Programmed ribosomal frameshifting is used in the expression of many virus genes and some cellular genes. In eukaryotic systems, the most well-characterized mechanism involves –1 tandem tRNA slippage on an X_XXY_YYZ motif. By contrast, the mechanisms involved in programmed +1 (or −2) slippage are more varied and often poorly characterized. Recently, a novel gene, PA-X, was discovered in influenza A virus and found to be expressed via a shift to the +1 reading frame. Here, we identify, by mass spectrometric analysis, both the site (UCC_UUU_CGU) and direction (+1) of the frameshifting that is involved in PA-X expression. Related sites are identified in other virus genes that have previously been proposed to be expressed via +1 frameshifting. As these viruses infect insects , plants  and vertebrates (influenza A virus), such motifs may form a new class of +1 frameshift-inducing sequences that are active in diverse eukaryotes. The most widely known frameshifting mechanism involves shifting to the –1 frame. In part, this is because of the relatively well-defined nature of the most commonly used shift site motif that allows two adjacent tRNAs to re-pair to mRNA in the –1 frame, and in part due to the prominence of the viruses and other mobile elements that use this type of frameshift. The other reading frame can be accessed by either a –2 or a +1 frameshift event, with the product of the former having an extra amino acid encoded by the shift site sequence relative to the latter.Schizosaccharomyces pombe, and –2 frameshifting when expressed in Saccharomyces cerevisiae [Tsh gene of several Listeria phages and Bacillus subtilis SPP1 phage, besides Escherichia coli yepP, and the pol gene of the S. cerevisiae retrotransposons Ty1 and Ty3 [In the majority of bacteria, frameshifting to the +1 frame is used as a sensor and effector of an autoregulatory circuit for the expression of release factor 2 ,4. In borevisiae . In thisrevisiae . Similarrevisiae . The onlrevisiae ,9. Prote and Ty3 –14. Give and Ty3 –18.Euplotes genes that use such frameshifting, but the transframe-encoded peptide that would demonstrate the nature of the shift remains elusive [Frameshifting, probably in the +1 direction, has also been reported in mitochondria from several diverse species, although functionally different cases of frameshifting used in human mitochondria are –1 –22. Pept elusive ,24. Furt elusive . Low-eff elusive –28. As a elusive .et al. identified a novel coding ORF (X) in influenza A virus [a). PA is a component of the viral polymerase, and the N-terminal domain carries an endonuclease activity that, as part of PA, cleaves capped RNA fragments from cellular pre-mRNAs to act as primers for viral transcription [Recently, Jagger  A virus . The X Ocription . As PA-Xcription . However3.b). Frameshift expression of the construct would result in the transframe fusion PA-X-GFP, which could be affinity-purified on GFP-TRAP beads, while non-frameshift expression would result in a product that does not contain GFP. The construct was expressed in 293T cells, and PA-X-GFP was affinity-purified from cell lysates and resolved by SDS-PAGE. An in-frame control, in which the predicted shift site UCC_UUU_CGU_C was mutated to UCC_UUU_GUC to force expression of PA-X-GFP, was also prepared to show the approximate size at which the frameshift protein should migrate in gels. The wild-type construct produced a specific band migrating at the expected size for PA-X-GFP. A gel slice containing this protein was excised, digested with trypsin, and the resulting peptides were analysed by nano-liquid chromatography tandem mass spectrometry (nano-LC/MS/MS).The efficiency of frameshifting at the PA-X shift site was previously estimated by translating reporter constructs in rabbit reticulocyte lysates and found to be around 1.3 per cent . When thfigure 1c; two of the peptides have overlapping sequence). Importantly, a peptide spanning the shift site itself was identified (figure 1d). This peptide, GLWDSFVSPR, defines the shift site (UCC_UUU_CGU) and direction (+1) of frameshifting (figure 1e). Molecular ions for GLWDSFVSPR were identified both with and without oxidation at the tryptophan, providing further support for the sequence assignment. No peptide compatible with –2 frameshifting was detected. Formally, the peptide GLWDSFVSPR is compatible with three different models for frameshifting: (i) +1 slippage with UUU in the P-site and an empty A-site; (ii) +1 slippage with UCC in the P-site and an empty A-site; and (iii) tandem +1 slippage with UCC in the P-site and UUU in the A-site. However, consideration of the potential for codon : anticodon re-pairings favours model (i). Both UUU and UUC are translated by a single tRNA isoacceptor whose anticodon, 3′-AAG-5′, has a higher affinity for UUC in the +1 frame than for the zero-frame UUU [Eight separate PA-X-GFP tryptic peptides were identified, including peptides encoded both upstream and downstream of the shift site  on stop codon readthrough implies that interactions at that site influence competition for A-site acceptance, but whether this influence acts via the P-site merits investigation [E. coli cell-free system, even partially mismatched P-site codon : anticodon interactions, which can be augmented by E-site mismatches, trigger retrospective editing and so influence events in the A-site [S. cerevisiae, but may exist and involve currently unidentified factors [The role of UCC in the E-site also remains uncertain. In analyses of codon usage in PA, it was observed that the motif UCC_UUU_CGU is extremely highly conserved at the 5′ end of the influenza A virus X ORF, despite the fact that five other codons could potentially be used to encode the serine ,38. Moretigation ,40. Notwtigation –44. In ae A-site . A count factors ,47. An E factors –51. On iAmalgamaviridae of plant viruses (RdRp)  [More generally, one might predict a class of +1 frameshift stimulators that comprise a UUU_C P-site slippery sequence and a restricted choice of A- and E-site codons. In eukaryote-infecting viruses, frameshifting by +1 nt has been predicted as the expression mechanism for non-5′-proximal ORFs in the closteroviruses (RdRp), leishmania RNA virus 1 (RdRp), chronic bee paralysis virus and the related Lake Sinai viruses 1 and 2 (RdRp), plant-infecting fijiviruses  and members of the proposed family iewed in ). Howeveiewed in . Interesnd 1.8%) . Whether4.4.1.Sequences encompassing the frameshift site (97 nt 5′+UCC_UUU_CGU+100 nt 3′) were generated using overlapping synthetic oligonucleotides and cloned into pDluc, a derivative of the dual luciferase reporter p2luc vector ,56. The 4.2.To create the PA-X-GFP expression construct, the nucleotide sequence corresponding to the coding region of PA-X, minus the X-ORF stop codon, was amplified from a A/Brevig Mission/1/1918 (H1N1) segment 3 reverse genetics plasmid  and clon4.3.ascot [Gel slices containing proteins of interest were excised, digested with trypsin, and analysed by nano-LC/MS/MS. All mass spectra were acquired with an LTQ-FT instrument (ThermoElectron). nano-LC with nano-electrospray was used with a 75 μm ID column (C18) and an acetonitrile gradient (0.1% formic acid). Primary mass spectra of peptide molecular ions, primarily observed at +2 charge states, were obtained in the FT-ICR (Fourier transform ion cyclotron resonance) part of the instrument. All peptide masses assigned were better than 2 ppm mass error compared with theoretical values. Both oxidized  and non-oxidized forms were identified for many peptides. Oxidation of peptides is a common occurrence observed during ionization with electrospray, but oxidation can also be present as a post-translational event. Peptide sequence information was acquired using MS/MS with the ion-trap part of the LTQ-FT instrument using collision-induced dissociation fragmentation of selected peptide masses. Peptides were assigned based on combined evidence of the molecular ions and MS/MS sequence. Searches of custom sequence databases were performed with Mascot , using s5.A.E.F. is supported by the Wellcome Trust (088789). J.F.A. is supported by Science Foundation Ireland (08/IN.1/B1889). P.D. is supported by Institute Strategic Grant Funding from the UK Biotechnology and Biological Sciences Research Council and the U.K. Medical Research Council (G0700815). B.W.J., P.D. and J.K.T. are also thankful for the support of the NIH-Oxford-Cambridge Research Scholars programme.


Cerebral endothelial cells (CECs) and astrocytes are the main cell components of the BBB. Although amyloid-β oligomers have been reported to mediate oxidative damage to the CECs and astrocytes and trigger the downstream MAPK/ERK pathway, the cell surface binding site for Aβ and exact sequence of these events have yet to be elucidated. In this study, the receptor for advanced glycation endproducts (RAGE) was postulated to function as a signal transducing cell surface receptor for Aβ to induce ROS generation from NADPH oxidase and trigger downstream pathways for the phosphorylation of extracellular-signal-regulated kinases (ERK1/2) and cytosolic phosphorilase ARAGE). To quantify the ROS production, we applied fluorescence microscopy of dihydroethidium (DHE) in the cells. Confocal immunofluorescence microscopy of double immunofluorescent labeled gp91-phox and p47-phox subunits was performed to characterize NADPH oxidase complex assembly. Western blot analysis was applied to characterize phosphorylation of cPLA2α.Immortalized CECs (bEnd3) and primary rat astrocytes were applied in this study. To confirm Aβ-RAGE binding, we examined the quantitative immunofluorescence microscopy (QIM) of cellular surface RAGE for primary astrocytes and bEnd3 cells pretreated with oligomeric Aβ1-42 at +4°C  and stained with antiRAGE primary antibody (Ab42 competed with the anti-RAGE antibody (AbRAGE) to bind to RAGE on the surfaces of CECs and primary astrocytes. In addition, AbRAGE abrogate Aβ42-induced ROS production and the co-localization between the cytosolic (p47-phox) and membrane (gp91-phox) subunits of NADPH oxidase in both cell types. AbRAGE, as well as NADPH oxidase inhibitor and ROS scavenger suppressed Aβ42-induced ERK1/2 and cPLA2 phosphorylation in CECs. At the same time, only AbRAGE, but not NADPH oxidase inhibitor or ROS scavenger, inhibited ERK1/2 and cPLA2α phosphorylation in primary astrocytes.We report that Aβ42 binding to RAGE on the CEC and astrocyte surfaces is required for NADPH oxidase complex assembling, resulting in subsequent ROS generation and phosphorylation of ERK1/2 and cPLA2 and suggested the presence of two different RAGE-dependent downstream pathways in CECs and astrocytes. Therefore, understanding precise molecular mechanisms underlying Aβ mediated oxidative damage may provide new insights into the development of preventive and treatment strategies for AD.This study demonstrated that Aβ


Calcium release activated calcium channels inhibitors have a potent role in treatment of autoimmune disorders mediated dysregulated T-lymphocyte and mast cell functioning. Herein, we describe the pre-clinical of RP3133, a novel and potent CRAC channel inhibitor with scope for development as a clinical candidate for asthma.In vivo efficacy of the compound was determined in experimental models of asthma in guinea pigs including PAF or ovalbumin induced eosinophil infiltration into lungs ovalbumin induced histamine release from mast cells, and airway hyper-responsiveness.Inhibition of CRAC channel activity in Jurkat cells, cytokine release from human whole blood or PBMC, and mast cell degranulation were estimated. 38 nM) besides reducing IL-4 (<550 nM) and IL-5 (<750 nM) release from human whole blood and PBMC. Additionally, the compound suppressed IgE-induced mast cell degranulation at nanomolar concentrations (139 nM). Oral administration of RP3133 in guinea pigs resulted in a dramatic reduction in eosinophil infiltration in an acute model of PAF-induced allergic asthma (50 = 0.2 mg/kg/p.oED) as well as in an experimental model of ovalbumin-induced chronic airway inflammation (50 = 2.5 mg/kg/p.o)ED. Additionally, RP3133 caused a significant reduction (P<0.05) citric acid, histamine, or methacholine induced airway resistance in sensitized guinea pigs. Consistent with in vitro findings, the compound caused a significant inhibition of mast cell degranulation manifested by a reduction in histamine release.RP3133 significantly inhibited calcium entry into Jurkat cells (Results demonstrate the potential of RP3133 as an anti-asthmatic agent as evidenced from pre-clinical data. Toxicological evaluation of the molecule is currently in progress.


Km, kcat, Ki values, enzyme half-life, product•enzyme dissociation constant, and the rate constant for the reversed reaction) has been developed. This tool provides comparison of the progress of uninhibited versus inhibited reactions for common inhibitory mechanisms, and guides the tuning of reaction conditions. Possible applications include: analysis of substrate turnover, identification of the point of maximum difference in product concentration (Δmax[P]) between inhibited and uninhibited reactions, determination of an optimal observation window unbiased for inhibitor mechanisms or potency, and interpretation of observed inhibition in terms of true inhibition. An important observation that can be utilized to improve assay signal strength and resolution is that Δmax[P] occurs at a high degree of substrate consumption (commonly >75%) and that observation close to this point does not adversely affect observed inhibition or IC50 values.A difficulty associated with high throughput screening for enzyme inhibitors is to establish reaction conditions that maximize the sensitivity and resolution of the assay. Deduction of information from end-point assays at single concentrations requires a detailed understanding of the time progress of the enzymatic reaction, an essential but often difficult process to model. A tool to simulate the time progress of enzyme catalyzed reactions and allows adjustment of reactant concentrations and parameters (initial concentrations,  With low substrate concentrations it often becomes necessary to allow reactions to proceed until a large proportion of substrate becomes depleted in order to obtain sufficiently high signals (i.e. a high S/B ratio). While such extended incubation times may obscure the effect of weak inhibitors, shorter incubation times give weaker signals that may adversely affect assay performance. Different modes of inhibition (e.g. uncompetitive and non-competitive) further complicates data interpretation and assay design. A further difficulty is that the underlying theory, which is based on rate-law equations for initial reaction velocity, becomes violated at extended reaction times and thus complicates data interpretation, particularly the relation between observed and true inhibitor potency A common experimental condition in HTS for enzyme inhibitors is to use low substrate concentrations ( equation 1). In practice, initial reaction rates can only be approximated since the real measurable quantity represents a concentration at a given time-point . With small enough time intervals, which is commonly used when determining kinetic parameters, the approximation improves, and for many experiments this is not a problem. However, conditions such as those commonly applied to HTS for enzyme inhibitors often violate this approximation and make interpretations based on the MM equation for initial reaction velocity less reliable. Furthermore, such assays are often associated with a high enough level of substrate turnover to render the phenomenon of product inhibition  significant, thus complicating interpretation of observed inhibition further. Consequently, interpretation of data from experiments such as HTS, as well as the design of HTS assay conditions, should ideally be founded on progress curve analysis. Since the MM rate law cannot be analytically integrated to explicitly express product concentration as a function of time and in terms of catk and Km, this has to be achieved by numeric approaches In experimental deduction of kinetic parameters, the initial reaction rate at different substrate concentrations is measured and data obtained fitted to the Michaelis-Menten (MM) rate law equation (Simulation Tool S1), or obtained from the author. Reaction variables  and parameters , enzyme half life) can be adjusted and the effects of entered values are directly coupled to graphs displaying the formation of product as a function of time ( & S1). The modes of inhibition included are; competitive (inhibitor·enzyme dissociation constant Kic), uncompetitive (inhibitor·enzyme dissociation constant Kiu), and mixed inhibition . This allows for non-competitive inhibition to be accounted for by setting the two inhibitor·enzyme dissociation constants for mixed inhibition to equal values (i.e. Kic = Kiu). The graphs also display the difference in product concentration between inhibited and uninhibited reactions (Δ[P]) as a function of time for the different types of inhibition, which is particularly useful for estimation of when the maximum difference in product concentration (Δmax[P]) occurs between uninhibited and inhibited reactions. For simple comparison, the time progress of Δ[P] is also shown in a separate graph for all three types of inhibition (P] as a function of inhibition (%) as the reaction proceeds, and Δ[P] as a function of the degree of substrate conversion .A tool in spreadsheet format in which progress curves of inhibited and uninhibited reversible enzyme reactions can be interactively adjusted and compared for various types of inhibition was developed. The tool can be downloaded as supplementary material  at that point, the associated amount of substrate conversion (%), and the value of Δmax[P] (µM) are given . There is also a possibility to directly enter specific time-points to see the resulting Δ[P], the percentage substrate conversion, and the degree of inhibition (%). Entered time points are coupled to two graphs displaying the degree of inhibition (%) as a function of time and substrate conversion (50) as a function of substrate consumption (Keq = VfKmb/VbKmf) is automatically deduced.Key data are extracted from the curves and presented in table format. For each type of inhibition the time point of Δnversion Fig. 2. S] = 2Km and [I] = 2Kic) and competitive inhibition the effect of different incubation times can be studied , Δ[P] is 19 µM, substrate conversion 24% (uninhibited reference reaction), and the observed inhibition 40%. Since the degree of inhibition is similar at these two points, but Δ[P] is more than doubled at Δmax[P], a time point closer to Δmax[P] is appropriate for read-out.Prior to experimentation, specific reaction conditions can be studied and compared by feeding different reaction parameters into the simulation tool. With a given set of conditions , the observed inhibition 32%, and substrate conversion 33%. Thus, the assay resolution becomes lower under these conditions.The effect of lowering substrate concentration to kcat = 1 s−1, Km = 1 mM and Ki = 200 nM), a good correlation between observed and simulated data was obtained; Δmax[P] was predicted to 1.23 mM and its time point to 77 minutes, which only deviates slightly from the observed Δmax[P] of 1.18 mM after 73–74 minutes. This demonstrates how a reasonable estimate of the progress curves can be directly obtained by using kinetic parameters pre-determined in an initial rate experiment, or based on literature values. It should be noted that full determination of kinetic parameters, inhibition mechanism, and error estimates, by progress curve analysis requires more data at different [S] and [I], as in initial rate experiments Km, the underlying rate constants, i.e. (k−1+kcat)/k1 = Km, remains undetermined if not other types of kinetic data is available. Furthermore, as for any method relying on progress curve analysis, the linearity and range of the response must be accounted for when interpreting and comparing experimental data with simulated data.Comparison with experimentally generated progress curves for LTA4H catalyzed peptide hydrolysis, with or without the competitive inhibitor bestatin, showed that simulated and experimental curves are in agreement. This suggests that predictions based on the simulation tool have significance Fig. 4. Km was determined to be 3.6 µM and kcat to be 0.43 sec−1 by progress curves analysis and to be 3.9 µM and 0.45 sec−1 by the initial rate method. (One reason for the successful determination of Km by the progress curve method was the possibility to use a relatively high [So] of ∼5 Km.) Comparing the experimentally observed Δmax[P] and the time point when it occurred, with the corresponding values predicted by the tool, gave deviations of only 0.17 µM (2%) and a 90 seconds (3%), respectively.To further evaluate the validity of the simulations, progress curves were also collected for another enzyme system, peptidolysis by presequence peptidase, with and without bestatin as an inhibitor Fig. 4. max[P] along with the associated time point, % Substrate, % Inhibition, and IC'50) that are automatically deduced in response to adjusted parameters. The corresponding data at defined time points can also be extracted by entering time points in the column headers of an adjacent table . This helps the user to identify an optimal window of observation. For each type of inhibition the corresponding reaction equations are given. Non-competitive inhibition is not directly included but can be accounted for setting Kic = Kiu, realizing that this is a special case of mixed inhibition.In the tool the user is presented to three differently colored blocks  in which reactions parameters and variables can be adjusted Fig. 5. Simulation Tool S1) or obtained from the author. The tool provides accurate simulation of experimental progress curves . The associated degree of substrate consumption is normally above 70% and is rather insensitive to changes in substrate and inhibitor concentrations. Furthermore, the observed inhibition does not significantly deviate from initial conditions until the substrate is almost completely depleted (>80%). An ideal point of observation should therefore be within a window of about 50–75% substrate consumption, which is in contrast to the common recommendation of <20% substrate depletion for determination of kinetic parameters.A general observation that emerges when using the simulation tool is that Δ50) as a function of substrate consumption  to true inhibition (IC50) at specific reaction conditions.Since interpretation of observed inhibition in terms of true inhibition can become confused at such high levels of substrate depletion, the tool clearly visualizes observed inhibition (%) and inhibitor potency . The fact that non-competitive and mixed inhibitors have two Ki values augmenting each other explains this effect. Consequently, identification of non-competitive and mixed inhibitors are generally favored.Comparing different modes of inhibition at corresponding reaction conditions indicates that non-competitive and mixed inhibition exhibit the largest Δ.As outlined above, an observation window close to the time point of Δvelocity Fig. 2, e.g. no more than 20 percent substrate conversion, or substrate conversion should be within the linear range, or substrate concentration must equal Km. Such rules may sometimes limit the successful design of experiments by giving low signal windows. Many of the rules that guide the set-up of HTS assays stem from a fear of violating MM conditions and the underlying assumptions for steady state kinetics. Comparative analysis of progress curves of uninhibited versus inhibited reactions helps to better understand when, and to what extent, these concerns should be accounted for and also helps in the final data interpretation. The tool presented here aids in this process and provides accurate simulations of experimental progress curves.In summary, HTS assays are often designed with a set of common rules of thumb in mind, Purified LTA4 hydrolase was a gift from Dr. Agnes Rinaldo Mattis, Karolinska Institute. Presequence peptidase was a gift from Prof. Elzbieta Glaser, Stockholm University. Mca-R-P-P-G-F-S-A-F-K(Dnp)-OH was from R&D Systems, UK. Alanine-4-nitroanlide and other standard chemicals were from Sigma-Aldrich, Sweden. equation 2) was numerically integrated according to Euler's method with an integration time step of 1 second ( equation 3). A function to estimate enzyme half life was also included as a mechanism-free model that accounts for enzyme inactivation by reducing its concentration with respect to its half-life ( equation 4). Because product inhibition and the reversed reaction may be prevalent as product accumulates, these phenomena were also taken into account in all rate equations ( equations 2 & 5–7). For inhibited reaction progress curves, the same approach was applied on the rate laws for the three common types of inhibition ( equations 5–7). Additional equations used in the simulation tool are shown in Equations S1 (equations 8–13).To simulate reaction progress curves expressing product concentration as a function of time, the reversible MM rate law for initial reaction velocity . Moreover, HTS assays are typically performed with [E] at orders of magnitude lower than [S]. A level with [S] approaching [E] is thus equivalent with almost complete substrate depletion and will therefore contribute very weakly to the overall reaction rate. For the reversed reaction, the equivalent condition is most pronounced at the very beginning of the reaction. However, since the product level at this stage is very low the overall contribution to the reaction rate will be small. Another reason to work with very low [E] is imposed by the fact that IC50 values lower than one-half of the [E] cannot be measured. Since compounds are typically screened at 1–100 µM it is common to work with [E] around 100 nM, which in most cases is much lower than [S].The underlying assumption is that the rapid equilibrium approximations holds true throughout the complete reaction and that the reactions are pseudo-first-order in  between inhibited and uninhibited reactions as a function of substrate conversion (left) or observed inhibition (right) for four types of inhibition: competitive (yellow trace), uncompetitive (green trace), non-competitive (orange trace), and mixed (blue trace). In the simulation tool, the graphs are directly coupled to user entered reaction parameters and variables. Entered reaction conditions were: [S] = Km = 0.25Kmp = 10[I] = 400[Eo] = 100 µM; [Po] = 0 µM; kcat = 0.5 s−1; enzyme t(1/2) = 24 hours; Kic = Kiu = Ki-non = 5 µM for competitive, uncompetitive, and non-competitive inhibition; and Kiu = 5Kic = 15 µM for mixed inhibition. Substrate conversion refers to the uninhibited reference reaction (left). Note the reversed x-axis (right).(TIF)Click here for additional data file.Figure S3Δ[P50 .] between inhibited and uninhibited reactions for competitive (yellow), uncompetitive (green), non-competitive (orange), and mixed (blue) inhibition as a function of IC' At initial reaction conditions, IC'50 equals IC50. In the tool, the graph is directly coupled to user adjustable reaction variables and parameters. Entered reaction conditions were: [S] = Km = 0.25Kmp = 10[I] = 400[Eo] = 100 µM; [Po] = 0 µM; kcat = 0.5 s−1; enzyme t(1/2) = 24 hours; Kic = Kiu = Ki-non = 5 µM for competitive, uncompetitive, and non-competitive inhibition; and Kiu = 5Kic = 15 µM for mixed inhibition.(TIF)Click here for additional data file.Figure S4Asymptotic behaviour of the Z-factor. The Z-factor is plotted as a function of the S/B ratio at different coefficients of variation (CV). The asymptotes are shown as horizontal dashed lines. Corresponding curves, asymptotes, and CV values are in identical colors. For a specific CV the Z-factor can only be improved by increasing the S/B ratio, with an upper bond defined by the asymptote.(TIF)Click here for additional data file.Figure S5Differences between progress curves generated from pseudo-first order and second order rate equations. Curves generated from the pseudo-first-order model are shown as thick lines and curves generated with the second-order model are shown as thin lines. A) Progress curves (solid lines) are plotted against the left Y-axis and the differences between the models (dotted lines) are plotted against the right Y-axis. Progress curves were generated by numeric integration of equation a and b for different Km values, as indicated in the graph. Other variables and parameters were as follows: [So] = 100 µM, [Etot] = 100 nM and kcat = 0.5 s−1. Differences between the two models are below 1% for the Km values tested. B) Progress curves  were generated by numeric integration of equation a and b for different values of [So], as indicated in the graph, and with other parameters as follows: Km = 20 µM, [Etot] = 100 nM and kcat = 0.5 s−1. Differences between the two models  are below 3% for the values tested. The results shown in A) and B) demonstrate that differences between progress curves generated from pseudo-first order and second order rate equations are small at conditions normally applied in HTS. The differences are of the same magnitude or smaller as experimental noise and should therefore only have a limited effect on the quality of predictions made by the tool. 1. Morrison JF (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim Biophys Acta 185: 269–286.(TIF)Click here for additional data file.Simulation Tool S1The simulation tool in Microsoft Excel binary format.(XLSB)Click here for additional data file.


Chlamydia in the bronchoalveolar lavage (BAL) fluid of pediatric patients with airway hyperresponsiveness. While specific IgG and IgM responses to C. pneumoniae are well described, the response and potential contribution of Ag-specific IgE are not known. The current study sought to determine if infection with Chlamydia triggers the production of pathogen-specific IgE in children with chronic respiratory diseases which might contribute to inflammation and pathology.Recent studies have confirmed the presence of viable Chlamydia-specific IgE was isolated from BAL and serum samples and their specificity determined by Western blot techniques. The presence of Chlamydia was confirmed by species-specific PCR and BAL culture assays.We obtained BAL fluid and serum from pediatric respiratory disease patients who were generally unresponsive to corticosteroid treatment as well as sera from age-matched control patients who saw their doctor for wellness checkups. Chlamydia-specific IgE was detected in the serum and/or BAL of 107/197 (54%) patients suffering from chronic respiratory disease, but in none of the 35 healthy control sera (p < 0.0001). Of the 74 BAL culture-positive patients, 68  tested positive for Chlamydia-specific IgE. Asthmatic patients had significantly higher IgE levels compared to non-asthmatics (p = 0.0001). Patients who were positive for Chlamydia DNA or culture had significantly higher levels of serum IgE compared to negative patients (p = 0.0071 and p = 0.0001 respectively). Only 6 chlamydial antigens induced Chlamydia-specific IgE and patients with C. pneumoniae-specific IgE had significantly greater levels of total IgE compared to C. pneumoniae-specific IgE negative ones (p = 0.0001).Chlamydial DNA was detected in the BAL fluid of 134/197 (68%) patients. Total IgE increased with age until 15 years old and then decreased. Chlamydia-specific IgE may prove significant in the exacerbation of chronic, allergic airway diseases, thus highlighting a direct role for Chlamydia in asthma pathogenesis.IgE antibodies play a central role in allergic inflammation; therefore production of  Inflammation of the airways is the most common finding in all asthma patients and today, most asthma experts consider airway inflammation a central feature of asthma pathogenesis,2. The gStreptococcus pneumoniae, Haemophilus influenzae and Moraxella catarralis[. More recently, Mycoplasma pneumoniae and Chlamydia pneumoniae have been identified in 5–25% of children with asthma exacerbations[Approximately 20% of wheezing children have serological evidence of an immune response to atarralis,10. Morerbations-13. Hahnrbations,15.et al.[H. influenzae and S. pneumoniae specific IgE antibodies have been found in the serum of approximately a third of atopic children and asthmatic adults, however, they were all related to a subject’s atopic status[Earlier studies by Welliver et al. reportedc status. The impc status.Chlamydia-specific IgE antibodies. We hypothesize here that the presence of Chlamydia-specific IgE antibodies explains, at least in part, the mechanism of chlamydial involvement in either initiating or exacerbating chronic allergic airway disease.In the current study we examined the BAL fluid and serum of a large cohort of children with chronic respiratory disease for the presence of Serum and BAL samples were analyzed from 197 patients between the ages of 0–20 who were patients at Baystate Medical Center. BAL samples were originally obtained from patients for diagnostic or treatment purposes. Children were considered for bronchoscopy only after a thorough, non-invasive evaluation did not yield a definitive diagnosis; symptoms were not improving with time; aggressive medical management had not been successful and the child was truly compromised i.e., was missing school, could not participate in physical activity and had frequent symptoms. Airway reactivity was monitored by spirometry in children capable of performing the test as well as clinically, by frequency and severity of symptoms and response to therapy. In children not capable of performing spirometry, clinical characteristics were assessed.We also obtained 35 age and gender-matched control patient sera as residual samples from patients undergoing wellness check for school records or general checkup at the University of Massachusetts Health Services complex over a similar period of time as the patient samples. We obtained IRB approval and patient consent to collect and use these residual samples for research from the Baystate Medical Center and University of Massachusetts Amherst IRB. In addition, a subset of 20 (of the 197) respiratory disease patients presented with physical airway anomalies including most commonly, tracheal bronchus, obstruction by a foreign object, laryngomalacia, tracheomalacia, and bronchomalacia with abnormal cough. These were considered non-inflammatory controls when compared with asthmatics and patients with pneumonia and bronchitis.Chlamydia viability as previously reported[Genomic DNA was isolated from BAL samples and PCR detection of chlamydial DNA was performed in the same manner as previously described for all samples in this cohort. All BALreported,19. DNA Total IgE was evaluated using the Elecsys IgE kit , with the electrochemiluminescence immunoassay according to the manufacturer’s instructions. Plates were read on the Roche Elecsys 1020 analyzer which automatically calculated the IgE concentration of each sample based on a standard curve. Elevated IgE levels were determined based on the manufacturer’s recommended threshold by age range.Chlamydia-specific IgE antibodies.Because the normal concentration for IgE in serum is approximately 0.0005 mg/ml, and is even less in BAL fluid, we utilized affinity beads in a similar manner as Kadooka et al to isolaTo test for IgE in BAL samples, recombinant protein A beads  were utilized in the same manner as the protein G beads above. In this case the protein A beads were used to adsorb any antibodies detectable in the BAL fluid. IgE antibodies present in the BAL fluid were bound by the protein A beads and the bound antibodies were eluted using a pH 2.5 glycine solution followed by centrifugation to obtain the supernatant containing IgE and other antibodies if present. The eluted antibodies were pH balanced using Tris–HCl and then used as the primary antibody in the Western blot procedure. Because of the limited volume of some BAL fluid samples available and the low concentration of IgE in each sample, BAL samples were initially assessed in batches of 4 to 7 samples each.C. pneumoniae (TW183) and C. trachomatis (serovar E) elementary bodies were purified by 20%–50% (vol/vol) Renografin gradient centrifugation as previously described[Chlamydia-specific IgE bands. All samples were assayed for the presence of specific IgE using identical conditions including sample dilution.As previously reported, Western blot is a highly sensitive and efficient technique in detecting specific IgE-25. C. pescribed and norm3. BAL differential counts were performed using Wright stained cytospin preparations of BAL and examination was performed under oil-immersion microscopy (50X or 100X). Results were expressed on the basis of a 100 cell count survey. Inflammatory asthma sub-phenotypes were determined based on BAL neutrophil and eosinophil cells counts, as previously reported[BAL cell counts and differentials were performed according to standard techniques in the hospital’s Hematology Clinical Laboratory at Baystate Medical Center  as previously reported,27. Briereported.p-value of 0.05 was considered significant and all tests were two-tailed.All statistical analyses among the different groups of the patient population were performed using Microsoft Excel®. Statistical correlations were determined with Fisher’s exact test and Chi-square with Yates correction (SPSS 15.0 software). A We obtained both BAL fluid and serum from 197 pediatric respiratory disease patients, average age 7.9 years old, who presented to a pediatric pulmonary practice in Springfield, MA with chronic respiratory disease and were generally unresponsive to corticosteroid treatment. The patient demographic  patient samples were positive for chlamydial DNA. Species-specific PCR revealed that 65 samples were positive for C. pneumoniae DNA only, 34 were positive for C. trachomatis DNA only and 35 patients harbored both C. trachomatis and C. pneumoniae DNA. In order to determine if the organisms detected by PCR were viable, all BAL samples were subjected to a modified tissue culture technique as previously described. The data showed that 74/197 (38%) patients assayed were culture positive for Chlamydia. Importantly, 62/74  culture positive patients were diagnosed with chronic, severe asthma according to GINA guidelines[Polymerase chain reaction (PCR) was utilized to determine if idelines. Analysep < 0.0001). Patients who were positive for Chlamydia by DNA or culture had significantly higher levels of total serum IgE compared to IgE negative patients  patients had elevated serum IgE levels, while 132/197 (67%) had normal IgE levels (Fisher’s exact T-test Chlamydia-specific IgE in both the serum and BAL fluid from each patient. Since BAL samples were assessed in batches of 4 to 7 samples each (because of limited sample volume), we cannot ascertain the exact percentage of patients with Chlamydia-specific IgE in their BAL fluid. However, qualitatively, BAL fluid samples from this patient cohort contained Chlamydia-specific IgE antibodies  patients suffering from chronic respiratory disease but none of the healthy control patient sera . In addition, a subset of 20 patient samples from our respiratory disease cohort who were diagnosed with structural anomalies rather than inflammatory airway disease, were also assessed for the prevalence of Chlamydia-specific IgE. The data shows that 1/20 (5%) had Chlamydia-specific IgE in their sera compared to the inflammatory patient cohort  tested positive for Chlamydia-specific IgE.C. trachomatis and C. pneumoniae-specific IgE antibodies were found in the BAL fluid . This was generally true however, for C. pneumoniae IgE antibodies, where 81% of the positive patients had IgE that recognized MOMP, compared to only 17% for C. trachomatis MOMP. Thirty three percent of BAL fluid positive patient samples displayed IgE antibodies against an un-annotated 250kDa C. trachomatis protein.Overall, 61% of the Chlamydia-specific IgE antibodies revealed bands on Western blot ranging in molecular weight from 6kDa to 250kDa . C. trachomatis - induced cLPS IgE antibodies were not detected in any serum sample. C. pneumoniae did not induce MOMP-specific IgE in serum, while 8% of IgE positive-patients had antibodies that recognized C. trachomatis MOMP  and the polymorphic outer membrane protein (POMP) did not induce IgE antibodies in the BAL fluid, however, they each accounted for the Chlamydia-specific IgE found in 20% of positive patient sera assayed. The frequency of MOMP-induced IgE in the BAL fluid was 61%, while the frequency in serum was only 5%, demonstrating possible site-specific differential cytokine and lymphocyte responses or antibody release  to activate T-cells and, thus, may not directly induce a Th2 response, this theory might not hold true in this case.The primary biological function of IgE is to provide immunity against multicellular parasitic pathogens. Howeveret al, that upC. pneumoniae has long been implicated in the inflammatory response experienced by chronic asthmatics; however, a specific mechanism of its involvement has remained elusive. While the role and regulation of IgE antibodies towards microbial antigens is far from being elucidated, results from the current study strongly suggest that this obligate intracellular pathogen can induce pathogen-specific IgE production and could therefore lead to mast cell degranulation and release of vasoactive agents. The presence of Chlamydia-specific IgE in the serum would suggest that the organisms play a direct role in asthma pathogenesis by continuous induction of IgE, since unlike most aeroallergens that a patient can avoid, the chlamydial organisms reside in the lower airways and are continuously secreting bacterial antigens. In addition, even if the organism is cleared by antibiotic use or natural immune responses, Chlamydia – specific IgE antibodies might still play an important role in the development of exaggerated airway responsiveness during subsequent subclinical or even asymptomatic infection.The authors declare that they have no competing interests.Chlamydia-specific Western blot method, performed data analysis and participated in the final manuscript review. KP and EA therefore share first author status. All authors read and approved the final manuscript.WW and PS conceived of the study and participated in its design, coordination, supervision, data analysis and drafted and the manuscript. PS was responsible for patient selection and sample collection. KP performed the cultures and PCR as well as assisting in the data analysis and drafting/revision of the manuscript. EA performed PCR on some samples, developed and optimized the


Cyanistes caeruleus and great tit Parus major. We found that blue tit nestlings had on average higher H/L than great tit nestlings, which corresponds with the ecological sensitivity of these species. In both species H/L was higher in a poor parkland habitat than in a high quality forest habitat. In nestling blue tits, higher H/L values occurred in years characterized by more extreme weather conditions and worse caterpillar availability. Such consistent patterns of variation in the H/L ratio of nestling blue tits and great tits suggest that, when age-dependent effects are controlled, the ratio can be used as an indicator of physiological stress that is generated by food-related stressors differing in space and time. In particular, elevated H/L ratios are indicative of human-induced changes in the structure of breeding habitats.Environmental factors affecting trophic conditions act as stressors on nestling altricial birds. Access of parental birds to a sufficient supply of food in a limited period of the nestling stage differ in time and space, depending on nesting habitat, prey density and weather conditions. Heterophil-to-lymphocyte ratio (H/L) is considered as a reliable indicator of prolonged stress reaction in birds. In this study we examine if variation in H/L shows consistent spatio-temporal patterns in nestlings of two parids, blue tit  A degree of specialization in utilization of habitats during breeding and non-breeding stages of life is variable among animals, including birds Unfortunately, variation in habitat quality is notoriously difficult to assess in the field As mediators in vertebrate stress response, glucocorticoid hormone concentration in blood provides direct metrics of stress Cyanistes caeruleus and great tits Parus major the involvement in parental care differs between the sexes but participation of both parents in feeding nestlings is essential for nestling growth and survival Nestlings of hole-nesting altricial birds grow in nests that are relatively safe and well isolated from external factors, which is considered to be the reason for a relatively long time of their post-hatching development Blue tits and great tits belong to the commonest and most numerous passerines in the Western Palearctic The main idea of this paper is that the environment subjects nestling tits to stress mostly through the food delivered by parental birds. Since trophic conditions differ among habitats and years, variation in physiological indicators of stress is expected to display a consistent pattern. Because blue tits are a little different from great tits in their degree of dependence on caterpillars, an inter-species difference in stress indices is also expected. General physical conditions, which constitute a background for nestling development, may also impact nestlings independently of food. Particularly, high temperature is a potential stressor that might induce elevated H/L ratios in nestlings and might differ between study sites.Consequently, the aim of this study was to examine the following predictions concerning the H/L ratio variation in nestling blue tits and great tits:- because of higher ecological sensitivity of blue tits in comparison with great tits, we expected that a corresponding difference should occur in chronic stress response, reflected in the H/L ratio,- because trophic conditions for growing nestlings are better in the forest site than in the parkland site, we predicted that the H/L ratio of nestlings should be higher in the parkland,- because food abundance and weather conditions during the nestling period tend to differ among years, we expected that the H/L ratio should also be variable between years,- in addition, since the study sites differ in tree cover and the degree of insolation, we checked if maximal temperatures differ between the sites.This study involved the sampling of a small amount of blood of free-ranging bird nestlings, c. 10 µL per nestling, taken from the ulnar vain. The work was conducted according to Polish legal guidelines. All procedures of bird handling and blood sampling were approved by the Local Ethical Committee for Animal Experiments in Łódź (Permit Number: 1/ŁB 530/2011) and the Director of State Office for Environment Protection (Permit Number: DOP-OZGIZ.6401.03.41.2011.dł), the latter being given at the national level. At each location, the study was carried out with permission of relevant land managers: the Director of Łódź Botanic Garden, the Director of Łódź Zoological Garden and the Director of Łódź Forestry Administration Unit. The permit to band birds was given by the National Central Office for Bird Ringing, Museum and Institute of Zoology, Warsaw.The study of variation in the H/L ratio of nestlings was carried out in 2005–2006 and 2010–2012 for blue tits and in 2010–2012 for great tits, as part of a long-term project (initiated in 1999) on the breeding ecology of nest-box populations of common hole-nesting birds in central Poland. The study system is localized around the city of Łódź and encompasses two 10 km-distant areas differing in habitat types: a parkland site with c. 200 nest-boxes and a woodland site with c. 300 nest-boxes. Wooden nest-boxes with removable front wall Carpinus betulus and oaks Quercus robur and Quercus petrea. The vegetation of the Zoological Garden descends from that forest, but it has been heavily fragmented by paths, buildings and fenced, deforested exposition spaces for animals. There are some small patches artificially wooded with Scots pines Pinus silvestris, birches Betula and some exotic tree species. Only some very limited fragments of the Botanic Garden consist of forest patches, mostly with birches as predominating trees. The vegetation of the most part of this garden has been formed artificially for the purpose of plant exposition, so that the tree cover is patchy with a large area of tree-free spaces. Tree patches are a mosaic of different deciduous and coniferous trees, in large part exotic species. Both gardens possess networks of pedestrian pathways and children playgrounds for visitors. They are very popular leisure time spaces for inhabitants of Łódź and tourists, and may be very crowded during spring and summer. South and south-east sides of the Botanic Garden border on highly urbanized area of the town with numerous blocks of flats.The parkland site  consists of two units, Łódź Botanic Garden (67 ha) and neighboring Łódź Zoological Garden (17 ha). Both the gardens belong to a large parkland area extending to the east and, partly, north of the study area, within the western part of the city of Łódź; the whole area is a remnant of an ancient deciduous forest dominated by hornbeam The woodland study site , the Łagiewniki Forest, is a rich deciduous forest of considerable size, c. 1250 ha, located N-E of Łódź Accipiter nisus breeding in this area in some years. A consistent feature of the study sites is that tree-crown-leaf-eating caterpillars are more numerous in the woodland than in the parkland In addition to the differences in the structure of the tree cover and human influence, the parkland and woodland study areas differ with respect to some other factors of ecological importance http://www.tutiempo.net/en/Climate/LODZ/124650.htm. In addition, the number of rainy days was calculated from records of daily rainfall in the same archive. The weather in May was highly variable among the years of the study . The ratio of the number of heterophils to the number of limphocytes (H/L ratio) was then calculated. Because some smears were faulty or non-readable, we finally analyzed smears for 649 nestlings from 175 blue tit broods (including only 7 late broods) and 607 nestlings from 206 great tit broods (including 41 late broods).On day 13–14 after hatching, the nestlings were individually banded, measured and sampled for blood, usually between 09∶00 and 14∶00 H. All nestlings in every brood were measured (wing length to the nearest 1 mm) and weighed (to the nearest 0.1 g), while only a random subsample of 3–5 nestlings blind-drawn out of same-age nestlings from every brood was designated for taking blood samples (c. 10 µL taken from the ulnar vain) that were used to prepare blood smears on individually numbered microscope slides. The smears were dried in the field and stained in the laboratory using a commercial Microscopy Hemacolor kit . The number of heterophils and lymphocytes for every blood smear was counted to sum up to 100 cells, applying a microscope under 1000x magnification with oil immersion. All microscope counts of blood cells were conducted by the same person (JS), with independent counts of heterophils and lymphocytes being highly repeatable ) to meet assumptions of analyses, but are presented in a non-transformed form in the figures and the text. Because the H/L ratios of nestlings from the same brood are not independent, the ln-transformed H/L ratios were analyzed using mixed linear models, with brood Id included as a random factor controlling for clustering; degrees of freedom were estimated by the Satterthwaite method Habitat-specific temperatures recorded with DS1921G Thermochron iButton loggers in 2012 were compared between the parkland site and the forest site using t-test, with average May temperatures for individual loggers being unit data points. Peak annual frassfall values during 2005–2012 were compared between the sites using paired t-test.All analyses were performed using IBM SPSS 20 software 1,384.599 = 10.458, p = 0.001) .The analysis of inter-species variation in first brood at the background of the year and site factors showed that nestling blue tits had 16% higher mean H/L ratio than nestling great tits (mixed model ANOVA: F= 0.001) . In bothIn first brood nestling blue tits, the H/L ratio differed between sites and years . BecauseIn first brood nestling great tits, the average H/L ratio was also notably higher, by c. 25%, in the parkland site than in the woodland site , Table 31,184.721 = 1.84, p = 0.18, with wing length as the significant covariant F1,417.588 = 6.588, p = 0.011 for blue tits, and mixed model ANOVA: F1,213.409 = 0.46, p = 0.50 for great tits).For both tit species we also compared first broods with late broods at the background of the habitat and year factors. The average H/L ratio did not differ between the two categories of broods in either species . A difference in mean maximum temperature was negligible and non-significant . Average weather conditions in May were similar and relatively mild in 2005, 2006 and 2011, with mean monthly temperature ranging from 13.5°C to 14.0°C, and total rainfall between 47.25 mm and 68.84 mm. By contrast, May weather in 2010 and 2012 deviated from average conditions in two opposite directions. May 2010 was cold (12.5°C) and rainy (158 mm), whereas May 2012 was warm (15.3°C) and dry (19.31 mm).The mean site-specific May 2012 temperature was c. 0.5°C higher in the parkland site than in the forest, the difference being statistically significant .The study sites notably differ in the abundance of caterpillars, as measured by peak frassfall , with caVariation in the heterophil-to-lymphocyte ratio reported in this study revealed significant inter-species, inter-site and year-to-year differences. The H/L ratio in nestling blue tits was higher than in great tits. In both species, the H/L ratio was higher in the urban parkland site than in the forest site. In the blue tit, but not in the great tit, the ratio was significantly variable between years. In addition to these patterns, a notable part of the total variance in the H/L ratio lies between broods, 89% for blue tits and 72% in great tits, as shown for the first clutch by estimation of intraclass correlations Although the H/L ratio was shown to be a reliable indicator of different kinds of stress influencing birds Falco tinnunculus the level of corticosterone responded with elevation to severe starvation and anthropogenic disturbances, while the H/L ratio increased in response to prolonged food-related, ectoparasitic, social and ecological stressors. Both these stress indicators responded to very severe stressors, potentially threatening to nestling survival. Because in studies on poultry it was found that intense stress may stimulate an increase in the number of heterophils and a decrease in the number of limphocytes, Maxwell Moreover, even if comparisons concern nestlings of the same age, the H/L ratio and the level of circulating corticosterone as stress indicators respond to different types of environmental stressors, at a different rate and on different time scales Several experimental studies on birds included the nestling H/L ratio as one of response variables Serinus serinus and the abundance of food in breeding territories and nestling growth indices, which leads to the conclusion that better nutrition results in lower stress indicators. Moreno et al. Certhia familiaris that broods in larger, less fragmented forest patches, where foraging is more efficient, were characterized by a lower H/L ratio. Because habitats and years tend to differ in the amounts and the availability of food in relation to specific abiotic and biotic conditions, it would be expected that indicators of stress should display corresponding patterns of spatio-temporal variation.Also ecological correlates of the H/L ratio suggest that food mediates in generating stress affecting nestlings. Hoi-Leitner et al. Accordingly, the patterns we report here seem reasonable. The higher average H/L ratio in nestling blue tits than in nestling great tits may suggest that the former are more sensitive to environmental stressors than the latter, which is consistent with the ecology of both these species. Although both species are commonly described as ecologically similar to each other, the blue tit is more than the great tit specialized in the preference for deciduous breeding habitats and in feeding nestlings with tree canopy caterpillars Lower average H/L values found in the forest site than in the park site are likely to be caused by the availability of food. Caterpillars, the key food of nestling tits Besides food, the inter-habitat difference in the H/L ratios could potentially result from some other habitat-specific effects. Because of tree-cover fragmentation, nest-boxes in our park habitat might be overheated as a result of more intense insolation than in the forest site. However, even in the very warm May 2012, mean maximum temperature did not differ between the sites, with mean monthly temperature being slightly higher in the parkland site than in the forest. This excludes a possibility of overheating as a major stressor.Although inter-habitat differences in physiological parameters seem more interesting than inter-year differences, the latter are also a notable components of natural variation that need to be examined. Variation in the H/L ratio among years, in this study found in blue tits, but not in great tits, may also be associated with differences in food availability. The availability of caterpillars for foraging adult birds depends not only on the prey abundance but also on prevailing external conditions, with rainfall being a hindering factor in prey collecting Though we expected that nestling H/L ratios should be lower in genuine first broods than in repeat and second broods (late broods), we did not find any difference. However, samples of late broods were small for both tit species, which reduced power of analyses. Timing of hatching did not affect the H/L ratio in another study on great tit nestlings To sum up, consistent patterns of variation in the H/L ratio of nestling blue tits and great tits support the idea that the ratio can be used as an indicator of physiological stress that is generated by food-related stressors differing in space and time. The inter-species difference in the H/L ratio reflects a difference in ecological sensitivity between blue tits and great tits.


Maternal antibodies inhibit seroconversion and the generation of measles virus (MeV)-specific antibodies  after vaccination whereas T cell responses are usually unaffected. The lack of seroconversion leaves individuals susceptible to vaccine-preventable infections. Inhibition of antibody secretion is due to the inhibition of B cells through a cross-link of the B cell receptor with the inhibitory FcγIIB receptor (CD32) by maternal antibody/vaccine complexes. Here, we demonstrate that a combination of TLR-3 and TLR-9 agonists induces synergistically higher levels of type I interferon in vitro and in vivo than either agonist alone. The synergistic action of TLR-3 and TLR-9 agonists is based on a feedback loop through the interferon receptor. Finally, we have identified CD21 as a potential receptor for interferon α on B cells which contributes to interferon α-mediated activation of B cells in the presence of maternal antibodies. The combination leads to complete restoration of B cell and antibody responses after immunization in the presence of inhibitory MeV-specific IgG. The strong stimulatory action of type I interferon is due to the fact that type I interferon uses not only the interferon receptor but also CD21 as a functional receptor for B cell activation. Maternal antibodies provide protection against infection with pathogens early in life but also interfere with vaccination. This interference is caused by a vaccine/maternal antibody complex which links the B cell receptor to the inhibitory CD32 molecule. Here, we show that this cross-link results in impaired B cell activation and proliferation which is correlated with diminished antibody responses. We also found that induction of large amounts of type I interferon restores the neutralizing antibody response in the presence of maternal antibodies. The best induction of type I interferon was accomplished by a combination of known activators of interferon secretion (a combination of TLR-3 and TLR-9 agonists). The strong stimulation by interferon is due to the previously unappreciated role of CD21 as functional receptor for interferon alpha. Our findings demonstrate that the dual receptor usage of type I interferon receptor and CD21 is crucial for B cell activation in the presence of maternal antibodies. This study suggests that measles vaccine, and potentially other vaccines, may induce optimal antibody responses when they are reconstituted with TLR-3 and TLR-9 agonists and thus these agonists may have great potential for clinical use. A fundamental unresolved issue in vaccinology is the inhibition of vaccination against infectious diseases of humans Worldwide, close to 200,000 children die of measles virus every year. During their first year of life, children are protected by neutralizing maternal antibodies against MeV infection. Over time, these antibody titers wane and eventually do not protect against wildtype virus infection  and Newcastle Disease virus (NDV)) which express measles virus hemagglutinin (H) can induce H specific neutralizing antibodies after vaccination in the presence of inhibitory MeV-specific IgG. In contrast to measles virus, both VSV 6 pDC (moi of 1). These results led us to try to improve the stimulation of the B cell response by using TLR-3 and TLR-9 agonists as inducers of type I interferon. We tested pI:C as TLR-3 agonist and human ODN type A, C and P as TLR-9 agonists on plasmacytoid dendritic cells individually and in combination. The combination of pI:C and human ODN 2216 (type A) proved to induce the highest levels of type I interferon in plasmacytoid dendritic cells  feedback loop, we blocked the signaling chain of the IFNR with antibody . This inIn order to test the ability of the pI:C/ODN2216 combination to stimulate B cell and antibody responses, the TLR agonists were used in an ELISPOT assay. Both pI:C and ODN 2216 stimulated higher B cell responses from the bone marrow of MeV-immune cotton rats but the combination was superior to either agonist alone . StimulaWhen cotton rats are immunized with MeV either intranasally or subcutaneously, the generation of neutralizing antibodies is suppressed by inhibitory MeV-specific IgG. If MeV was supplemented with either pI:C, ODN 2216, or a combination of both, the induction of neutralizing antibodies was detected . After iAfter immunization in the presence of maternal antibodies, the generation of neutralizing antibodies is inhibited. This could be due to a lack of B cell generation, or a lack of antibody secretion by B cells. In order to determine B cell development after immunization with MeV, cotton rats were immunized i.n., and mediastinal lymph nodes , spleen and bone marrow were tested for the presence of MeV-specific B cells on day 5, 8, 12 and in weekly intervals between week 3 and 10 by ELISPOT . AntibodIn order to test the stimulatory effect of the pI:C/ODN 2216 combination on MeV immunization, one set of cotton rats was immunized i.n. and B cell numbers in lymph nodes, spleen and bone marrow were measured on day 12 and day 35 after immunization . A seconSimilarly, s.c. immunization with MeV led to early activation of B cells in spleen and bone marrow at day 12, with increased numbers in spleen and bone marrow, and neutralizing antibodies at day 35. After s.c. immunization with MeV and pI:C/ODN 2216, B cell numbers were comparable to MeV immunization alone but the titer of neutralizing antibodies was increased on day 35. After s.c. immunization with MeV in the presence of inhibitory MeV-specific IgG, no neutralizing antibodies were induced, and B cell numbers in spleen and bone marrow were similarly low as after i.n. immunization with MeV in the presence of inhibitory MeV-specific IgG. After immunization with MeV and pI:C/ODN 2216, B cell numbers in spleen and bone marrow were reduced at day 12 compared to immunization with MeV in the absence of inhibitory IgG. However, at day 35 B cell numbers and neutralizing antibodies were comparable to immunization with MeV alone. In summary, these data demonstrate that MeV with the combination of pI:C/ODN 2216 restores the B cell and antibody response in the presence of inhibitory antibodies after both i.n. and s.c. immunization.In this and other studies, interferon α proved to be a stronger stimulator of the B cell response than other cytokines including IL-6. A potential explanation might be provided by recent data which demonstrate that interferon α binds to CD21 with the same avidity as its natural ligand, C3d, which also stimulates B cell responses The inhibition of seroconversion after immunization in the presence of maternal antibodies is a well-documented phenomenon. The lack of generation of antibody suggests an impairment of B cell activation and development, although this was never proven. Our data demonstrate clearly that the inhibition in the generation of antibody correlates with the inhibition of B cell development, and particularly with early activation of B cells in draining lymph nodes. We have shown previously that the inhibitory action of maternal IgG is mediated through a complex of MeV vaccine/IgG cross-linking the B cell receptor (BCR) with the inhibitory receptor CD32 in vivoet al compared cytokine responses of all the possible combinations of known TLR ligands in human PBMCs Previously, we have shown that the provision of type I interferon by immunization with NDV-H was able to partially restore the neutralizing antibody response in the presence of maternal antibodies. NDV interacts with TLR-7 and the RIG-I pathway which leads to the induction of type I interferon One possible application of our data would be the direct inoculation of IFN α with MeV vaccine in order to stimulate a better B cell and antibody response. In mice, the inoculation of a high dose of IFN α and influenza vaccine induced good T cell and B cell responses In summary, we have demonstrated that a combination of TLR-3 and TLR-9 agonists induces higher levels of type I interferon than either agonist alone. Subsequently, IFN α utilizes both IFN-R and CD21 as functional receptors for B cell stimulation and leads to restoration of B cell responses after immunization in the presence of inhibitory MeV-specific IgG.Animal use protocols for the experiments reported in this manuscript have been approved by the Institutional Animal Care and Use Committee of The Ohio State University in accordance with the Animal Welfare Act of the United States of America.5 pfu of MeV (Schwarz strain) intranasally or subcutaneously with TLR agonists as indicated in each experiment. We have chosen human MeV-specific antibodies to standardize our experimental approach and because of experimental advantages: 1. the level of natural maternal antibodies in pups varies (presumably because of the suckling hierarchy), human antibodies metabolize faster  and can be distinguished by ELISA from cotton rat antibodies.Inbred cotton rats (Sigmodon hispidus) were purchased from Harlan Laboratories, Inc. For immunization experiments in the presence of inhibitory antibody, MeV-specific IgG (NT of 100) was injected intraperitoneally (i.p.) into cotton rats to replace natural maternal IgG. One day later, animals were immunized with 10The Schwarz vaccine strain of measles virus was grown and titrated on Vero cells as described Human MEV-specific polyvalent IgG (Carimmune) with a neutralization titer (NT) of 640 was purchased from ZLB Behring. Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems. Rabbit IgG specific for human/mouse/rat CD21 was purchased from Santa Cruz Biotechnology. Goat antibodies specific for human interferon receptor 1 and chicken antibodies specific for human interferon receptor 2 with neutralizing capacity were purchased from Thermo Scientific.Spleen cells were stimulated with 10 µg/ml LPS (Sigma) or 2.5 µg/ml Concanavalin A for 72 hours and lymphoblasts purified by centrifugation over a ficoll gradient. Subsequently, cells were stained with antibodies specific for CD21, or interferon receptor 1 or 2. The respective secondary antibody labeled with FITC was pre-absorbed with cotton rat serum for 1 hour on ice. Subsequently cells were analyzed by flow cytometry .5′-ggGGGACGATCGTCgggggg-3′ , type P ODN (CpG ODN 21798 5′-tCGtCGaCGatCGgcgCGcgccg-3′) and type C ODN  were purchased from Invivogen. Poly (cytidylic-inosinic) acid potassium salt (poly I:C) was purchased from Sigma.CpG ODN 2216 (Bone marrow derived dendritic cells were generated as described 3 pfu of recombinant vesicular stomatitis virus expressing green fluorescent protein (rVSV-GFP). After 48 hours of incubation at 37°C, plates were evaluated for the presence or absence of green fluorescence on an Olympus 1 IX51 fluorescent microscope. IFN-α/β concentrations in samples were expressed as units with 1 unit being the equivalent of 1 pg of IFN-α. The threshold of detection was 16 units.Samples from three wells were pooled and treated with 0.1M hydrochloric acid to inactivate IFN-γ for 2 hours and the acid was neutralized with sodium bicarbonate. Cotton rat osteosarcoma cells CCRT were incubated with serially diluted samples (in duplicate) or recombinant cotton rat IFN-α (R&D Systems) as standard for 24 hours. Subsequently, CCRT cells were infected with 105′-TTGGCACACTTAGGCACAGC-3′ (forward) and 5′-CAAAAGGACTGGCCGAGGAC-3′ (reverse), and the plasmid pCR-script Amp SK(+)-Cotton rat IL-6 Total RNA from cotton rat lung tissue was extracted using Qiagen RNeasy kit (Qiagen) and treated with DNase (Ambion). To generate cDNA, AffinityScript multiple temperature reverse transcriptase (Agilent technologies) and random primers (Promega) were used. Quantitative polymerase chain reaction was performed using a LightCycler RNA amplification kit with SYBR green I (Roche). The primer sequence for IL-6 was 4 Vero cells were added per well. Five days post-infection cytopathic effect (CPE) was determined microscopically. The titer was defined as the reciprocal of the last protective serum dilution, as calculated from duplicate measurements.Cotton rat serum samples were two-fold serially diluted and incubated with 50 pfu MeV strain NSE for one hour at 37°C in a 96-well. One hour post-incubation 105 pfu of MeV (Schwarz strain) were used. 96 well plates (Millipore) were coated with gradient-purified, UV-inactivated MeV antigen in sodium carbonate buffer (pH 9.6) overnight at 4°C. Serially diluted lymphoid cells were plated and cultured at 37°C in an incubator. After overnight incubation, plates were washed with PBS/0.05% Tween 20. Plates were incubated with rabbit anti-cotton rat IgG (Virion Systems) and subsequently with goat anti-rabbit alkaline-phosphatase conjugated IgG (Zymed) in PBS/10% cotton rat serum. For development of spots, plates were washed three times with PBS and TMB-HK  substrate was added. Plates were incubated at room temperature for 5 minutes and spots were counted using an ELISPOT plate reader (CTL).For the B cell ELISPOT assay, mediastinal lymph node cells, bone marrow cells or spleens from measles virus immune cotton rats four to eight weeks after s.c. immunization with 10Multiple comparison analysis was done by one-way analysis of variance (ANOVA) using Graph Pad InStat 3 for Windows (GraphPad Software). The number of asterisks denotes the level of statistical significance .


To investigate the role of tyrosine 23 (Tyr23) phosphorylation of Annexin A2 (Anxa2) in regulating the proliferation and invasion of human breast cancer SK-BR-3 cells.A panel of lentivirus plasmids expressing Anxa2-wide type (Ana2-WT), Anxa2-Y23A, and Anxa2-Y23D was generated and infected with SK-BR-3 cells. The monoclonal strains were screened. The expression of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D was determined by Western blot analysis. The ability of the cells to proliferate was detected through an MTT  test. Boyden chamber assays were employed to examine migration and invasion abilities. The interaction between Anxa2 and Stat3 was analyzed by immunoprecipitation analyses. Nucleoprotein and cytosolic protein were extracted from SK-BR-3, Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D cells to analyze the expression and localization of Stat3 phosphorylation.P<0.05). Immunoprecipitation analysis revealed that Anxa2 and Stat3 interacted with each other, and the expression of Stat3 phosphorylation in the nucleus was enhanced by Anxa2-Y23D.The monoclonal strains constitutively expressing Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D were screened. Both Anxa2-WT and Anxa2-Y23D enhanced the proliferation, migration and invasion abilities of SK-BR-3 cells (Tyr23 phosphorylation of Anxa2 promotes the proliferation and invasion of human breast cancer SK-BR-3 cells and the phosphorylation of Stat3 in the nucleus. In China the morbidity of breast cancer rises year by year, and diagnosis is usually made at an early age. High metastatic potential and drug resistance account for the failure of breast cancer treatments. Annexin A2 (Anxa2), a member of the Annexin family of calcium-dependent phospholipid binding proteins, was reported to be abundantly expressed in many cancer tissues and is believed to play an important role in tumorigenesis and breast cancer progression. Structurally, Anxa2 consists of a highly conserved C-terminal core domain and a variable N-terminal domain with p11 proteins and t-pA binding sites and phosphorylation sites for different kinases, for example, Ser25 for protein kinase C (PKC) phosphorylation and tyrosine 23 (Tyr23) for Src kinase phosphorylation. Tyr23 phosphorylation of Anxa2 mediates cell scattering and branching morphogenesis, and regulates Rho-mediated actin rearrangement and cell adhesion. The dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration. A more recent study demonstrated that Tyr23 phosphorylation-dependent cell-surface localization of Anxa2 is required for the invasion and metastases of pancreatic cancer. However, little is known about the effects of Tyr23 phosphorylation of Anxa2 on the proliferation and invasion abilities of breast cancer cells and on the localization of the phosphorylation signal transducer and activator of transcription (Stat3). In the present study, Tyr23 was mutated to an alanine residue, creating a non-phosphorylatable mutant or an aspartic acid mimicking constitutive phosphorylation. A panel of experiments was performed on the two mutants, Anxa2-Y23A and Anxa2-Y23D, as well as on Anxa2-WT cells to detect the probable function of Tyr23 phosphorylation in regulating the migration and metastases of SK-BR-3 cells and the localization of Stat3 phospohrylation in SK-BR-3 cells.According to the global cancer statistics, breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females, accounting for 23% of the total cancer cases and 14% of the cancer deathsRabbit monoclonal and mouse monoclonal anti-Annexin A2, mouse monoclonal anti-phospho-Annexin A2, and anti-actin and anti-GFP antibodies were bought from Santa Cruz . Rabbit monoclonal anti-Stat3 antibodies and anti-Phospho-Stat3 antibodies were purchased from Cell Signaling Technology . Rabbit monoclonal and mouse monoclonal anti-IgG as well as the secondary antibodies, goat anti-rabbit or anti-mouse IgG or donkey anti-goat IgG conjugated to fluorescein isothiocyanate (FITC) or Texas Red, were obtained from Santa Cruz. Lipofectamine 2000 was purchased from Invitrogen . All other chemicals were purchased from Sigma .-WT was referred to as Anxa2-WT in this study. The mutants of pEGFP-N3-Anxa2-Y23A and pEGFP-N3-Anxa2-Y23D from this cDNA, in which Tyr23 was replaced by alanine and aspartic acid, were cloned by sequential site-directed mutagenesis reactions using Quik-Change site-directed mutagenesis (Stratagene) according to the manufacturer’s instructions. The recombinant plasmids were confirmed by a testing sequence, and the quality of the recombinant protein was checked with a fluorescence microscope and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).The full-length human Anxa2 cDNA was amplified through reverse transcription polymerase chain reaction (RT-PCR). The PCR products (1033bp) were cloned in the Xho I and Bam HI sites of pEGFP-N3 vector. pEGFP-N3-Anxa2. The SK-BR-3 cells were infected with the lentivirus plasmids expressing Anxa2-WT, Anxa2-Y23A and Anxa2-Y23D or an empty vector using Lipofectamine 2000 according to the manufacturer’s procedures. Stable transfectants were selected with 800 µg/mL G418. Clones were picked and maintained in a complete medium containing 100 µg/mL G418. The expression of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D was analyzed by Western blot with GFP and green fluorescence.Breast cancer SK-BR-3 cells were cultured as described previouslyg for 5 min. The supernatant was saved as a cytoplasmic fraction. The nuclear pellet was resuspended by a fractionation buffer again, passed through a 25 G needle several times, and centrifuged again at 800 g for 10 min. The buffer was removed, and the nuclear pellet was suspended in the nuclear buffer (standard lysis buffer with 10% glycerol and 0.1% SDS).The cells were collected in a 1.5 cm Eppendorf tube and suspended by a fractionation buffer. The cells were passed through a 25 G needle 10 times using a 1 mL syringe. The cells were left on ice for 20 min. The lysate was then centrifuged at 800 g for 20 min at 4°C. The supernatants were used for immunoprecipitation and immunoblot analysis. The obtained supernatant was mixed with protein A/G beads (Invitrogen) at 4°C, incubated for 1 h, and centrifuged for 30 s at 10,000 g. The supernatant was then incubated overnight with Stat3/Anxa2 antibody and protein A/G beads and centrifuged for 30 s at 10,000 g. Proteins were prepared by whole-cell lysis and subcellular fractionation for immunoblot. A SDS-PAGE sample buffer was boiled for 5 min to 10 min. The proteins were then separated by electrophoresis on 12% SDS-PAGE gels. The proteins were transfected onto a polyvinylidene fluoride membrane, which was blocked with 5% skim milk and incubated overnight with primary antibodies at 4°C. After incubation in an Odyssey blocking buffer with secondary antibodies for 1 h at room temperature, the immunoreactive bands were then determined by image scanning on an Image Station LI-COR Odyssey imaging system. The bands were analyzed using the imageJ software.Briefly, cells were placed on ice and incubated for 30 min in a lysis buffer . The lysed cells were then centrifuged at 12,000 2 round glass coverslips (6×103/coverslip). The cells were fixed with 4% paraformaldehyde at room temperature for 10 min labeling. After fixation, the cells were permeabilized with 0.2% Triton X-100 in PBS and quenched with 50 mM NH4Cl/PBS. Several washing steps later, the coverslips were blocked with 3% BSA , then incubated in primary antibodies (1:100) and secondary antibodies . The cells were incubated for 10 min with 1 µg/mL DAPI  for cellular nuclei staining. Finally, the coverslips were mounted on slides with mowiol and observed under an Olympus BX40 epifluorescence confocal microscope. The images were captured with MagnaFire software and processed using Photoshop.Various cells were grown on 18 mm.Proliferation, migration, and invasion assays were performed as described previouslyt-test was used to determine whether a significant difference exists between two means (P<0.05). In multiple means, the difference was determined by one-way analysis of variance (P<0.05). The significant differences are marked in the graphs. Triplicate experiments were done for all the samples.Student’s WT, pEGFP-N3-Anxa2Y23A, and pEGFP-N3-Anxa2Y23D  to determine the exact expression of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D proteins in the SK-BR-3 cells . The SK-BR-3 whole-cell lysates, and protein G-Sepharose beads, and antibodies against Anxa2 and Stat3 were employed to carry out the experiment. in vitro led to the hypothesis that Stat3 and the phosphorylation Stat3 (P-Stat3) may be up-regulated in Anxa2-WT or Anxa2-Y23D cells. Western blot results suggested no significant differences in the expression of Stat3 and phosphorylation Stat3 (P-Stat3) in both SK-BR-3 and Anxa2-WT cells among different groups exposed to EGF for 0, 5, and 10 min 2-(p11)2 heterotetramer formation or stabilization. Anxa2 can be phosphorylated on Tyr23 by Src kinase both in vivo and in vitro[-]. And constitutively phosphorylated with Tyr23 leads to the cell-surface localization of Anxa2, which is essential for the invasion and metastases of pancreatic cancer. Moreover, Src kinase-mediated phosphorylation of Anxa2 on 23Tyr regulates the cytoskeletal dynamics, such as cell scattering and the epithelial mesenchymal transition (EMT).Anxa2 is up-regulated in various tumor types and plays multiple roles in cellular functions, including tumorigenesis, proliferation, cell migration, invasion and adhesionin vitro proliferation and invasion of SK-BR-3 cells. We generated a panel of lentivirus plasmids expressing Anxa2-WT, Anxa2-Y23A in which Tyr23 was altered into an alanine residue to create non-phosphorylatable mutant, or Anxa2-Y23D in which Tyr23 was altered into an aspartic acid residue to mimick constitutive phosphorylation. Since Anxa2 is overexpressed in invasive tumors and plays an important role in cancer progression and invasion. Constitutive phosphorylation of Anxa2 also enhances the proliferation, migration and invasion abilities of SK-BR-3 cells.In this study, we establish three new findings that elucidate the role of Anxa2 in the proliferation and invasion of breast cancer SK-BR-3 cells, and the effect of Anxa2 on the stimulation of Stat3 pathways. First, the Tyr23 phosphorylation of Anxa2 contributes to the . The tyrosine phosphorylation protein Anxa2 can also been activated by Src-kinase pathways. In the present study, we find that Anxa2 and Stat3 interact with each other, and they have a colocalization in the nucleus of SK-BR-3 cells.Second, Anxa2 and Stat3  interact with each other, and have a colocalization in the nucleus. EGF  binding to its receptor (EGFR) induces dimerization and autophosphorylation of the receptor at multiple tyrosine residues, which serve as docking sites for recruitment of proteins with SH2 (Src homology 2) domains that activate multiple downstream signaling pathways. The adaptor protein Grb2 (growth factor receptor-binding protein 2) binds to EGFR, which leads to activation of Stat3 pathways. Here, we provide evidence that the expression of phosphorylation Stat3 does not change when the exposure time of the SK-BR-3 cells to EGF are prolonged, or the Anxa2 is up-regulated or constitutively mimicking phosphorylation. However, the phosphorylation Anxa2 is increased with prolonged exposure to EGF, and Stat3 is up-regulated in nucleus when Anxa2 constitutively phosphorylated on Tyr23.Third, Tyr23 phosphorylation enhances Stat3 phosphorylation and localization in the nucleus. The proline-rich tyrosine kinase (pyk2) along with c-Src, facilitates epidermal growth factor (EGF)-mediated Stat3 reporter gene activation and Stat3 phosphorylation on amino acid residues Tyr-705 and Ser-727. The activation of these proteins trigger proliferation, invasion, migration and oncogenesis. Hepatocyte growth factor (HGF) induces Src kinase activation phosphorylates Anxa2 on Tyr23, and further mediates cell scattering, branching, and the cytoskeletal dynamics, which are necessary for the epithelial mesenchymal transition (EMT). Both Anxa2 and Stat3 can be activated by Src kinase pathway. The present study finds an interaction between Anxa2 and Stat3, and phosphorylation Anxa2 on Tyr23 enhances Stat3 phosphorylation and localization in nucleus. We are reasoned to believe that Anxa2, especially phosphorylation Anxa2 participates in the EGF and Src kinase induced Stat3 activation and Stat3 triggered oncogenesis. The exact machanism of the interaction between Anxa2 and Stat3 will be our future research objective.The epidermal growth factor (EGF) and Src kinase induced Stat3 activation mediate series proteins including cell-cycle regulators such as FBJ murine osteosarcoma viral oncogene homolog, mitogen-activated protein kinase 5, c-Myc, and inducers of tumor angiogenesis in invasive breast cancer tissue such as vascular endothelial growth factor, cyclooxygenase-2, matrix metalloproteinase (MMP)-2, MMP-10, and MMP-1Overall, these findings identify the relationship between phosphorylated Anxa2 and phosphorylated Stat3 in the nucleus. Tyr23 phosphorylation of Anxa2 could activate Stat3 and phosphorylation Stat3 localization in the nucleus of breast cancer SK-BR-3 cells.


Keratins are cytoplasmic intermediate filament proteins expressed in tissue specific and differentiation dependent manner. Keratins 8 and 18 (K8 and K18) are predominantly expressed in simple epithelial tissues and perform both mechanical and regulatory functions. Aberrant expression of K8 and K18 is associated with neoplastic progression, invasion and poor prognosis in human oral squamous cell carcinomas (OSCCs). K8 and K18 undergo several post-translational modifications including phosphorylation, which are known to regulate their functions in various cellular processes. Although, K8 and K18 phosphorylation is known to regulate cell cycle, cell growth and apoptosis, its significance in cell migration and/or neoplastic progression is largely unknown. In the present study we have investigated the role of K8 phosphorylation in cell migration and/or neoplastic progression in OSCC.To understand the role of K8 phosphorylation in neoplastic progression of OSCC, shRNA-resistant K8 phospho-mutants of Ser73 and Ser431 were overexpressed in K8-knockdown human AW13516 cells . Wound healing assays and tumor growth in NOD-SCID mice were performed to analyze the cell motility and tumorigenicity respectively in overexpressed clones. The overexpressed K8 phospho-mutants clones showed significant increase in cell migration and tumorigenicity as compared with K8 wild type clones. Furthermore, loss of K8 Ser73 and Ser431 phosphorylation was also observed in human OSCC tissues analyzed by immunohistochemistry, where their dephosphorylation significantly correlated with size, lymph node metastasis and stage of the tumor.Our results provide first evidence of a potential role of K8 phosphorylation in cell migration and/or tumorigenicity in OSCC. Moreover, correlation studies of K8 dephosphorylation with clinico-pathological parameters of OSCC patients also suggest its possible use in prognostication of human OSCC. Keratins (K) are largest subgroup of intermediate filament (IF) proteins preferentially expressed in epithelial tissues K8 and K18 is the prototype keratin pair of simple epithelia and are predominantly expressed in epithelial components of glandular tissues, including the pancreas and intestine, with other keratins such as K7, K19 and K20 K8 and K18 expression is not observed in stratified adult epithelial tissues. However, they are often aberrantly expressed in carcinomas including oral SCC and their expression is correlated with invasion and poor prognosis 2-terminal “head” and COOH-terminal “tail” domains All IF proteins, including keratins, consist of a conserved central coiled-coil α-helical- “rod” domain and flanking non–α-helical NHIn the present study we have shown that overexpression of shRNA-resistant K8 phospho-mutants in K8-knockdown OSCC cells demonstrate increase in cell motility and tumorigenicity compared with K8-wild type clone. Furthermore, K8 dephosphorylation was also observed in human OSCC samples which significantly correlated with size, lymph node metastasis and stage of the tumor.In order to express K8 phpspho-mutants and K8-wild type in K8 knockdown AW13516 cells, shRNAK8.2 resistant GFP tagged K8 phospho-mutants and K8-wild type (described above) were generated by site directed mutagenesis. The resistance of resulted mutants against shRNAK8.2 was checked by cotransfecting them with shRNAK8.2 in HEK-293 cells followed by measurement of fluorescence intensity and western blot analysis  during tumor progression in OSCC, shRNA-resistant K8-Ser73Ala and K8-Ser431Ala phospho-mutants were transfected in K8 deficient AW13516 C1-shRNAK8.2 cells. Stably over-expressed GFP tagged K8 wild type (K8WT-1and -2), K8-Ser73Ala mutant (K8S73A-1 and -2) and K8- Ser431Ala mutant (K8S431A-1 and -2) clones were selected in 1000 ug/ml G418-sulphate  using semi-quantitative IHC analysis , lymph node metastasis (P = 0.011) and stage (P = 0.013) (P = 0.002), and stage (P = 0.009) (P = 0.047), while phosphorylation at Ser431 did not correlate with patient survival  . K8 deph= 0.009) . K8 deph= 0.009) . Status survival . Thus, oIn summary, overexpression of K8 Ser73Ala and Ser431Ala mutants in K8-knockdown OSCC cells resulted in significantly increased cell migration and tumorigenicity. Furthermore, our in vitro results correlated well with IHC analysis of OSCC samples. Dephosphorylation of K8 (both at Ser73 and Ser431) was observed in many of the tumor samples. Loss of K8 phosphorylation (both at Ser73 and Ser431) correlated with tumor size, lymph node metastasis and stage. Moreover, dephosphorylation of K8 at residue Ser73 significantly correlated with poor patient survival.IF form an important tissue specific compartment of cellular cytoskeleton We have used an OSCC derived cell line AW13516 as model system in this study Dynamics of K8 and K18 filament assembly is controlled by their post translational modifications mostly by phosphorylation under various patho-physiological conditions Progression of tumor cells is dependent on their migratory ability Next, to understand the prognostic significance of K8 dephosphorylation in human OSCC, we studied correlation of K8 dephosphorylation with clinico-pathological parameters of the patients. Here we observed loss of K8 phosphorylation at both Ser73 and Ser431 in K8 positive OSCC samples ; Table 1Prior studies suggest that keratin phosphorylation is regulated in vivo by many kinases and phosphatases during various patho-physiological conditions In summary, our results clearly suggest that loss of K8 phosphorylation leads to increased cell migration and tumorigenicity in OSCC cells. In addition, K8 dephosphorylation was also observed in human OSCC samples which correlated with tumor size, stage and lymph node metastasis. Furthermore, loss of K8 Ser73 phosphorylation significantly correlated with poor patient survival indicating its significance in prognosis. Our results support Mizuuchi et al's hypothesis that phosphorylation dependent reorganization of keratins accelerate cell motility and metastatic potential of carcinomas This study was approved by the “Tata Memorial Hospital Human Ethics Committee”. The written informed consent was obtained from the patients as well as healthy individuals. All protocols for animal studies were reviewed and approved by the “Institutional Animal Ethics Committee (IAEC)” constituted under the guidelines of the “Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)”, Government of India .2 atmosphere The cell line AW13516 derived from the SCC of tongue 5′-GGAGGCATCACCGCTGTGACGGTCAACCAGAGC-3′ was synthesized as per manufacturer's instructions, containing 2 silent mutations in the shRNAK8.2 binding site To generate shRNA resistant K8-GFP wild type (K8-GFP-WT-R), K8-Ser73 mutant (K8-GFP-S73A-R) and K8-Ser431 mutant (K8-GFP-S431A-R), Quick change Site directed mutagenesis kit (Stratagene) was used  To investigate the effect of K8 phosphorylation on cell migration and tumorigenicity, the shRNA resistant K8-GFP wild type (K8-WT-GFP-R), K8-Ser73 mutant (K8-S73A-GFP-R) and K8-Ser431 mutant (K8-S431A-GFP-R) were overexpressed in K8-knockdown clone shRNAK8.2-C1. The stable K8-knockdown clones were generated as described previously Whole cell lysates were prepared in SDS lysis buffer  with protease inhibitors cocktail . Equal amount of protein was loaded and run on SDS-PAGE. The gels were transferred on PVDF membrane (Hybond Amersham) and probed with the primary antibody followed by secondary antibody conjugated with HRP (Amersham). The primary antibodies used were as follows: K8 , K18 , GFP , Keratin8-pSer73 , Keratin8-pSer431  and β-actin . The signals were detected using ECLplus detection system (Amersham) according to manufacturer's protocol.To detect the localization of proteins and filament organization of keratins in cells, immunofluorescence assay was performed as described previously 5′-AGATGAACCGGAACATCAGC-3′, reverse 5′-AGCAGCTTCCTGTAGGT- 3′ and GAPDH forward 5′-GAAGGTGAAGGTCGGAGTC-3′ reverse 5′-GAAGATGGTGATGGGATTTC-3′. PCR conditions were as follows: denaturation at 94°C for 30 s annealing at 56°C and 58°C for Keratin 8 and GAPDH respectively for 30 s and extension at 72°C for 60 s. PCR products were run on agarose gel electrophoresis to compare RNA levels.RNA was isolated by TRI reagent (Sigma) and RT-PCR was conducted using RevertAid™ First Strand cDNA synthesis Kit (Fermentas) according to manufacturer's protocol. The following primers were used, Keratin 8-forward To check the effect of loss of K8 phosphorylation on cell motility, differential distribution of K8 phospho-mutants and wild type expressing cells was analyzed using inverted fluorescence microscopy. The cells were seeded in 35 mm tissue culture plates in selection medium and allowed to grow for 48 hrs. The GFP expression was analyzed and images of 5 different fields of the clones were taken using an AxioCam MRm camera. Wound-healing assay was performed as described previously. Briefly, the cells were grown in 35 mm plates to 95% confluency and were replaced with fresh medium containing 20 µg/ml mitomycin C to inhibit cell proliferation. After 3 hr incubation, medium was discarded and wounds were scratched with the help of sterile 2 µl pipette tip. The cells were fed with fresh medium and observed by time lapse microscopy, and images were taken every 10′ for 20 hr. Migration was measured using Axiovision software version 4.5 (Zeiss).6 cells were injected sub-cutaneously in the dorsal flank of 6–8 weeks old NOD-SCID mice. Six mice were injected for each clone and observed for 60 days for tumor formation. The ellipsoid volume formula 1/2 x L x W x H was used to calculate the tumor volume To test the tumorigenicity of the cells the NOD-SCID mice (6–8 weeks old) were used. AW13516 clones (both K8 wild type and phospho-mutants overexpressed cells) were suspended in plane IMDM without serum, 3×10Tumor tissues were collected from the Tata Memorial Hospital (TMH), Parel, Mumbai, India. A total of 52 K8 positive primary oral SCC  were selected for IHC analysis. Five punch biopsy samples of patients suffering from inflammatory fibrous hyperplasia of oral cavity  were obtained from Nair Dental College, Mumbai, India. The patients had no history of malignancy. These tissues were used as controls. Clinico-pathological information like tumor site, size, histological grade, perineural invasion, perineural extension, cut margins, bone and lymph node involvement, loco-regional recurrence, metastasis and survival was collected from the clinical and pathology records of Tata memorial Hospital  for histological diagnosis. Immunohistochemical staining was performed using Elite ABC Kit  as described earlier Chi Square test was used. Univariant analysis was performed using the Kaplan-Meier method and statistical significance between survival curves was assessed by the log rank tests. The data were analyzed with the Statistical Package, SPSS 16.0 for Windows . Two groups of data were statistically analyzed by t test using Graphpad Prism5 software. A P value less than 0.05 was considered statistically significant.To assess correlations between clinico-pathological parameters and results of IHC, the Figure S1IHC analysis of K8 expression in malignant and non-malignant human oral tissues. Representative images of H&E and IHC staining using antibody against K8 on paraffin embedded sections of human oral tumors and non-malignant tissues. Note that non-malignant oral tissues showed negative staining while malignant tissues showed positive staining of K8.(TIF)Click here for additional data file.Figure S2IHC staining of phosphorylated K8 in human OSCC. Representative images of H&E staining along with positive IHC staining with antibodies specific to phosphorylated Ser73 and Ser431 of K8 on paraffin embedded sections of human OSCC.(TIF)Click here for additional data file.Table S1Clinico-pathological parameters of OSCC patients (n = 52).(DOC)Click here for additional data file.