Leishmania major infection in a susceptible mouse strain. Thus, for the first time, using CD40 signaling as a model, we show how a bi-modular assembly of kinases imposes reciprocity to a receptor signaling. The findings unravel that the signalling plasticity is inherent to a reciprocal system and that the principle can be used for designing a therapy.Depending on the strength of signal dose, CD40 receptor (CD40) controls ERK-1/2 and p38MAPK activation. At low signal dose, ERK-1/2 is maximally phosphorylated but p38MAPK is minimally phosphorylated; as the signal dose increases, ERK-1/2 phosphorylation is reduced whereas p38MAPK phosphorylation is reciprocally enhanced. The mechanism of reciprocal activation of these two MAPKs remains un-elucidated. Here, our computational model, coupled to experimental perturbations, shows that the observed reciprocity is a system-level behavior of an assembly of kinases arranged in two modules. Experimental perturbations with kinase inhibitors suggest that a minimum of two trans-modular negative feedback loops are required to reproduce the experimentally observed reciprocity. The bi-modular architecture of the signaling pathways endows the system with an inherent plasticity which is further expressed in the skewing of the CD40-induced productions of IL-10 and IL-12, the respective anti-inflammatory and pro-inflammatory cytokines. Targeting the plasticity of CD40 signaling significantly reduces  A receptor can trigger physiologically distinct cellular fates in response to different strengths of an identical stimulus. For example, in T cells, accumulation of cytotoxic T lymphocyte antigen-4 (CTLA-4) at the immunological synapse is proportional to the strength of the T cell receptor (TCR) signal Logically, as the signal flows from the receptor to the nuclear targets st module (M1) were maximally phosphorylated at lower doses of the stimulus whereas the kinases in the 2nd module (M2) were maximally phosphorylated at higher doses of the stimulus. The experimental perturbation studies revealed the intrinsic plasticity that guides the systems signalling direction: inhibition of any kinase of M1 leads to inhibition of rest of the kinases of M1 and activation of all the kinases in M2, and vice versa. The mathematical model built to reproduce the dose-dependent reciprocal phosphorylation of the bi-modular assembly of kinases suggests that two negative feedback loops are indeed required to reproduce the reciprocal effects observed in the experimental perturbations. For the biological significance of the model, we argued that as the protozoan parasite Leishmania major exploits the plasticity by skewing the CD40 signaling towards ERK-1/2 phosphorylation at all doses, targeting the systems plasticity to redirect the signal in a reverse way to p38MAPK can be an effective immunotherapeutic strategy. We verified our hypothesis with the model-guided experimental perturbations in L. major-infected macrophages and in susceptible BALB/c mice.Here, our CD40 signal dose-response experiments with macrophages show that based on their phosphorylation profiles, the kinases can be clustered into two modules: the kinases in the 1Syk and lyn are the kinases recruited first to the ligand bound CD40 Next, to understand the dynamics of phosphorylation in a reciprocal system, we studied phosphorylation kinetics of syk, lyn and the terminal MAPKs, ERK-1/2 and p38MAPK . The dynUpon ligand binding, CD40 recruits syk and lyn in two different membrane domains in macrophages Both the equations represent that CD40 is not an enzyme but it acts as an essential activator of syk and lyn. This assumption is empirically driven from our previous observations that CD40 is required for the phosphorylation process of syk and lyn, but it cannot phosphorylate the kinases itself and requires various adaptor molecules (TRAFs) for the assembly of the kinases following which phosphorylation occurs Experimentally, three different doses of anti-CD40 antibody were used as standardized earlier As syk concentration is much higher than that of lyn The proposed feedback loop is based on the observation  and 2 thThe negative feedback was considered as a hyperbolic modification where n1 is Hill coefficient and KI1 is the kinetic parameter associated with the negative feedback. The Hill coefficient in the negative feedback considers the effect of plausible intermediates whose overall effect is the observed feedback between M1 and M2 kinases. The bi-modular system with one negative feedback responded to signals of different strengths in a fashion similar to that observed in the experiments. Although we largely reproduced the signal dose-dependent reciprocity in the CD40 signaling with the model with one negative feedback loop, the proposed architecture called for further experimental verification to tightly establish such a regulatory design. According to the systems design with M2→M1 feedback loop , inhibitIn the experimental inhibition of these kinases, we chose to use only the intermediate signal dose (3 µg/ml), as both M1 and M2 kinases had intermediate phosphorylation amplitudes at this dose  and 2; aThe negative feedback was similar to the feedback loop from M2 to M1 and was considered as a hyperbolic modifier (n13 is Hill coefficient and KI13 is the kinetic parameter associated to the negative feedback). Simulation of the updated model captured the signal strength mediated reciprocal phosphorylation of the bi-modular kinases .To understand how the inhibitions affect the dynamics of the reciprocal system, we experimentally stimulated the system for different periods when either ERK-1/2 or p38MAPK phosphorylation was inhibited. To verify the presence of the proposed feedback loops, effect of inhibition of the MAPKs on the phosphorylation amplitude of earliest activated/membrane kinases syk and lyn was measured. The experiments show that inhibition of ERK-1/2 phosphorylation enhances lyn phosphorylation and subsequent suppression of syk phosphorylation. The reverse effect on syk and lyn phosphorylation was observed when p38MAPK phosphorylation was inhibited. All the perturbation studies taken together, an inherent plasticity in the reciprocal system is discovered, owing to which the system can skew the direction of signal flow from M1 to M2 or vice versa. The plasticity emerges as a global property of the system due to its structural requirements for exhibiting the observed reciprocal behavior. To understand the significance of the systems plasticity, we used the updated model to make further predictions that could be compared to the experiments.Effect of experimental depletion of total concentration of three kinases Raf-1, PI3-K and p38MAPK is shown through siRNA inhibition studies. Leishmania-infected macrophages, ERK-1/2 phosphorylation is enhanced whereas p38MAPK phosphorylation is inhibited at any doses of anti-CD40 stimulation L. major plausibly targets the plasticity of the system to redirect the signal flow towards ERK-1/2 for ensuring its own survival. But, as our perturbation studies showed, flow of signal could be modulated to either direction due to systems plasticity. We examined whether we could experimentally target the plasticity to restore the ratio of IL-12 to IL-10.In Leishmania infected macrophages. Sensitivity plots  was used for infection.BALB/c mice from Jackson Laboratories  were bred in our experimental facility. All animal experimentations were performed following an approved animal use protocol [IAEC  approved protocol number EAF-110. This is under the central control of the “Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)” permit no. 7/1999/CPCSEA-09/03/1999]. P388D1, a macrophage-like cell line Thioglycolate-elicited macrophages were treated with an agonistic anti-CD40 antibody 5 cells/well were cultured in antibiotic-free RPMI-1640 supplemented with 10% FCS. Following manufacturer’s protocol, the macrophage cell line P388D1 was transfected with the siRNA by using Santa Cruz transfection agent (Sc-29528) and transfection medium (Sc-36868). The cells were lysed after 24 hr, 36 hr and 48 hr to assess the maximum inhibition of the protein expression using Western blot. The time point showing the maximum inhibition was chosen for a particular kinase. The siRNA effect was tested on the CD40-induced phosphorylation of kinases by Western blot and expression of IL-10 and IL-12 by reverse transcription-PCR using the primers: GAPDH sense, 5′GAGCCAAACGGGTCATCATC3′, GAPDH anti-sense, 5′CCTGCTTCACCACCTTCTTG3′; IL-10 sense, 5′CTGCTATGCTGCCTGCTCTT3′, anti-sense IL-10, 5′CTCTTCACCTGCTCCACTGC3′; IL-12p40 sense, 5′CTGGCCAGTACACCTGCCAC3′ and IL-12p40 anti-sense, 5′ GTGCTTCCAACGCCAGTTCA3′. Each sample was first amplified for mouse GAPDH to ensure equal and good quality cDNA input.In a 6-well tissue culture plate, 2×10The tyrosine kinase encoding cDNA was released from pCALyn plasmid by digestion with BamHI/XbaI (polished) and cloned into the BamHI/BglII (polished) sites of pSG5 expression vector . An expression cassette consisting of the SV40 promoter, an intervening β-globin intron, the lyn coding region and polyA was released from pSG5-Lyn upon SalI digestion. The said cassette was further sub-cloned at SalI site of the lentiviral vector LV-Neo, derived by incorporation of neo gene in the previously described self-inactivating (SIN) vector construct, and virus particles containing the transgene were generated by multi-plasmid transfections in HEK-293 cells Peritoneal macrophages were infected with Leishmania promastigotes at a 1∶10 ratio for six hours 6L. majorrd day of infection subcutaneously, with or without intra-peritoneal α-CD40 (50 µg/ml) every alternate day. Weekly footpad measurements and the parasite load upon sacrifice were performed as described earlier BALB/c mice were infected with 2×10The experiments were performed three times and the data from a representative experiment are shown. Western blot data was subjected to densitometry analysis and phosphorylation ratio (PR)  = , for individual kinase was calculated for each of the applied signal doses L, M and H . For theSignal flow through coupled phosphorylation and dephosphorylation in the kinase network were constructed and simulated following standard approaches The method used for sensitivity analysis was partial rank correlation coefficient analysis (PRCCA), a rank transformed linear regression analysis that is routinely used for analysis of systems with a nonlinear and monotonic relationship between the system inputs and outputs Equations given below captured the reciprocal regulation in the CD40 signaling. Differential equations in the right hand side of the equations capture the time evolution of the system. In the left hand side to the differential equations is the flux equations constructed assuming that a steady state was reached in the enzyme substrate complex during signal propagation.Thus for the reaction E + S = [ES] → P + E, the flux equations were constructed for the condition when [ES] = constant. Here, E = Kinase, S = substrate, P = phosphorylated kinases.Activation of lyn, syk, Raf-1 and PI3-K was considered to be activated upon single phosphorylation following the previous guidelines Derivation of the flux equations for syk and lyn phosphorylation was empirically driven. Exact mechanistic steps involved in CD40 triggered phosphorylation of syk and lyn are currently unclear, but it is known from our earlier studies that CD40 activates syk and lyn in different signalosome complexes in the cell membrane The biochemical steps that were assumed to capture syk phosphorylation in presence of its essential activator CD40 bound to the signalosome complex E1 is given as followsUnder the steady state assumptions of the enzyme-substrate complexes,Thus the flux equation of syk phosphorylation is given asv1 is the flux of syk phosphorylation and V1 is the Vmax associated to signalosome complex E1. The ki, i = 1–5 are the elementary reaction rates for the above reaction schema and in the equations above, In the equation above or as in the equation 1 above, Activation of MEK-1/2, MKK-1/2, ERK-1/2 and p38MAPK requires double phosphorylation The equations below show the dynamics of phosphorylation and dephosphorylation of all of the model kinases. The mass conservation equations for each kinase were given below the differential equations of the same. For example differential equation capturing Raf-1 phosphorylation is followed by the mass conservation equation for Raf-1.T is related to the amount of unphosphorylated kinase: K1-1(t) at any time t as K1-1(t) = K1T - K1_P (t).Since the absolute concentration of each kinase was assumed constant during the simulation time mass conservation equation could be used to determine the time evolution of the dependent variable. So, any phosphorylated kinase K1 at time t: K1_P (t), and its total concentration: K1Flux equations were constructed assuming steady state in the enzyme substrate complexes during the signal propagation Figure S1Phosphorylation profile of model kinases for different doses of CD40 stimuli, without any feedback loop. (A) Steady sate ERK-1/2-PP and p38MAPK phosphorylation profiles as a function of CD40 stimuli. (B) Steady state ERK-1/2-PP and p38MAPK phosphorylation profiles plotted against the steady state phosphorylation profile of syk-P and lyn-P, simulated for different strengths of CD40 stimuli. (C) Steady state phosphorylation profiles of syk-P and lyn-P as functions of CD40 signal strengths(BMP)Click here for additional data file.Figure S2The kinetics of phosphorylation of top layer kinases syk, lyn and bottom layer kinases ERK-1/2 and p38MAPK are shown from the model simulations with two trans-modular negative feedback loops. Ratio of phosphorylation  of the kinases at 5, 15, 30, 60 minutes is shown for the three doses L, M and H of CD40 signal strength.(BMP)Click here for additional data file.Figure S3Effect of experimental depletion of lyn concentrations by siRNA inhibition in P388D1, a macrophage cell line is shown. (A) The western blots show the phosphorylation ratio  of the kinases at uninhibited and siRNA mediated inhibition of the kinases. In the experiments, BALB/c-derived thioglycolate-elicited peritoneal macrophages were treated with the anti-CD40 antibody (3 µg/ml). (B) Densitometric analysis of the western blot results are shown to compare the relative phosphorylation of syk, lyn, ERK-1/2 and p38MAPK in unperturbed condition  and for the lyn siRNA condition .(BMP)Click here for additional data file.Figure S4Densitometric analysis of kinases overexpression and inhibition and cytokines production in uninfected and Leishmania infected macrophages. (A) Lyn overexpression changed syk, p38MAPK and ERK-1/2 phosphorylation in macrophages. (B) Lyn overexpression led to inhibition of IL-10 and enhancement of IL-12 productions in both uninfected (UIM) and infected macrophages (IM). (C) Syk inhibition using Piceatannol (PC) enhanced IL-12 production but reduced IL-10 production; in uninfected and infected macrophages. (D) Lyn inhibition using PP1 resulted in enhanced IL-10 and reduced IL-12 production.(BMP)Click here for additional data file.Figure S5CD40 signaling in macrophages phosphorylates different kinases reciprocally clustering them into two modules. Thioglycolate-elicited BALB/c-derived peritoneal macrophages, was stimulated with the indicated doses of agonistic anti-CD40 antibody (clone 3/23) for 15 minutes, followed by the indicated Western blots with the cell lysates for eight different phosphorylated and total kinases. Based on their dose-response behaviour they are categorized in to the two modules M1 and M2. The experiments were repeated thrice and one representative dataset are shown below. (TIF)Click here for additional data file.Figure S6The kinetics of phosphorylation of top layer kinases syk, lyn and bottom layer kinases ERK-1/2 and p38MAPK. Figures show that reciprocity is a dynamically conserved property during signal processing by the CD40 receptor. (TIF)Click here for additional data file.Figure S7Pharmacological inhibitor-mediated inhibition of the kinases recapitulates the role of reciprocity in skewing CD40 signaling. (A). CD40-induced phosphorylation profiles of the kinases in peritoneal macrophages after treatment with anti-CD40 antibody (3 µg/ml) and inhibitors of lyn (PP1), Syk (Piceatannol (PC)), PI3-K (Ly294002), Raf-1 (Radicicol (Rad)), ERK-1/2 (PD098059) or p38MAPK (SB203580) were assessed. Densitometry units from the western blots are summarized showing the reciprocal relationship between the kinases in two modules. The figures show the extent of inhibition achieved in one representative dataset. (TIF)Click here for additional data file.Figure S8Kinetics of syk and lyn phosphorylation during ERK-1/2 and p38MAPK inhibition. The blots clearly show that due to its inherent plasticity the reciprocal system enhances syk phosphorylation and inhibits lyn phosphorylation during p38MAPK inhibition, whereas, during ERK-1/2 inhibition the reverse effect was observed. Inhibitor doses used for the study were SB = 5 µg/ml and PD = 50 µg. SD–S3 shows the densitometric analysis values for the three studies performed.(TIF)Click here for additional data file.Figure S9siRNA-mediated inhibition of expression of the kinases recapitulates plasticity of the reciprocal system. Inhibition of kinase expression by siRNA reemphasizes the reciprocity in activation of kinases in the two modules. Inhibition of (A) Raf-1, (B) PI-3K, (C) p38MAPK by siRNA regulates the CD40-induced phosphorylation of the indicated kinases reciprocally in two modules in P388D1, a macrophage cell line. (D) Expression of the respective kinases subjected to siRNA inhibition. In the experiments, BALB/c-derived thioglycolate-elicited peritoneal macrophages were treated with the anti-CD40 antibody (3 µg/ml). (E) The extent of inhibition achieved for the three kinases during the siRNA studies is shown. (TIF)Click here for additional data file.Text S1Model building, parameters and concentrations of kinases used for the simulations of the reciprocal system.(DOC)Click here for additional data file.Dataset S1Results of Densitometric analysis is shown for 1] Dose-response studies 2] Pharmaceutical inhibitor mediated studies for 15 minutes stimulation 3] siRNA mediated kinase inhibition studies 15 minutes stimulation. The excel sheet shows the densitometric values of the “phosphorylated” and “total kinase” obtained from the western blots and the respective ratios for dose-response/inhibitor/siRNA treatment. Subsequently, mean of three studies is shown in sheet no.2. Results from sheet no. 2 are plotted. For the pharmaceutical inhibitors studies, the dose of inhibitor for which best results were observed was plotted, as highlighted with red colour. The table can be found as a separate excel datasheet attached with this manuscript.(XLS)Click here for additional data file.Dataset S2Results of Densitometric analysis for kinetics of phosphorylation at L, M and H of the applied strengths of CD40 stimulation are shown with their mean of three experiments and the corresponding standard deviations.(XLS)Click here for additional data file.Dataset S3Results of Densitometric analysis for kinetics of phosphorylation of syk and lyn when either ERK-1/2 or p38MAPK is inhibited. The inhibitor doses used were SB = 5 µg/ml, PD = 50 µM. Experiments carried out at strength M of CD40 stimulation are shown with their mean of three experiments, and their corresponding standard deviations.(XLS)Click here for additional data file.


O-mannosyltransferases (PMTs) catalyze the initial reaction of protein O-mannosylation by transferring the first mannose unit onto serine and threonine residues of a nascent polypeptide being synthesized in the endoplasmic reticulum (ER). The PMTs are well conserved in eukaryotic organisms, and in vivo defects of these enzymes result in cell death in yeast and congenital diseases in humans. A group of rhodanine-3-acetic acid derivatives (PMTi) specifically inhibits PMT activity both in vitro and in vivo. As such, these chemical compounds have been effectively used to minimize the extent of O-mannosylation on heterologously produced proteins from different yeast expression hosts. However, very little is known about how these PMT-inhibitors interact with the PMT enzyme, or what structural features of the PMTs are required for inhibitor-protein interactions. To better understand the inhibitor-enzyme interactions, and to gain potential insights for developing more effective PMT-inhibitors, we isolated PMTi-resistant mutants in Pichia pastoris. In this study, we report the identification and characterization of a point mutation within the PpPMT2 gene. We demonstrate that this F664S point mutation resulted in a near complete loss of PMTi sensitivity, both in terms of growth-inhibition and reduction in O-mannosylglycan site occupancy. Our results provide genetic evidence demonstrating that the F664 residue plays a critical role in mediating the inhibitory effects of these PMTi compounds. Our data also indicate that the main target of these PMT-inhibitors in P. pastoris is Pmt2p, and that the F664 residue most likely interacts directly with the PMTi-compounds.Protein  O-mannosylation, is an evolutionarily conserved form of post-translational modification for many proteins that enter the secretory pathway O-mannosylglycan biosynthesis pathways have been linked to congenital disorders like Walker–Warburg syndrome O-mannosylation is essential for cell wall integrity and cell viability O-mannosylation process occurs in the endoplasmic reticulum (ER), where a family of well-conserved protein-O-mannosyltransferases (PMTs) transfers the initial mannose residue from dolichol-phosphate-mannose (Dol-P-Man) to the serine or threonine residues of the nascent polypeptide chains PMT1, PMT2, and PMT4 subfamilies. Saccharomyces cerevisiae, the organism in which the O-mannosylation process has been studied most comprehensively, contains seven PMT genes (PMT1-7). Candida albicans and P. pastoris both have five PMT genes . In order to carry out their enzymatic functions, members of the PMT1 subfamily  needed to form heterodimers with members of the PMT2 subfamily , whereas Pmt4p, the sole member of the PMT4 subfamily, forms a homodimer In vitro, the Pmt1p-Pmt2p heterodimer is responsible for the majority (>80%) of the PMT enzymatic activities N-glycosylation scanning), a topology model for ScPmt1p has been proposed by Strahl-Bolsinger and Scheinost The attachment of mannose residues to the hydroxy oxygen groups of serine or threonine residues of the nascent polypeptide chains, commonly referred to as et al. reported the identification of a group of chemical compounds that inhibit PMT enzymatic activity in vitroC. albicansS. cerevisiae, they seem to block PMT activities of all three subfamily members P. pastoris also suggested that these rhodanine-3-acetic acid derivatives affected multiple PMT members, with preference to PMT1 and PMT2 gene products S. cerevisiae and C. albicansO-mannosylglycan modifications on heterologous proteins expressed in Ogataea minuta and P. pastorisIn 2004, Orchard P. pastoris strains, which are capable of producing recombinant proteins with human-like N-linked glycosylation, have become very attractive hosts for the production of therapeutic proteins N-glycosylation, many recombinant proteins expressed in P. pastoris and other yeasts are also subject to O-mannosylation whereby Ser and/or Thr residues are decorated with mannoses in chain lengths ranging from one to five O-mannosylglycans may interfere with heterologous protein expression, folding, stability and, more importantly, could give rise to altered pharmacokinetic profiles due to increased clearance O-mannosylation process needs to be tightly controlled. For this purpose, we have identified several chemical analogs with much improved PMT-inhibition potency, and have routinely used them in our fermentation processes to maximally reduce O-mannosylglycan modifications on our heterologously expressed protein products P. pastoris. In this study, we report the identification and characterization of a point mutation within the PMT2 gene. We demonstrate that this F664S point mutation resulted in a near complete loss of PMTi susceptibility, both in terms of growth-inhibition and O-mannosylglycan site occupancy reduction. Our results provided strong genetic evidence indicating that the PMT-inhibitors directly interact with Pmt2p, and that the region surrounding the F664 residue likely plays an important role in PMT function.Glycoengineered P. pastoris strain y19376 was grown in 40 ml YSD  liquid medium overnight at 24°C. Upon reaching an OD600 of 5, a 10 mL aliquot of culture was transferred into an empty 100 mm sterile Petri dish and treated, with the lid off, with 12 mJ/cm2 of UV irradiation using a Stratagene UV Stratalinker 2400 . After the UV treatment, the Petri dish was immediately covered with aluminum foil to prevent photo-induced DNA repair and the mutagenized cells were allowed to recover at 24°C for 3 hours in the dark. Two mL of the recovered y19376 was then centrifuged at 2000 rpm for 5 min in a SORVALL Legend XTR centrifuge (Thermo Scientific USA). The cell pellet was then re-suspended in 400 µL of 2% BMGY , 2% peptone, 0.34% yeast nitrogen base w/o amino acids and ammonium sulfate (YNB), 1%(NH4)2SO4 (w/v) and 4×105% biotin in pH 6.0 100 mM potassium phosphate buffer) media, and subsequently plated onto YSD agar plates containing 1 µg/mL, 2 µg/mL, and 4 µg/mL of PMTi inhibitor. After a 7-day incubation at 24°C, colonies were picked and re-streaked onto fresh PMTi-containing plates. Only the clones that displayed a continued PMTi-resistance were kept for further evaluation as PMTi-resistant mutants.UV mutagenesis was performed as described previously Early stationary phase cultures of each strain were first diluted in fresh YSD liquid media to OD600 of 0.05. Subsequently, 400 microliters of the diluted cell suspensions were transferred into a 96-deep-well plate, with each well containing a final concentration series of 100, 33.3, 11.1, 3.7, 1.2, 0.4, 0.14, 0.046, 0.015, 0.005, 0.0017, and 0 µg/ml of either PMTi-3 or PMTi-4 inhibitor. These PMTi-containing cultures were then incubated at 24°C in a shaking incubator  at 840 rpm, and after 32 hours of growth, the OD600 values were determined for each culture. Percent growth inhibition was defined as [OD600 at the particular PMTi concentration]÷[OD600 at 0 µg/ml PMT-inhibitor]×100.7 cells from each strain were mixed in a 50 mL Falcon tube for each mating reaction and then collected on the membrane surface of a vacuum filtration apparatus . Each filter was transferred with cells facing up, to a mating agar plate  and incubated for 5 days at 24°C. The mating reaction was stopped on day 8 by transferring each filter to a 50 mL Falcon tube, washing the mating pairs off with 4 mL YSD. Cells were incubated in a rotating shaker for 3 h at 24°C. The cells were then plated onto selective plates (YSD with 100 µg/ml zeocin and 0.5 mM arsenite) to select for zeocin-resistant and arsenite-resistant diploid strains.To generate diploid strains, zeocin-resistant y17156 and y17157 were mated with y19661 (arsenite-resistant) as previously described To make clones haploid, sporulation was performed. In preparation for sporulation, positive (diploid) mated clones were patched onto YSD plates and incubated at 24°C for 3 days. Thereafter, cells were patched onto a sporulation plate  and incubated for 4 days at 24°C. Subsequently, cells were scraped off and resuspended in 700 µL diH2O in 1.5 mL Eppendorf tube for each mated clone. For each tube, 700 µL of diethyl ether was added, the mixture was vortexed for 5 min and centrifuged at 6000 rpm for 1 min in a SORVALL Legend XTR centrifuge (Thermo Scientific USA). Approximately 1 mL of liquid was removed from the top, leaving ∼0.5 mL diH2O and the haploid cell pellet. The cell pellet was resuspended in the remaining diH2O. 50 µL and 200 µL of the sporulation mixture was plated out onto YSD plates and incubated at 24°C. To determine if clones retained PMTi resistance, the sporulated clones were replica plated onto YSD plates containing 4 µg/mL PMTi.PMT2 locus and contains the P. pastoris URA5 gene flanked by a pair of direct lacZ repeat sequences. On one side of the lacZ-URA5-lacZ cassette is a DNA fragment containing the open reading frame (ORF) encoding the PMT2 (F664S) point mutation, and on the other side by a DNA fragment from the 3′ region of the PMT2 gene. After SfiI digestion, the linearized pGLY5931 plasmid is transformed into a ura−P. pastoris strain. By double-crossover homologous recombination, the SfiI fragment of the pGLY5931 vector will delete the wild-type PMT2 ORF and replace it with the mutated PMT2 (F664S) gene.Plasmid pGLY5931 is an integration vector that targets the O-mannosylglycan site occupancy was quantified using the high-performance anion exchange chromatography with pulsed amperometric detection (HPAED-PAD) method described by Stadheim et al.O-mannose chains were: 1) chemically cleaved from the mAbs using alkaline elimination; 2) hydrolyzed into monosaccharide units (i.e. mannose and mannitol); 3) assayed by HPAEC-PAD to quantify the levels of the monosaccharide residues released from the mAb molecules. The alkaline elimination reaction converts, exclusively, the reducing terminal mannose (i.e. the mannose molecule attached to the Serine or Threonine residue) to a mannitol molecule. Therefore, there is a strict 1:1 relationship between mannitol molecules and the O-mannose chain numbers, regardless of the chain-lengths of the mannosylglycans. Determination of the average number of mannitol groups per mAb (H2L2) molecule gives a precise and accurate assessment of the O-mannosylation site occupancy.et al.P. pastoris strains used in this study are listed in Yeast transformations were performed using the standard electroporation method as described by Cregg in vitro inhibitors against CaPmt1p P. pastoris, we evaluated the susceptibility of two different P. pastoris strains against two different PMT inhibitors. The two strains are y8316, which has all the PMT genes  intact, and y19376, which is a pmt4Δ single mutant. The first inhibitor, PMTi-3 (5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid), is one of the original rhodanine-3-acetic acid derivatives described by Orchard et al.PMT inhibitors are rhodanine-3-acetic acid derivatives originally identified as P. pastoris. To this end, we randomly mutagenized y19376 by UV irradiation, and subjected the mutagenized cells to growth-inhibitory concentrations of PMTi-4. From approximately 107 UV-treated cells, we identified two independent mutants: y17156 and y17157. The y17156 mutant appeared to be extremely resistant to PMTi-4, increasing the IC50 by more than 5000-fold (from 0.02 µg/ml to greater than 100 µg/ml). In comparison, y17157 displayed a modest, but easily detectable improvement in its tolerability to PMTi-4 with a 25-fold increase in its IC50 values.To better understand the molecular mechanism of PMT-inhibition, and to gain potential insights for developing more effective PMT-inhibitors, we decided to identify PMTi-resistant mutants in C. albicans strains isolated from patients contained mutations in ERG11, or FKS1, the molecular targets of fluconazole or caspofungin, respectively PMT1, 2, 5, and 6 in both PMTi-resistant mutants. No mutations were found in any of these PMT genes for y17157, demonstrating that the resistance mechanism of this mutant did not involve mutations in the PMT genes. Since overexpression of efflux pumps to minimize intracellular drug accumulation is another common mechanism for drug resistance PMT2 coding region for y17156, the strain that exhibited the very strong PMTi-4 resistant phenotype  PMT2 F664S mutation might be the causative mutation responsible for the PMTi-resistant phenotype.To determine whether PMTi-resistance was dominant or recessive, we crossed y17156 with a sensitive, and non-mutagenized, strain y19661, and examined the diploid for its susceptibility to the PMTi-inhibitors. Interestingly, the diploid strain displayed an IC50 value comparable to the haploid y17156 for both PMTi-3 and PMTi-4, demonstrating that the diploid strain retained the strong PMTi-resistant phenotype. This suggests that the PMTi-resistant phenotype of y17156 is fully dominant. To evaluate the connection between this PMTi-resistant phenotype and the observed F664S mutation, we performed a random-spore analysis on the diploid strain, and followed the segregation patterns of pmt4Δ, LmSTT3D  to over 100 µg/ml (y28432). For PMTi-3, the IC50 values increased from 9 µg/ml to 59 µg/ml. These results demonstrated that the PMT2-F664S point mutation alone is sufficient for causing the PMTi-resistant phenotype. Furthermore, identification of the PMTi-resistant F664S substitution within the PMT2 gene strongly suggested a direct interaction between the PMTi inhibitor and the Pmt2p protein, most likely within a region very close to the F664 residue.To evaluate if the identified s strain . As expePMT2-F664S mutation affects the O-mannosylation process, we examined O-mannosylglycan modifications of two different recombinant monoclonal antibodies (mAbs) known to be O-mannosylated in yeast. These mAbs were expressed in a P. pastoris host that contained the F664S PMT2 gene. To this end, we utilized 1L-scale bioreactors to express both mAbs, using a standard P. pastoris fermentation process in the presence, or absence, of the PMTi-4 inhibitor. Upon completion of the fermentation, secreted mAbs were purified from the supernatant by protein A affinity chromatography, and then subjected to O-mannosylglycan site occupancy (average number of mannose chains attached to each mAb H2L2 molecule) and product titer determinations.To further evaluate how the PMT2 were heavily O-mannosylated with an average of 56 and 48 O-mannosylation events per each H2L2 molecules of mAb#1 and #2, respectively  in O-mannosylglycan site occupancy in vivo activity of the mutated Pmt2p (F664S) protein. In the presence of PMTi-4, the inhibitor dramatically reduced the O-mannosylglycan site occupancy for mAbs derived from the PMT2 wild type strains . Increasing the PMTi-4 dose (5-fold higher) reduced the O-mannosylglycan site occupancy slightly further to 1.8 for mAb#2. In contrast, PMTi-4 inhibitor exhibited very limited effects in strains containing the PMT2-F664S mutation with O-mannosylglycan site occupancy moving from 42 and 31 to 37 and 30 for mAb#1 and #2, respectively. With 5-times of the normal PMTi-4 dosage, mAb#2 produced in the F644S mutant strain remained extensively O-mannosylated, with an O-mannosylglycan site occupancy of 19 per molecule of mAb. These results demonstrated that the F664S mutation led to a near complete loss of susceptibility to the inhibitory effects of PMTi-4. Interestingly, we also observed a clear negative correlation between O-mannosylglycan site occupancy and mAb product titer , and to 600 mg/L (5x PMTi-4 dosage) for mAb#2. For strains containing the F664S mutation, PMTi-4 addition had a much less profound impact on product titer changing from 60 to 75 mg/L for mAb#1, and from 563 to 622 mg/L with 1x PMTi-4 dosing, and to 765 mg/L with 5x PMTi-4 dosing for mAb#2. It is of interest to note that in the absence of PMTi-4 inhibition, strains with the F664S mutation displayed higher product titers than the corresponding PMT2 wild type hosts, with 30% and 75% increase for mAb#1 and #2, respectively.In absence of PMTi-4, mAbs produced from strains with wild type ectively . From thct titer . The addO-mannosylation process. There are up to seven PMT genes encoding Pmt homologues in yeast, and the Pmt1p-Pmt2p heterodimer accounts for greater than 80% of the PMT enzymatic activities in vitroPMT2-F664S substitution as the causative mutation for the PMTi-resistant phenotype, and report the characterization of the effects of this mutation on the O-mannosylation of selected monoclonal antibodies expressed in these PMTi-resistant host strains.PMTs play essential roles in the PMT2 gene is the causative mutation for the PMTi-resistance phenotype.Mutant y17156 displayed an extremely strong PMTi-resistant phenotype: the UV-mutagenesis has led to a greater than 5000-fold increase in the IC50 values against the PMTi-4 inhibitor. As a matter of fact, we were unable to determine the minimum inhibitory concentration (MIC) for y17156, because at the highest PMTi-4 concentration tested (100 µg/ml), it still exhibited very robust growth. Because acquiring mutations within the target gene is one of the most common mechanisms for pathogenic fungal organisms to become resistant to anti-fungal drugs O-mannose chains attached on mAbs molecules. However, in the presence of 8 mM PMTi-4, the dosage that effectively eliminated ∼95% of the O-mannosylglycan site occupancy in strains containing wild type PMT genes, the F664S substitution completely blocked any inhibitory effect the PMTi-4 had on O-mannosylation. The O-mannosylglycan site occupancies observed from mAbs expressed from the F664S-containing hosts exhibited no detectable changes either without or with 8 mM PMTi-4 inhibitor . Consistent with this lack of reduction in O-mannosylglycan site occupancy levels, strains harboring the F664S mutation became extremely resistant to the growth inhibitory effects of PMTi-4 with IC50 values increasing to at least 100-fold in the wild type strain background, and over 5000-fold in the pmt4Δ strain background. Collectively, these results suggested that the F664S point mutation led to a partial loss of the enzymatic function of Pmt2p, and simultaneously resulted in a near complete loss of susceptibility to PMTi-4 inhibition. Furthermore, our data provided strong genetic evidence that, in P. pastoris, the PMTi-4 compound inhibited the O-mannosylation process predominantly through Pmt2p inactivation, and the observed strong PMTi-resistant phenotype is a direct consequence of the F664S mutation preventing or disrupting the Pmt2p-PMTi-4 interactions. Lastly, our results also indicated that the Pmt2p enzyme, or more precisely the Pmt1p-Pmt2p heterodimer complex, is responsible for the majority of O-mannosylglycan modifications for mAbs heterologously expressed from P. pastoris strains, because inhibition of the Pmt2p enzyme by PMTi-4 blocked most of the O-maanosylglycan modifications on the mAb molecules.We have shown that, in the absence of PMT-inhibitors, the F664S point mutation led to modest reductions (between 26 to 35%) in the average number of PMT genes are very well conserved from yeast to human. Members of the PMT1, 2, and 4 subfamilies are all multi-span integral membrane proteins, sharing very similar hydropathy profiles. Multi-span membrane proteins are very difficult to isolate in sufficient quantities and purity to permit crystallization. Consequently, like most multi-span membrane proteins, no three dimensional structures of any PMT enzymes have been obtained so far. Therefore, very little is understood about the active-site composition and substrate-binding characteristics of PMT-family enzymes, the structural elements participating in PMTi interactions, and how these inhibitor-protein interactions translate into enzymatic inactivation. To our knowledge, results reported in this study provide the first experimental evidence indicating that the region surrounding amino acid F664 participates in the interaction between the PMT-inhibitors and the Pmt2p enzyme. Based on the topology model proposed by Strahl-Bolsinger and Scheinost in vivo function.C. albicans, PMTi-3 inactivated the CaPmt1p enzyme in vitroPMT gene family in P. pastoris suggested that PMTi-3 preferentially inhibited PpPMT1 and PpPMT2 gene products in vivoS. cerevisiae PMTi-3 did not appear to have any preference to any PMT genes, rather, it broadly inhibited all PMT family members quite equally. Although the PMT2-F664S mutation resulted in significant resistance to both PMTi-3 and PMTi-4 inhibitors, it is worth noting that the F664S mutation responded to inhibitions by PMTi-3 or by PMTi-4 quite differently. For strains containing intact PMT genes , the PMTi-4 compound is a much more potent inhibitor than PMTi-3 ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid). It has been shown that, in n PMTi-3 . However


Systemic anthrax disease is characterized by vascular leakage pathologies. We previously reported that anthrax lethal toxin (LT) induces human endothelial barrier dysfunction in a cell death-independent manner with actin stress fiber formation and disruption of adherens junctions (AJs). In the present study, we further characterize the molecular changes in the AJ complex and investigate whether AJ structure and barrier function can be preserved by modulating key cytoskeletal signaling pathways. Here, we show that LT reduces total VE-cadherin protein and gene expression but the expression of the key linker protein beta-catenin remained unchanged. The changes in VE-cadherin expression correlated temporally with the appearance of actin stress fibers and a two-fold increase in phosphorylation of the stress fiber-associated protein myosin light chain (p-MLC) and cleavage of Rho-associated kinase-1 (ROCK-1). Co-treatment with ROCK inhibitors (H-1152 and Y27632), but not an inhibitor of MLC kinase (ML-7), blocked LT-induced p-MLC enhancement and stress fiber formation. This was accompanied by the restoration of VE-cadherin expression and membrane localization, and attenuation of the LT-induced increase in monolayer permeability to albumin. Together, these findings suggest the ROCK pathway may be a relevant target for countering LT-mediated endothelial barrier dysfunction. Bacillus anthracis. Many of the symptoms of systemic inhalational anthrax can be attributed to the action of anthrax toxin, which is made up of three secreted proteins: protective antigen (PA) and lethal factor (LF), which combine to form lethal toxin (LT), and edema factor (EF) which combines with PA to form edema toxin [in vivo by virtue of its 11-fold higher affinity for PA [Inhalational anthrax is a disease caused by inhaling spores of the gram-positive bacterium ma toxin ,2. PA bima toxin . Once inma toxin . LF is ama toxin ,6. Whiley for PA .in vivo [Systemic anthrax infection is often accompanied by profound vascular pathologies including edema, hemorrhage, pleural effusion, and vasculitis in animals and humans ,9,10,11.in vivo . This fiin vivo . Consistent with the vascular pathologies of anthrax, we previously reported that LT induces cell death-independent barrier dysfunction in primary human lung microvascular endothelial cell culture characterized by actin stress fiber formation and altered adherens junction (AJ) structure . VE-cadhE687C were kindly provided by Dr. Stephen H. Leppla  [Phosphate-buffered saline (PBS) and Hank’s balanced salt solution with calcium and magnesium (HBSS) were obtained from Invitrogen . The MLC kinase (MLCK) inhibitor ML-7, the ROCK inhibitors H-1152 (Rki) and Y-27632 (Y27) were purchased from EMD Chemicals . LF, PA, and inactive mutant LFsda, MD) ,25. ToxiGoat polyclonal antibodies to VE-cadherin  and ROCK-1 (C-19) , and rabbit polyclonal antibodies to ROCK-1 (H-85) , ROCK-2 , beta-catenin  and tubulin  were purchased from Santa Cruz Biotechnology . Rabbit polyclonal antibody to p-MLC (Thr18/Ser19)  was purchased from Cell Signaling Technology . Rabbit polyclonal antibody to MEK1  was obtained from Millipore . E687C and 500 ng/mL PA). Individual toxin components LF or PA did not alter endothelial morphology or barrier function [Primary human lung microvascular endothelial cells were obtained from Lonza  and cultured as described previously . For inhfunction .2), the FITC-HSA concentration was multiplied by the volume of the lower chamber and divided by the membrane area and the FITC-HSA incubation time.Cells grown to confluence on porous membrane inserts  were treated as described above. After 72 h, 50 µL of culture medium from the upper chamber was replaced with an equal amount of medium containing 5 mg/mL FITC-HSA . After 2 h, 20 µL samples were drawn from the lower chamber and diluted 10-fold. Data were collected from duplicate inserts per treatment in each experiment. Fluorescence measurements were obtained using a microplate reader  with excitation and emission filters of 485 and 535 nm, respectively. FITC-HSA concentrations were calculated using a FITC-HSA standard curve. To quantify the trans-membrane flux  and anti-VE-cadherin (1:200) for 1 h at room temperature. Immunofluorescence analysis using Alexa Fluor 555-labeled secondary antibodies (1:800 dilution) and Alexa Fluor 488-labeled phalloidin (1:100 dilution) to stain filamentous actin (Invitrogen) was performed as described previously . Nuclei CDH5 (VE-cadherin), GAPDH, and CTNNB1 (beta-catenin). Reactions were performed in triplicate and run on the Applied Biosystems 7900HT real-time PCR system. Fold gene expression was calculated using the 2-∆∆CT method using GAPDH as the reference gene [RNA extraction and gene expression procedures were described previously . Gene exnce gene .Whole cell extracts were prepared as described previously using ice cold RIPA lysis buffer  containing protease inhibitors  and the phosphatase inhibitors NaF (1 mM), sodium orthovanadate (1 mM), sodium pyrophosphate (2.5 mM), and beta glycerophosphate (1 mM) . Reduced samples were run on NuPAGE 4-12% gradient Bis-Tris gels in MOPS SDS running buffer. Proteins were transferred to PVDF membranes and detected as described previously . Densitopost-hoc Student’s t-test using the JMP (version 7) software . p < 0.05 was considered statistically significant.Data are represented as means ± SE for replicate experiments. Statistical analysis was performed by ANOVA with We previously reported that LT disrupts AJ integrity in endothelial cells . To furtWe previously showed that LT-induced changes in AJs were accompanied by actin stress fibers . Actin sMLC phosphorylation is primarily catalyzed by myosin light chain kinase (MLCK) and ROCK . To examC-terminus domain of ROCK-1 has previously been shown to generate a constitutively active 130 kDa fragment [C-terminus of ROCK-1. This antibody recognized the 160 kDa band, but not the cleavage product in LT-treated cells. Notably, the cleavage band was not detected at 24 h suggesting a temporal correlation between the appearance of cleaved ROCK-1 and enhanced p-MLC. MEK1 cleavage analysis confirmed intracellular LT activity at both time points . At 72 h, a cleavage band was detected in LT-treated cells when using an antibody raised against an internal sequence of ROCK-1 (H-85). This cleavage fragment was not observed in non-treated cells or mutant LT-treated cells. Proteolytic cleavage of the inhibitory fragment ,32. To fC. At 72 We hypothesized that modulating the formation of actin stress fibers could preserve VE-cadherin contacts. To test this hypothesis, we examined the effect of ROCK and MLCK inhibitors on VE-cadherin localization and expression. In non-treated cells, VE-cadherin localized to intercellular junctions exhibiting a continuous and intense cortical staining pattern A. After In separate experiments, we investigated whether LT-induced AJ disruption could be ameliorated by inhibiting ROCK activity after LT exposure. Cells were treated with LT for 24 h prior to the addition of Y27. Similar to the inhibitor pretreatment experiments, Y27 prevented morphological changes and rescued VE-cadherin localization after 72 h (data not shown). The protective effect of ROCK inhibition on endothelial morphology and VE-cadherin contacts was particularly evident at time intervals beyond 72 h. After 120 h, LT-treated monolayers and VE-cadherin contacts were mostly disintegrated . Rki andWe next examined whether the preservation of AJ integrity by ROCK inhibition translates to attenuation of barrier dysfunction. LT produced a two-fold increase in monolayer permeability to albumin. LF, PA, or mutant LTm had no effect on albumin flux. Rki and Y27, but not ML-7, significantly inhibited the LT-mediated increase in monolayer permeability to albumin . TogetheVascular leakage pathologies such as pleural effusion, edema, and hemorrhage are hallmarks of anthrax pathogenesis ,9,10,11.Endothelial AJs play a major role in regulating barrier function . We founDue to the dynamic and selectively permeable nature of the endothelial barrier, several intrinsic signaling pathways function to modulate the barrier in cases where controlled passage of proteins and leukocytes is beneficial. Infectious and/or inflammatory agents often trigger these same mechanisms to induce pathological barrier dysfunction. One of these mechanisms involves rearrangement of the actin cytoskeleton from barrier-protective cortical actin to stress fibers, a common consequence of bacterial cytotoxins . MLCK anet al. reported no differences in p-MLC in LT-treated cells over the course of 4-24 h. In our cell culture system, LT produced no changes in p-MLC after 24 h, but a two-fold enhancement after 72 h. While the present findings suggest a role for ROCK in LT-mediated endothelial dysfunction, the precise mechanisms have yet to be defined. An important experimental limitation in our study was that the ROCK inhibitors alone produced profound effects on the actin cytoskeleton. Moreover, under certain conditions, these inhibitors may nonspecifically inhibit other protein kinases such as PKA and PKC . Thus, tThe ROCK family consists of two closely related serine/threonine kinases, ROCK-1 and ROCK-2, both of which are highly expressed in the endothelium ,37. The Rolando and colleagues showed that LT does not activate the small GTPases RhoA, Rac and Cdc42 . HoweverAntibiotic therapy is currently the primary option to treat anthrax. However, antibiotics are not effective in treating the later stages of an infection due to substantial toxemia ,43,44. Tin vitro. Here, we show that inhibition of ROCK ablates LT-induced morphology changes and dysregulation of AJs, a major barrier-regulating structure. Importantly, ROCK inhibition also provided significant protection against LT-induced barrier dysfunction. Together, these findings provide important insight into the profound endothelial dysfunction associated with anthrax and may be helpful in designing vascular-targeted therapies to halt disease progression and prolong the therapeutic window for countering the bacteremia.Vascular leakage pathologies such as pleural effusion, edema, and hemorrhage are hallmarks of anthrax pathogenesis. Emerging evidence points to the endothelial lining of blood vessels as a critical pathogenic target. Anthrax LT, the major virulence factor of anthrax, triggers human endothelial barrier dysfunction


Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae) was first released against Bactrocera latifrons (Hendel) (Diptera: Tephritidae) in Hawaii in March 2003. Over a three month period, eight releases, totaling 7,696 females and 3,968 males, were made in a turkeyberry, Solanum torvum Swartz  patch known to have a well established B. latifrons population. The establishment of D. kraussii was assessed through fruit collections conducted over a three-year period beyond the last release. D. kraussii was recovered 2 weeks, 31 months, and 39 months after the last parasitoid release, with collections not only from the release site, but also from a control site about 5.0 km distance from the release site. Recovery from fruit collections three years after the last parasitoid release confirmed that D. kraussii had become established in Hawaii. Parasitism rates were low, only 1.0–1.4%, compared to rates of 2.8–8.7% for the earlier established egg-larval parasitoid, Fopius arisanus (Sonan). Bactrocera latifrons (Hendel) (Diptera: Tephritidae) is the fourth and most recent invasive tephritid fruit fly species of economic importance to become established in Hawaii. It was first reported in Hawaii in 1983 , Ceratitis capitata (Wiedemann), and B. dorsalis (Hendel)) , the primary parasitoid found, only about 5.2% (Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae), a new parasitoid species with the potential to improve biological control of B. latifrons.A number of classical biological control programs were conducted against the first three invasive tephritid fruit fly species established in Hawaii )) e.g., , but no  Hawaii. . Prelimiout 5.2% . TherefoD. kraussii is an Australian parasitoid of Bactrocera tryoni (Froggatt) and several other endemic tephritids , a plant known to be a good host of B. latifrons  was shipped to the quarantine facility of the Hawaii Department of Agriculture in Honolulu, Hawaii, from a laboratory colony in Queensland, Australia. From this cohort, a small colony  of D. kraussii was maintained in quarantine for about three years until approval to release was given in 1998. D. kraussii was then removed from quarantine and reared in insectaries on the islands of Kauai and Oahu, using B. latifrons as a rearing host. B. latifrons larvae were provided by the USDA-ARS Pacific Basin Agricultural Research Center, Honolulu, Hawaii. Adult parasitoids were maintained in the insectary at 28 ± 2° C, 60–80% RH, with a 12:12 L:D photoperiod, in 25 × 25 × 25 cm wooden screened cages, and given water and spun pure honey ad libitum. Subsequent parasitoid colonies were initiated using oviposition units made of 9 cm diameter plastic petri dishes with tight-fitting lids covered with organdy, through which the wasps could oviposit  with 150 g fresh wheat diet  were collected from the rearing cages at the Kauai Agricultural Research Center and the Honolulu laboratory on Oahu by aspiration into 20 cm3 plastic vials, each containing a strip of filter paper with a drop of pure spun honey. Each vial held an average of 25 individuals (range: 12–31). The vials were closed with a gauze cover. If necessary , they were stored overnight at 6 ± 2° C before being transported in insulated boxes to the release site. All releases were done within one to two hours after boxes were picked up from the air cargo station at the airport in Kahului, Maui, or two to three hours after packaging (unless overnight storage was needed).Adult B. latifrons in Hawaii . Additional patches of turkeyberry were present in the vicinity of the release site. Patch size averaged about 457 m2, ranging from about 93 m2 to 2190 m2. Relative size and locations of all patches are illustrated in The release patch at the release site j-11 in , where aD. kraussii individuals released were from about the 85th generation from the initial cohort received in Honolulu. The numbers of parasitoids released on each of the eight shipment dates are presented in D. kraussii releases were made (“release patch”) is indicated in All releases were conducted between 11 March 2003 and 20 May 2003 in the thicket of turkeyberry plants at the release patch j-11 in . ReleaseB. latifrons populations were monitored at 13 separate turkeyberry patches at the release site, and 10 separate turkeyberry patches at the control site, through the use of Jackson traps baited with 2.0 mL alpha-ionol -3-buten-2-ol, obtained from Bedoukian Research, Inc., http://www.bedoukian.com/) and 1.0 mL rectified cade oil  deployed on separate 3.8 cm long × 1.0 cm diameter cotton wicks, each enclosed in separate plastic baskets hung from the middle of the trap hanger. The bait is attractive to sexually mature male B. latifrons adults, with males exhibiting 50% and 75% of peak response at 7 and 14 days old, respectively .Adult ectively . A singlD. kraussii became established, regular fruit collections of yellow (ripe) turkeyberry fruits were initiated on 14 January 2003 (control site) and 11 February 2003 (release site), eight and four weeks, respectively, before the first D. kraussii release, and were continued every two weeks until 10 August 2004 (over one year after the last release). Green (unripe) turkeyberry fruits were found not suitable for infestation by B. latifrons . Fruits were counted, weighed, and then placed in screened containers, which held sand as a pupation medium. The sand was sieved through a strainer weekly for four successive weeks in order to recover pupariating larvae and puparia, which were then held individually in separate containers for recovery of emerging flies and parasitoids. All species of flies and parasitoids that were recovered were identified and counted. Parasitoid identifications were initially made in the laboratories of Aimé H. Bokonon-Ganta and Grant T. McQuate based on locally established keys (unpublished) and prior experience, and were subsequently confirmed by Mohsen Ramadan, Hawaii State Department of Agriculture.In order to assess whether atifrons  and wereD. kraussii individuals, of which 65% were female, were released between 11 March 2003 and 20 May 2003. Mortality during transport was very low . B. latifrons populations were typically highest in late summer to early winter, and lowest in late winter to early spring, when there was a lapse of flowering and fruiting of turkeyberry. B. latifrons populations were low, but present, over the course of the D. kraussii releases at both the release patch and at the other turkeyberry patches at the release site  and 23 February in 2004 (1149 puparia/kg fruit). Peak pupal recovery averaged over all turkeyberry patches at the release site came a bit earlier in 2003 (285 puparia/kg ripe fruit on 25 February) and a bit later in 2004 (717 puparia/kg ripe fruit on 5 April)  . At the rol site .D. kraussii were observed searching on turkeyberry leaves and fruits at the release site. Further evidence of D. kraussii presence came through larval recoveries from ripe fruit collections. B. latifrons, other tephritid fruit fly species, D. kraussii, and other parasitoid species. The six D. kraussii reported in D. kraussii release, with two females and one male recovered from patch j-7, one female recovered from patch j-8, and two females recovered from patch j-9. No D. kraussii were recovered again until 7 December 2005, when one male was recovered from the control site, located about 5.0 km from the release site. Because of the 7 December recovery, it was apparent that additional collections were needed to further verify establishment. Consequently, three additional collections were made in 2006. In the third collection (15 August), one male and one female D. kraussii were recovered from patch j-8 at the release site, and three males and two females were recovered from the control site. Based on the 2005 and 2006 recoveries, it was concluded that D. kraussii was established at multiple sites as a parasitoid of B. latifrons. The parasitism rate, though, is quite low at both sites. Using data from the 2006 collections where D. kraussii was recovered at both treatment and control sites, the parasitism rate of D. kraussii  was 1.0% (5/494 B. latifrons puparia) at the control site and 1.4% (2/138 B. latifrons puparia) at patch 8  at both sites (0.8% (4/494 B. latifrons puparia) at the control site and 1.4% (2/138 B. latifrons puparia) at patch 8 at the release site). However, the rate was considerably less than that of F. arisanus (2.8% (14/494 B. latifrons puparia) at the control site and 8.7% (12/138 B. latifrons puparia) at site 8 at the release site).Two weeks after the first release, adult female  8 j8 in  at the rD. kraussii for biological control of B. latifrons in Hawaii. The success was achieved on turkeyberry, which is known to be a good host and is present as an invasive weed over extended areas. Although a number of other B. latifrons hosts are known  released in Taiwan, which resulted in the successful recovery of the wasp  were also reported for D. kraussii following the larger releases made in Israel against C. capitata  fruit sampling may have caused a considerable reduction in the site density of fly larvae that could have been available for the parasitoids and resulted in low estimates of subsequent parasitism , which were not sampled   sampled . The cloe ground . In thatD. kraussii was recovered 5 km from the release site. This dispersal might have been achieved through movement by the wind or transportation of fallen turkeyberry fruits by water or birds. This dispersal was in the range of 0.8 km/yr to 6.7 km/yr recorded by various authors for Cotesia rubecula, an introduced parasitoid of Pieris rapae (L.) , and Diadromus collaris (Gravenhorst) spread 100 km/yr, 110 km/yr, and 105 km/yr, respectively. Other examples of rapidly dispersing microhymenoptera include the encyrtid parasitoids Apoanagyrus (Epidinocarsis) lopezi De Santis, which covered 100 km per season  . Howeverwo years . Additiowo years  reportedr season , and Gyr km/year .D. kraussii over the period extending three years after the last release suggests the need to reassess the status of establishment continuously in order to determine whether the low level of parasitism observed increases with time and starts to have significant impact on B. latifrons populations. Beyond making future assessments at the sites used in this study, additional releases could be tried under alternative conditions that may be conducive to more successful establishment, such as making releases at times of high B. latifronspopulation levels (B. latifrons host plant areas, or making releases in localities in Africa where B. latifrons has recently colonized (B. latifrons may better be achieved through augmentative releases of another, already established, parasitoid species, such as F. arisanus, which naturally established itself as the predominant parasitoid in Hawaii (The observed low level of parasitism by n levels , releasiolonized  and othe


We estimate that the carbon sequestration potential between 1990 and 2050 would be 9.4–35.7 Mg ha−1 under the current high manure application at the three sites. Analysis of SOC in each carbon pool indicates that long-term fertilization enhances the slow pool proportion but decreases the passive pool proportion. Model results suggest that change in the slow carbon pool is the major driver of the overall trends in SOC stocks under long-term fertilization.Soil organic matter models are widely used to study soil organic carbon (SOC) dynamics. Here, we used the CENTURY model to simulate SOC in wheat-corn cropping systems at three long-term fertilization trials. Our study indicates that CENTURY can simulate fertilization effects on SOC dynamics under different climate and soil conditions. The normalized root mean square error is less than 15% for all the treatments. Soil carbon presents various changes under different fertilization management. Treatment with straw return would enhance SOC to a relatively stable level whereas chemical fertilization affects SOC differently across the three sites. After running CENTURY over the period of 1990–2050, the SOC levels are predicted to increase from 31.8 to 52.1 Mg ha Soil organic carbon (SOC) is one of the most important terrestrial pools for C storage. It is estimated that the total soil carbon pool is around 1400–1500 Pg C, which is approximately three times greater than the atmospheric pool (750 Pg C) Long-term experiments are crucial for determining fundamental crop, soil and ecological processes and their impacts on the environment Process-based models are an effective way to evaluate SOC changes across a broader set of environmental conditions and management practices Here, we evaluate the CENTURY with data from three long-term experiments with wheat-corn cropping rotations and different fertilization practices. Specifically, our objectives were (i) to evaluate the performance of CENTURY with evaluation of modeled SOC stocks for different fertilizations and under acidic soil; (ii) to study the effect of fertilization practices on different SOC pools in the modeling framework; and (iii) to predict soil carbon potential under long-term fertilization.Three long-term experiments were utilized for this study, which were located at Changping , Yangling  and Qiyang  in China. Climate conditions varied from semi-humid (Changping site) to humid warm-temperate (Yangling sites) to humid subtropical climate (Qiyang site). Annual mean temperature was 13.1°C at the Changping site, 14.9°C at the Yangling site, and 18.1°C at the Qiyang site. Annual precipitation was generally low at Changping (515 mm) and Yangling (525 mm) sites but 1445 mm at Qiyang. However, annual evaporation was much higher, varying from 993 mm to 1470 mm th, and harvested around June 1st. The wheat seeding rates ranged from 165 to 225 kg ha−1. Summer corn was sown around June 10th, with the rate of 63 000–75 000 seeds per hectare, and harvested around October 1st at most sites. For the Qiyang site, summer corn was sown in holes between the wheat strips in early April, and harvested in the middle of July. Thus, there was a short period of inter-cropping for wheat and corn at the Qiyang site. However, in the CENTURY modeling, wheat was harvested in April and corn was sown in May at the Qiyang site since inter-cropping could not be fitted in the model. We believed that there would be little impact on the belowground biomass of wheat and corn, and thus to the soil organic carbon turnover.The experimental sites had double cropping systems, i.e., winter wheat and summer corn. Winter wheat was seeded in early November and harvested in early May at the Qiyang site. For the other two sites, winter wheat was seeded around October 202–400 m2) at each study site. The durations of experiments were from 1990 to 2005 at the Changping site, from 1990 to 2008 at the Qiyang sites, and from 1990 to 2009 at the Yangling site.Seven treatments were selected in this study: (i) control (no fertilizer); (ii) mineral nitrogen (N); (iii) mineral nitrogen and phosphorus combination (NP); (iv) mineral nitrogen, phosphorus and potassium combination (NPK); (v) NPK combinations with livestock or farmyard  manure (NPKM); (vi) 1.5 times’ application rate of NPKM (hNPKM); and (vii) mineral NPK combined with crop straw (NPKS). The total nitrogen applied  was equal  in each of the fertilizer treatments . The NPKM and hNPKM treatments had 30% of total N applied from mineral fertilizer and the remaining 70% from organic manure. The treatment plots were initially randomized and isolated by 100-cm-cement baffle plates. There was no replicate for the treatments at these sites due to field availability. However, the plot size was relative large (196 m−1 yr−1) of both wheat and corn straw was incorporated at the Qiyang site, whereas 2250 kg ha−1 yr−1 and 4500 kg ha−1 yr−1 of corn straw were incorporated at the Changping and Yangling sites, respectively Urea, calcium superphosphate, and potassium chloride were used as sources of mineral N, P, and K, respectively. All P and K fertilizers and nearly half of the mineral N fertilizer were applied as basal fertilizers prior to seeding. The remaining mineral N fertilizer was applied as top dressing during the growing season. Manure was generally applied once each year before wheat sowing. However, at the Qiyang site, 30% of manure was applied before wheat seeding and remaining 70% was applied before corn seeding. The sources of organic manure were farmyard manure mixed with crop residue at Changping site, cattle manure at Yangling site, and pig manure at Qiyang site There was no irrigation at the Qiyang site since sufficient precipitation occurs during the growing season . However, at the other two sites, irrigations were applied 2–3 times during wheat growing season: 5 mm before seeding, 4 mm for the over wintering stage if needed, and 5 mm at jointing stage. Irrigation was applied once during corn growing season, with 5 mm before seeding.Plots were ploughed to a depth of 20 cm twice a year, usually in early June at most sites after wheat harvest and early October after corn harvest. In contrast, at the Qiyang site, the whole plot area was ploughed (20 cm depth) in late October before wheat seeding. Then, area between the wheat belts was ploughed (20 cm depth) in early April before the corn seeding.Aboveground biomass was removed with negligible stubble left in the field. Thus, organic carbon input was mainly from manure application , residue addition  and from root system during crop growing season. All harvested biomass was removed from the plots for determining crop grain and residue yields. The grain and residue samples were air-dried, threshed, oven-dried at 70°C to a uniform moisture level, and then weighed separately.−1 was calculated by multiplying the SOC content (g kg−1) by averaged soil bulk density (g cm−3) and depth (20 cm).During the experimental periods, surface soil (0–20 cm) was collected in each plot annually approximately 15 days after corn harvest. Five cores of soil from each plot were randomly taken, and soils were mixed thoroughly, and air dried for seven days after removing discrete plant residues and visible soil organism. Air-dried soil was sieved through 2 mm screen to determine pH (1∶1w/v water). Representative subsamples were crushed to 0.25 mm for measurements of SOC 2 evolved in the transformations was indicative of the microbial growth efficiencies. The first-order decay rates for each of the pools corresponded to turnover times of roughly 3 and 0.5 years for the structural and metabolic components; 1.5 years for the active fraction, 25 years for the slow pool, and 1000 years for the passive pool The simulation of SOC dynamics was performed with the CENTURY model (version 4.5) which was described by Parton and Rasmussen −1 before the long-term experiment. Then carbon input varied based on measured data at different sites.Model simulations were set-up based on the historical crop rotations and farm practices’ investigation from the local farmers at these sites. Five distinct periods were modeled , includihttp://cdc.cma.gov.cn/). For the future simulations , we used monthly mean climate data from the 1990–2010 period. Soil properties parameters were soil texture , soil pH, and bulk density ) defined for each crop and 0–1 scalars depending on soil temperature, moisture status, shading by dead vegetation, and seedling growth. The maximum potential production of a crop, unlimited by temperature, moisture or nutrient stresses, was primarily determined by the level of photosynthetically active radiation, the maximum net assimilation rate of photosynthesis, the efficiency of conversion of carbohydrate into plant constituents, and the maintenance respiration rate pHf) in the crop growth sub-model to account for this effect:δ and β were the maximum  and minimum  pH value that effected plant growth, respectively.The CENTURY model had no function for pH effects on aboveground growth. Based on the 20 years of field measurement at the Qiyang site, soil pH had no impact on crop growth as long as the value remains above pH 5.5, and would completely stop growth in wheat and corn if the value declined to less than pH 4.0 i) the sample coefficients of determination (2R), (ii) the normalized-root mean square error  the index of agreement  with field measured values. Both visual examination of graphic output and several statistical tests were used to evaluate the CENTURY model performance. Four statistical parameters were selected  n-RMSE, , and  provided the percentage of data variance that was accounted for by the model. The RMSE evaluated the difference between observed and modeled values in the original units of the data, while the normalized-RMSE  ranging from 0.57 to 0.92 (p<0.05), indicating that CENTURY model simulates the grain yields with reasonable accuracy.We evaluated the model results for both grain yields and the SOC stock predictions. We were focusing on the SOC stock predictions of the model. In summary for the grain yield results , we founThe modeled SOC follows the trend of the measurements reasonably well in all treatments across the sites , and 6. P<0.001) under various fertilization treatments. Approximately 77%–84% of the variability in the observed SOC stocks can be explained by the simulation. Therefore, in our experiment, the CENTURY model performs well and is suitable for predicting SOC dynamics for different types of N fertilizer  and N rates. The coefficients of determination (2R) are higher at the Yangling and Qiyang sites than the Changping site.Linear regressions of observed vs. simulated SOC stocks  are all RMSE is useful for evaluation of precision in model results n-RMSE ranges from 4% to 7% at the Changping site, 9%–14% at the Yangling site, and 6%–11% at the Qiyang site, respectively. Similarly, Fallon and Smith n-RMSE ranging from 1.8% to 16.4% when they modeled SOC in several long-term experiments in Europe. Also Álvaro et al. n-RMSE (below 6%) when they used the CENTURY model to evaluate tillage effects on SOC. Overall, the n-RMSE value is less than 15% for all the treatments in our study, demonstrating that the model is relatively precise in predicting the trends for different fertilization practices that are common in Chinese agricultural systems.d values and coefficients of determination (2R) are highest in the NPKM, hNPKM and NPKS treatments at most sites. However, the 2R value was much lower in the control (∼0.07) or N (∼0.19) treatments. The reason was that inter-annual fluctuations in crop yield were larger in the control and N treatments due to the long-term nutrient deficiency The −1 across the sites .Long-term fertilization has different effects on soil organic carbon across the three sites . For exa−1 and 52.1 Mg ha−1 under the NPKM and hNPKM treatments, respectively. With straw return, the SOC level reaches 34.3 Mg ha−1. Balanced chemical fertilization (NPK) also yields relatively high SOC contents at 24.6 Mg ha−1 by 2050. There is little difference in SOC stocks between N and NP treatments. SOC increases to 20.1 Mg ha−1 in 1994, then declines to 15.3 Mg ha−1 in 2050 for the control treatment.For the Yangling site , SOC con−1, 44.2 Mg ha−1, and 28.2 Mg ha−1 for the NPKM, hNPKM, and NPKS treatments, respectively. For all chemical fertilization treatments, we assume no decrease in pH after year 2010. Therefore, soil pH has not negative impact on carbon input due to a reduction in crop production associated with pH, and applying NPK fertilizers maintain SOC levels at 21.7 Mg ha−1.Similar to the Yangling site, SOC levels increase through the entire period of simulation under the NPKM, hNPKM, and NPKS treatments for the Qiyang site . By 2050−1) and lowest at the Yangling site (16.4 Mg ha−1). In contrasts, the highest simulated SOC level in 2050 happens at the Yangling site and lowest at the Changping site. Compared with the NPKS treatment, carbon sequestration potential is greater with hNPKM management, ranging from 9.4 Mg ha−1 to 35.7 Mg ha−1. For the NPKS treatment, SOC increases to 17.9 Mg ha−1 at the Yangling site but only 7.1 Mg ha−1–8.1 Mg ha−1 at the other two sites.The SOC trends are relatively stable by the end of 2050 in the different treatments . In ordeStudies has proved that optimizing fertilization managements can raise crop yield as well as biomass, which would enhance biomass input into the soil from crop straw and roots −1 yr−1 to 298 kg ha−1 yr−1 with straw returning during the period of 1990–2050 under current returning rate of straw residue . The results are lower than the estimated rate (108–728 kg ha−1 yr−1) in the similar region by Lu et al −1 yr−1).Straw incorporation would also increase carbon input to the soil. Li et al −1–31.0 Mg ha−1) by 2013 . Analysis of the proportion of organic carbon in each of the soil organic matter pools indicates that long-term fertilization enhances the slow pool and leads to a decline in the proportion of carbon in the passive pool. Model results suggest that changes in slow organic matter drive the overall trends in SOC stocks associated with long-term fertilization.We used the CENTURY model (version 4.5) to simulate soil organic carbon (SOC) dynamics for long-term fertilization experiments with wheat-corn cropping systems in China. Our study indicated that CENTURY can simulate fertilization effects on SOC trends for different climatic conditions and soil properties that are common for wheat-corn systems in China. For most sites, the model was more precise in predicting the SOC trends for treatments with balanced nutrient additions  than the control and unbalanced fertilization treatments (N and NP). After simulating SOC dynamics from 1990–2050, the SOC levels increased from 31.8–52.1 Mg ha


IgE recognition of panallergens having highly conserved sequence regions, structure, and function and shared by inhalant and food allergen sources is often observed.We evaluated the IgE recognition profile of profilins , PR-10 proteins  and tropomyosins  using the Immuno-Solid phase Allergen Chip (ISAC) microarray system. The three panallergen groups were well represented among the allergenic molecules immobilized on the ISAC. Moreover, they are distributed in several taxonomical allergenic sources, either close or distant, and have a route of exposure being either inhalation or ingestion.+), 1,420 (45.6%) to PR-10 (92.5% Bet v 1 IgE+) and 632 (20.3%) to tropomyosins (68% Der p 10 IgE+). A significant direct relationship between different representative molecules within each group of panallergens was found. 2,688 patients (86.4%) recognized only one out of the three distinct groups of molecules as confirmed also by hierarchical clustering analysis.3,113 individuals  were selected on the basis of their reactivity to profilins, PR-10 or tropomyosins. 1,521 (48.8%) patients were reactive to profilins (77.6% Mer a 1 IgEUnless exposed to most of the allergens in the same or related allergenic sources, a preferential IgE response to distinct panallergens has been recorded. Allergen microarray IgE testing increases our knowledge of the IgE immune response and related epidemiological features within and between homologous molecules better describing the patients' immunological phenotypes. The prevalence of allergic diseases has dramatically increased in last decades, mostly in developed countries A major problem is represented by the possibility that positive results, employing extracts, could be caused by IgE recognition of homologous molecules instead of molecules that represent the genuine marker of sensitization to a given biological source Fagales order, whereas homologous from foods, like apple, carrot, celery, kiwi, are from quite distant taxonomical species. Tropomyosins are animal structural muscle proteins with a molecular weight of about 35 kDa. Tropomyosins are well described food allergens from crustaceans, but homologous allergens are found in arthropods not commonly used as food, like mites and cockroaches, causing allergic diseases by the inhalation route. Up to now 93 tropomyosins have been described as allergenic, 77 from shellfishes. All data on panallergens have been retrieved from Allergome , accessed April 30, 2011. This web-based platform can be used for more detailed and up to date descriptions of each allergen reported in the present study.Adverse reactions after the ingestion of animal- or plant-derived foods caused by IgE cross-reactive molecules shared by inhalant and food allergen sources are often seen in clinical practice The aim of our study was to bring a further and more in deep insight into the distribution of the IgE reactivity to three panallergen groups  in 3,113 individuals with a history of food allergy, rhinitis, asthma or atopic dermatitis, either looking at the reciprocal IgE positivity of each group compared to the others, or within each of the three panallergen groups. The study population is from Italy, extracted from a reported study The study was approved by the Institutional Review Board of Istituto Dermopatico dell'Immacolata – IDI-IRCCS, Rome, Italy (n. 106-CE-2005). The Institutional Review Board approved that the study is based on the oral informed consent for blood sampling obtained from patients or caregivers during the allergy consult, as IgE detection by microarray testing was part of the routine procedures in each patient diagnostic work-up. All patients, being informed by the specialist performing demographic and clinical data collection and suggesting them the IgE testing as reported in the present study, approved the study itself.Data herein reported have been extracted and further analyzed from the total of 23,077 subjects previously reported by us Three thousand, one hundred and thirteen individuals  were selected on the basis of their IgE reactivity detected using the Immuno-Solid phase Allergen Chip (ISAC) system . Candidates had to be IgE positive to at least one allergen of the following group of panallergens: profilins ; PR-10 ; tropomyosins . Selected subjects thus acted as our study group.Escherichia coli, with the exception of Ole e 2, Pen m 1, Pen i 1, which were naturally purified preparations. www.allergome.org) Glass microscopy slides, each bearing four reaction sites bearing immobilized allergens, as provided by the manufacturer (VBC-Genomics), were washed for 60 min in TBS-T buffer , rinsed with deionised water, and dried. Slides were placed into a humid chamber and 20 µl of undiluted serum from each patient was applied to the reaction site. After incubation with patient's sera for 120 min at R.T., slides were rinsed and washed for 15 min in TBS-T, for 5 min in deionised water and dried. To detect bound IgE antibodies, allergen chips were incubated for 60 min at room temperature with 20 µl of an Alexa Fluor 546 fluorescence-labeled  anti-human IgE antibody  diluted 1∶1000 in TBS-T containing 5% milk powder. Afterwards, slides were washed twice for 10 min with TBS-T, rinsed with deionised water, dried, and stored in the dark until scanning.A/l by interpolating the mean fluorescence value with a previously established reference curve Images were acquired by scanning allergen chips with a ScanArray Gx Microarray Analysis System  with two different laser power settings. In the first scan, 70% of the laser power (50 db) was employed to obtain the highest possible signal intensities with acceptable background noise. In a second scanning step, laser power was reduced to 35 db in order to prevent saturated signals (65.535). Both images of each spot were analyzed using the ScanArray Express 3.0 software (PerkinElmer). Captured images saved as a TIFF file were imported into the ISAC software (VBC-Genomics) which specifically process the raw fluorescence value expressed as fluorescence index of each spot. From the three fluorescence values obtained for each allergen on triplicate spots, the mean value was calculated and used for further analysis. IgE values were expressed as kUThough several ISAC biochips with different number of spotted allergens have been used during the study timeframe , we analyzed results from the 23 homologous molecules immobilized on all of them. For comparative purposes we used results available the same microarray testing for Phl p 1, Par j 2, and Der p 1, all considered genuine markers of sensitization to grass and parietaria pollen, and to house dust mite, respectively. The only route of exposure for all the selected genuine allergens is inhalation.To verify the molecule IgE recognition profiles, we took advantage of the Genesis software version 1.7.2, originally developed for cluster analysis of genomics data Relatedness of IgE recognition profiles was tested by applying unsupervised Eisen's hierarchical cluster methods Data collected in the InterAll database were also analyzed using the SPSS/PC + statistical package . The levels of the variables of interest in the serum (IgE) from allergic patients were summarized showing the mean ± SD, in order to provide the most easily interpretable summary measure. However, differences between the levels of these variables were also tested using the nonparametric Mann–Whitney U-test. The degree of relationship between the quantitative variables studied was analyzed using Pearson Correlation test (r). Chi-square test has been used for prevalence comparisons. Statistical significance cut-off level has been set for p<0.01.vs 30.4±16.1, Mann-Whitney Rank Sum Test p <0.0001). No major differences were recorded comparing to the median age of the entire studied population (30.8±18.3 years), whilst the gender distribution was different when compared to the original general population  from which our study group was extracted.A total of 3,113 subjects , showing an IgE reactivity to at least one molecule belonging to profilin, PR-10, and tropomyosin allergen groups, represented our study group. The median age was 30.8±17.04 years, but male subjects were significantly younger (28.3±17.5 2 test 4.8 p = 0.027), whereas the opposite was observed in the case of tropomyosin reactivity (χ2 test 5.36 p<0.021). A slight, not significant, prevalence of male population (51.4%) was observed in profilin reactive patients, as well. Unless profilin sequence identity were above 75%, IgE recognition ranked between 76.6% and 36.2%. A broader sequence identity was recorded within PR-10 molecules, having Dau c 1 (39%) the lowest sequence identity value. IgE prevalence was the broadest as well, ranging between 92.5% and 4.2%. Tropomyosins behaved as profilins, having the lowest sequence identity above 64%, but IgE prevalence ranged between 68% and 43%.As shown in The panallergens IgE-positive cohort has been divided in eight consecutive age groups . PatientTwo thousand, six hundred and eighty-eight patients (86.4%) recognized just one out of the three distinct groups of panallergens, with a preferential recognition behavior , Panel AThe association among the different molecules studied without distinction between independent and dependent variables was analyzed using the Pearson Correlation test. As shown in In the case of profilin and tropomyosin molecules, all the Pearson correlation coefficient resulted to be higher than 0.70, thus confirming a strong structural and immunological relationship between the tested molecules.Supervised two-way hierarchical clustering analysis, sorting for both allergenic molecules and IgE recognition values, generated distinct clusters of IgE recognition of profilin, PR-10, and tropomyosin molecules, as shown in In the present study we perform an in deep analysis of the prevalence of IgE reactivity to panallergens in a large cohort of allergic patients, evaluated by means of a microarray IgE detection system. We studied three groups of molecules belonging to different biological sources and tissues , involved at the same time in respiratory and food allergy. Such allergens have been reported as representative examples of IgE “cross reactivity” between otherwise unrelated organisms We found that panallergen IgE recognition is exclusive in a large part of our Italian cohort. More than 86% of patients produce specific IgE to a group of panallergens but not to the others. As a result, in the vast majority of cases, the multiple reactivity to inhalant or food allergenic sources was caused by the sensitization to at least one representative molecule of a single panallergen group. Similarly, tropomyosin reactive individuals did not raise an IgE response to other panallergens, as also shown by the hierarchical clustering analysis . ComparaThe use of the same approach, originally designed for genomic evaluation, in the epidemiological assessment of IgE reactivity to panallergens, might help in the management of large amount of data generated by serial proteomic evaluation of hundreds of molecules at the same time, in the same group of patients Mercurialis annua pollen is rarely found in the atmosphere Mercurialis profilins could act just as the best “profilin” reagents for specific IgE detection among the many profilins already identified. Better conclusions on this issue could be drawn when some profilins, like Cuc m 2, the muskmelon profilin, commonly causing patients' reported symptoms Furthermore, as in the case of profilin IgE sensitization, molecules not commonly found in the environment such as Hev b 8, hardly found in latex gloves Discovering that the production of IgE is preferentially addressed to a panallergen rather than another is relevant also considering the different clinical phenotypes that these molecules could determine. Clinical reactions of panallergens, in fact, might range from very mild symptoms in the case of profilin reactivity Another topic addressed by our study is represented by the possibility to evaluate the IgE reactivity to several representative molecules within each group of panallergens by means of the microarray Overall, allergy diagnosis based on allergenic molecules is crucial for a correct evaluation of patients sensitized to panallergens. Molecular analysis represent the only way to establish the presence of new profiles represented by profilin, tropomyosin or PR-10 reactivity, conditions that are addressed to a preferential recognition of one panallergen, in the vast majority of cases, as suggested by our observations.In conclusion, protein microarray techniques applied in allergy diagnosis allow the identification of several IgE reactivity patterns and possibly could lead to a better knowledge of the relationship between basic immunological mechanisms and clinical symptoms. We would put much emphasis on the opportunity that micro-technologies give us to broad our allergy testing and, at the same time, get detailed and in deep views of unselected patient's IgE immune recognition


Drosophila visual system as an in vivo model to examine how the toxicity of Tau varies with phosphorylation status. This system relies on overexpression of Tau from transgenes but is susceptible to position effects altering expression and activity of the transgenes. We have refined the system by eliminating position effects through the use of site-specific integration. By standardising Tau expression levels we have been able to compare directly the toxicity of different isoforms of Tau and Tau point mutants that abolish important phosphorylation events. We have also examined the importance of human kinases in modulating Tau toxicity in vivo. We were able to confirm that human GSK3β phosphorylates Tau and increases toxicity but, unexpectedly, we identified that preventing phosphorylation of Ser404 is a protective event. When phosphorylation at this site is prevented, Tau toxicity in the Drosophila visual system is increased in the presence of GSK3β. Our data suggest that not all phosphorylation events on Tau are associated with toxicity.Hyperphosphorylation of the microtubule associated protein, Tau, is the hallmark of a group of neurodegenerative disorders known as the tauopathies which includes Alzheimer's disease. Precisely how and why Tau phosphorylation is increased in disease is not fully understood, nor how individual sites modify Tau function. Several groups have used the  The tauopathies are a group of neurodegenerative diseases characterised by the accumulation of intra-neural aggregates of the microtubule-associated protein, Tau. They include Alzheimer's disease (AD), fronto-temporal lobar degeneration (FTLD-Tau), Pick's disease, progressive supranuclear palsy and corticobasal degeneration. The microtubule-binding properties of Tau were identified more than 30 years ago but, and despite intense research, its true physiological role remains unclear, thus the cellular mechanisms underpinning the tauopathies are also poorly understood. More recently, additional properties of Tau have been identified, including an ability to bundle actin filaments, and multiple Tau-binding partners have been identified, including tyrosine kinases. Together, these have led to the suggestion that Tau may act as a signalling scaffold  and through a multitude of post-translational modifications. These include multiple phosphorylation and acetylation events, glycosylation, ubiquitylation and sumolyation amongst others . Four of the five lines  were homozygous viable insertions and none of these showed an eye phenotype in the absence of the GMR-GAL4 driver. Line 3 exhibited the most severe degeneration . We concluded that the Tau-mediated degeneration is highly dependent on positional effects and we would need to control for these if we were going to compare the toxicity of different forms of Tau.We identified the genomic insertion site of UAS-Tau for each line . We next generated four UAS-hGSK3β transgenic lines by random insertion and found each line was expressed to varying amounts (Drosophila heads and found no change in the ratio of soluble to insoluble Tau in these fractions when hGSK3β was co-expressed together with Tau in Drosophila (In order to examine whether human GSK3β (hGSK3β) could also enhance Tau-mediated toxicity in  amounts  yet expr amounts . When hG amounts . The deg amounts . hGSK3β  amounts . Sarcosy amounts . We extrosophila .Drosophila expressing the 2N4RTau transgene inserted into the 68A landing site  recognise known GSK3β target sites in Tau , whereas labelling by antibody AT100 requires phosphorylation of both Thr212 and Ser214 by GSK3β in combination with another kinase . Solubleing site . We founxpressed ; PHF1. Rxpressed ; AT270. xpressed ; AT8. Phxpressed ; AT100. in vitro and acts as a priming kinase for GSK3β .Our failure to detect phosphorylation of Thr212/Ser214 by hGSK3β to generate the AT100 epitope may indicate a requirement for priming at adjacent upstream Ser residues by an additional kinase . One canor GSK3β . The DYRor GSK3β . We genein vivo are essential for increased toxicity, we replaced the individual residues, Ser202, Ser205, Thr212 or Ser404 in 2N4R Tau with non-phosphorylatable alanine. In a fifth mutant, all four of these residues were substituted by alanine (termed Tau4xA). We examined the effect of these mutations on the ability of hGSK3β to phosphorylate 2N4R Tau in Drosophila cells. When 2N4R Tau is expressed in cells, multiple Tau bands were detected using a phospho-independent antibody. These likely correspond to different phosphorylated forms mediated by endogenous kinases. Co-expression of hGSK3β resulted in a retardation of the Tau bands in the gel, indicating increased phosphorylation , with or without a point mutation, R406W, which is found in some autosomal dominant forms of FTLD-Tau . Previoutoxicity . We confransgene . Howeverransgene , the R40s of Tau . In an iotubules  and an i oocytes , Tau R40 oocytes .in vitro but it is not yet clear how each phosphorylation event contributes to Tau toxicity . Details of genetic markers and balancer chromosome are described at Flybase (http://flybase.org). GMR-GAL4 was used to drive expression of UAS-Tau and UAS-kinases in the visual system as previously described  or GenetiVision . All transgenic flies produced were confirmed by sequencing and Splinkerette PCR was used to identify genomic DNA flanking regions . The conescribed .Full length open reading frames (ORFs) of cDNAs for Tau and each kinase were amplified with proof-reading Taq polymerase, cloned into pENTR (Invitrogen) and verified by sequencing. Constructs were recombined into the Murphy collection of Destination vectors supplied by the Drosophila Genomic Resource Centre . The kinases were epitope-tagged with Myc or Flag sequences, while Tau was not tagged.The ORF of 2N4R human Tau cloned into pTW was mutated using the QuikChange Multi kit (Stratagene) and confirmed by sequencing.TM Reverse Transcription System (Promega) following the manufacturer's instructions. 500 ng RNA was used per reaction.RNA was extracted from 5–10 adult fly heads using Tri Reagent (Sigma) and used immediately for cDNA synthesis with the ImProm-IIhttp://www.qstandard.co.uk). Transcript levels for the following genes were quantified: Drosophila actin5c (CG4027), Drosophila GAPDH2 (CG8893), Drosophila EIF-4a (CG9075), mouse CD8a (geneID: 12525), human Tau (geneID: 4137).Quantitative PCR was performed by QStandard  to decapitate. Fly heads were separated from thoraces and abdomens by shaking through a fine sieve.A two-stage, neutral-alkaline extraction procedure was used to maximise recovery of Tau protein. Fly heads were homogenised (20 heads/100 µl) in 100 µl ice-cold homogenisation buffer , pH 6.8, and garnet beads in a FastPrep24 homogeniser . Heads were homogenised twice at 6.5 m/s for 20 s and centrifuged at 20,000 g for 10 min at 4°C. The supernatant was removed and stored on ice. A second homogenisation was then performed by adding 100 µl of ice-cold homogenisation buffer at pH 9.2 to the pellet and centrifuging as above. The supernatants were combined, the pH adjusted to 8.0 and then centrifuged at 20,000 g for 30 min at 4°C. The final supernatant was stored at −80°C.Ice-cold Tris buffer  was added to the fly heads (30 heads/60 µl buffer) and garnet beads. Fly heads were homogenised three times at 6.5 m/s for 20 s as above, then centrifuged at 1,000 g for 5 min at 4°C. The supernatant was removed and stored at −80°C and referred to as the sarcosyl-soluble fraction. N-lauroylsarcosinate (Sigma) was added to the pellet to give a final concentration of 1% (w/v) and the suspension was incubated for 1 h at ambient room temperature with gentle shaking. The suspension was centrifuged at 100,000 g for 1 h at 4°C. The insoluble pellet was resuspended in 50 mM Tris-HCl, pH 7.5, stored at −80°C and referred to as the sarcosyl-insoluble fraction.SDS-PAGE, Western blotting, membrane blocking and probing were all performed by standard protocols. The membrane used was supported nitrocellulose (BioRad) which was blocked for 1 h in 5% milk in Tris-buffered saline (TBS) without Tween-20. Antibodies were diluted in 5% milk in TBS, 0.1% Tween-20 (TBST). Blots were washed with TBST, followed by two washes in TBS without Tween prior to imaging.Primary antibodies raised against: total human Tau (phosphoinsensitive), used at 1/10,000 (Dako); PHF1 human Tau pS396/pS404, used at 1/5,000 ; AT8 human Tau pS202/pT205 at 1/2,000 (Innogenetics); Tau1 human Tau 197–208 at 1/1,000 (Chemicon); AT100 human Tau pT212/pS214 at 1/1,000 (Innogenetics); AT270 human Tau pT181 at 1/1,000 (Innogenetics); 9E10 c-Myc at 1/1,000 (Santa Cruz); M2 Flag-tag at 1/1,000 (Sigma); β-actin at 1/5,000 . Secondary antibodies: Alexa-680 mouse anti-IgG at 1/15,000 (Invitrogen); Alexa-800 mouse anti-IgM at 1/15,000 (Rockland); Alexa-800 rabbit anti-IgG at 1/15,000 (Rockland).Membranes were scanned and quantified using an Odyssey infra-red scanner (Licor Biosciences) and Prism 5 software (GraphPad).A constant amount of recombinant Tau was included in an individual lane on each Tau protein gel to act as a normalisation control.Statistical analysis was performed using Prism 5 (GraphPad). Transcript and protein quantification data were analysed for significance by one-way ANOVA with Bonferroni correction. Resulting p values lower than 0.05 were deemed significant.