing and processing of mRNA.To further interpret the data in a biological context, the list of proteins whose phosphorylation was significantly changed was analyzed using the Ingenuity program which identifies networks and biological functions which are most significant to the data set. The top two networks involved were (i) Cell Morphology, Cellular Assembly and Organization, Nervous System Development and Function and (ii) RNA Post-Transcriptional Modification, Cell Cycle, Cellular Movement. The proteins attributed to the networks are shown in Proline-directed, basophilic and acidiphilic are the three main Ser/Thr protein kinase groups classified based on the substrate residues that govern kinase-substrate recognition. The 43 phosphorylation sites from the 41 peptides with significantly increased phosphorylation and the 81 phosphorylation sites from the 77 peptides with significantly decreased phosphorylation were organized into these general sequence categories. Proline-directed phosphorylation (60.5%) was found dominant in the group with increased phosphorylation , while ihttp://helixweb.nih.gov/CPhos/) to identify evolutionarily conserved functional phosphorylation sites High-throughput mass spectrometry has identified an overwhelming number of novel protein phosphorylation sites. However, the biological functions of most of these sites remain elusive. One alternative way to potentially address the functional relevance of a discovered phosphosite is to assess the evolutionary conservation of these sites across species since functionally relevant sites would be expected to be highly conserved Analysis of PANTHER pathway terms for the 118 peptides whose phosphorylation was significantly changed revealed that a number of signaling pathways are regulated by CSPGs . These i2(CSPG/Control) value of −0.83 at 30 min after CSPG treatment. We performed another iTRAQ-8 plex phosphoproteomics experiment using the same procedure except without introducing SCX fractionation. In this experiment, the phospho-Serine3 of cofilin showed a consistent decrease among all three biological replicates, with a log2(CSPG/Control) value of −0.47, −0.87 and −0.33, respectively. To further confirm the regulation of cofilin phosphorylation by CSPGs, we examined the time course of cofilin phosphorylation in CGNs by western blotting with an antibody against Serine-3 phosphorylated (pS3) cofilin as well as an antibody against total cofilin. Cofilin phosphorylation was slightly decreased at 1 min and 5 min after CSPG treatment, and was further decreased at 30 min  value of −0.63 for pS215 and −0.30 for pY216, although the differences were not statistically significant likely related to the small sample size. To confirm these changes, we performed western blotting using an antibody against pY216 of GSK-3β and an antibody against total GSK-3β. The level of GSK-3β phosphorylation of pY216 was unchanged at 1 and 5 min after CSPG treatment, and modestly decreased by 30 min  and approved by the National Heart, Lung, and Blood Institute Animal Care and Use Committee .This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. Primary CGNs isolated from P5-8 day old mice were plated onto 10 cm poly-L-lysine (PLL)-coated dishes and cultured in neurobasal-A medium supplemented with B27 . CSPGs  or the same volume of PBS was added to the culture at day 2 and incubated for 30 min. Cells were then washed twice with PBS and lysed in 0.5 mL lysis buffer (8 M urea/50 mM Tris-HCl/75 mM NaCl) containing 1× protease inhibitor cocktail  and 1× phosphatase inhibitor cocktail (Thermo Scientific) Each protein sample was reduced with 10 mM DTT for 1 h, followed by alkylation with 40 mM iodoacetamide for 1 h under dark conditions. Samples were diluted with 50 mM ammonium bicarbonate to a less than 1 M final urea concentration before protein digestion with trypsin (Promega) at a mass ratio of 1∶20 (trypsin:protein) overnight at 37°C. Following tryptic digestion, peptide samples were desalted on Oasis HLB 1 cc (10 mg) Extraction cartridges . The eluted peptides were dried in a SpeedVac and then labeled with iTRAQ reagents according to the manufacturer’s instructions. Briefly, the dried peptides were reconstituted in 80 µl dissolution buffer (20 µl/100 µg protein). Each of the six samples  was labeled separately with 4 vials of iTRAQ isobaric reagent , respectively. After incubation for 2 h at room temperature, the reaction was stopped by acidification with formic acid (1%). The 6 iTRAQ-labeled peptide samples were then combined and desalted on Oasis HLB1 cc (10 mg) Extraction cartridges (Waters). The eluted peptides were dried in a SpeedVac.2PO4, 25% ACN, pH 2.67) and injected onto a PolySulfoethyl A SCX column . SCX chromatography was carried out on an Agilent HP1100 system at 1 mL/min flow rate using the following gradient: 100% buffer A and 0% buffer B for 2 min; 0–14% buffer B for 33 min; 14–100% buffer B for 1 min; 100% buffer B held for 4 min . UV absorbance at 214 nm was monitored while fractions were collected every 1.5 min and pooled to create 26 fractionated samples. Each fraction was desalted on Oasis HLB 1 cc (10 mg) Extraction cartridges prior to phosphopeptide enrichment.SCX fractionation was performed as previously described Phosphopeptide enrichment was performed using immobilized metal affinity chromatography (IMAC) with a Fe-NTA Phosphopeptide Enrichment (Thermo Scientific) according to the manufacturer’s instructions. Briefly, dried peptide samples were resuspended in 200 µL of binding buffer prior to loading onto Fe-NTA columns and incubated for 20 min with rotation. Columns were washed twice with 100 µL of wash buffer A, twice with 100 µL of wash buffer B followed by another wash with 100 µL of ultrapure water to equilibrate resin for elution. Elution of phosphopeptides was carried out using 50 µL of elution buffer each for three times.Combined eluents were acidified with 100 µL of 2.5% trifluoroacetic acid. The samples were then desalted with the Pierce Graphite Spin Columns (Thermo Scientific) and resuspended in 0.1% formic acid prior to analysis by LC-MS/MS.http://helixweb.nih.gov/ESBL/PhosSA/) which has been optimized for HCD fragmentation Samples were analyzed on an Eksigent nanoflow LC system  connected to an LTQ-Orbitrap Velos mass spectrometer  Proteome Discoverer and Quari, in-house algorithms for quantification of iTRAQ labeled peptides. To minimize contaminating near isobaric ions, only the peptides with isolation specificity more than 75% were quantified. For redundant peptides, the CSPG/Control ratio was calculated from the pair with the highest summed reporter ion intensity. The phosphopeptide ratios were normalized by dividing by the median ratio of all peptides identified. The log2 value of the ratio was used as the basis for the calculation of the mean and standard deviation (SD) for each peptide across all three biological replicates. An unpaired t-test was used to determine whether changes in phosphopeptide abundances were significant.Abundance ratios (CSPG/Control) were quantified by http://www.pantherdb.org/). For the pathway analysis, the total identified phosphoproteins were used as background. The fold enrichment for each pathway term relative to the background was calculated by dividing the proportion of each Panther pathway term in the total changed phosphoproteins by the proportion of that particular Panther pathway term in the background data.Phosphoproteins that were identified were classified by the PANTHER classification system (http://www.ingenuity.com/) was used to identify protein networks and biological functions which are significantly over-represented in the list of changed phosphoproteins.Ingenuity pathway analysis  to generate logos showing over-represented amino acids at specific positions relative to the phosphorylated amino acid.For phosphorylation motif analysis, the list of increased or decreased phosphopeptides was first aligned to 13-mer sequences with the phosphorylation site located in the center using an in-house program. Classification of phosphorylation motif was performed using a binary decision tree as previously described 6 cells/well. At day 2, cells were treated with PBS or 15 µg/mL of CSPGs for 1 min, 5 min and 30 min. Cells were then washed twice with PBS and lysed with 2× SDS sample buffer in the presence of 1× phosphatase inhibitor cocktail . The protein concentration was determined using the BCA assay. The cell lysates were then subjected to SDS-PAGE and immunoblotted with antibodies against phospho-cofilin (pS3) , cofilin , phospho- GSK3β (pY216) , and GSK3β . Horseradish peroxidase-conjugated secondary antibodies against rabbit (to detect cofilin) and mouse (to detect GSK3β) were used and visualized with an enhanced chemiluminescence (ECL) reagent.Dissociated CGNs were first plated on PLL-coated 6-well plates at a density of 1.5×10Table S1List of all identified and quantified phosphopeptides.(XLSX)Click here for additional data file.Table S2List of phosphopeptides changed by CSPG treatment and its gene ontology analysis using the PANTHER classification system or Ingenuity program.(XLSX)Click here for additional data file.Table S3Conservation analysis of the changed phosphosites using CPhos program.(XLSX)Click here for additional data file.