Ciliary neurotrophic factor (CNTF) expression is repressed in astrocytes by neuronal contact in the CNS and is rapidly induced by injury. Here, we defined an inhibitory integrin signaling pathway.The integrin substrates laminin, fibronectin and vitronectin, but not collagen, thrombospondin or fibrinogen, reduced CNTF expression in C6 astroglioma cells. Antibodies against αv and β5, but not α6 or β1, integrin induced CNTF. Together, the ligand and antibody specificity suggests that CNTF is repressed by αvβ5 integrin. Antibodies against Thy1, an abundant neuronal surface protein whose function is unclear, induced CNTF in neuron-astrocyte co-cultures indicating that it is a neuroglial CNTF repressor. Inhibition of the integrin signaling molecule Focal Adhesion Kinase (FAK) or the downstream c-Jun N-terminal kinase (JNK), but not extracellular regulated kinase (ERK) or p38 MAPK, greatly induced CNTF mRNA and protein expression within 4 hours. This selective inhibitory pathway phosphorylated STAT3 on its inhibitory ser-727 residue interfering with activity of the pro-transcription Tyr-705 residue. STAT3 can activate CNTF transcription because it bound to its promoter and FAK antagonist-induced CNTF was reduced by blocking STAT3. Microinjection of FAK inhibitor directly into the brain or spinal cord in adult mice rapidly induced CNTF mRNA and protein expression. Importantly, systemic treatment with FAK inhibitors over 3 days induced CNTF in the subventricular zone and increased neurogenesis.Neuron-astroglia contact mediated by integrins serves as a sensor to enable rapid neurotrophic responses and provides a new pharmacological avenue to exploit the neuroprotective properties of endogenous CNTF. Endogenous CNTF regulates the development of oligodendrocytes  and someThe expression of CNTF is rapidly and robustly induced in astrocytes upon brain injury  and stroIntegrins are a group of 24 heterodimer receptors with alpha and beta subunits binding extracellular matrix (ECM) proteins as adhesion partners . The neuWe set out to identify the CNTF-repressing signaling pathway from neuronal ligand to astroglial transcription factor, and whether its pharmacological inhibition would increase functional CNTF using adult SVZ neurogenesis as an outcome measure.To identify which integrins repress CNTF, we first tested various ECM ligands with known differential integrin binding partners ,34,35 inThe surface protein Thy-1 is enriched in neurons throughout the CNS ,41 and bFAK is the best-known kinase associated with integrin signaling ,44. C6 cDownstream targets of FAK include ERK , JNK 4646,47 andhttp://www.cisreg.ca]; Motifmap, [http://motifmap.ics.uci.edu]) starting 25 nucleotides upstream of the CNTF initiation point. We also found a consensus sequence at −1954 nucleotides (TTCTGGGAA); ). This may allow CNTF-induced proliferation until the neuroblasts reach their target in the olfactory bulb which is rich in Thy-1. Integrins such as α6β1, αv and β8, and ligands such as laminin, play a key role in neuroblast migration  protease inhibitor cocktail. Cells were washed in ice cold PBS (Gibco) before cells were scraped from the surface with an inverted p1000 pipette tip in RIPA buffer. Lysate was transferred to Eppendorf tubes and placed on ice. The lysate was then triturated using a 1 ml syringe (BD) and 26½ gauge needle before samples were returned to ice and incubated for 30 minutes. Samples were centrifuged at 12,300 rpm at 4°C for 15 minutes. Lysate was then transferred to fresh Eppendorf tubes and stored at −80°C or prepared for protein quantitation with Pierce’s BCA protein assay as per manufacturer’s instructions. Proteins were separated by SDS-PAGE and blotting was then performed with specific antibodies for CNTF , or with FAK , pFAK-Tyr397 , JNK , pJNK-Tyr185 , pSTAT3-Ser727 , pSTAT3-Tyr705 . Concentration used was 1/1000 STAT3 , αTubulin , all from Cell Signaling Technology. Briefly, after transfer, PVDF membranes were blocked in 5% non-fat milk in Tris buffered saline with 0.05% tween (TBST) for 1 hour then incubated overnight (4°C) in primary antibody (5% milk:TBST). Blots were washed with TBST before incubation  with appropriate Horse Radish Peroxidase conjugated secondary antibodies  in 5% milk; TBST. Blots were washed and ECL substrate used to visualize antibodies according to standard procedures.Mice were transcardially perfused with ice-cold PBS followed by 4% paraformaldehyde. Their brains were extracted, post-fixed overnight and cryoprotected in 30% sucrose in PB for 24 hours. Coronal 30 μm thick sections were cut on a sliding freezing microtome. Starting at a random point along the rostrocaudal axis of the brain, every sixth section through the SVZ was immunostained for doublecortin (dcx) to detect neuroblasts. Briefly, sections were incubated in 5% donkey for 1 hour followed by overnight incubation (4°C) with goat anti-DCX . Secondary antibodies were anti-goat IgG  for 1 hour at room temperature. Sections were incubated with Hoechst (1 μg/ml) before cover slipping for imaging. Confocal images were taken on a Nikon D-Eclipse C1 confocal microscope. The images of 1024 × 1024 x-y pixel and 8.4 μm z-stack were taken using a 100x oil objective.t test or One Way Analysis of Variance (ANOVA) with a Dunnett’s or Bonferroni post-hoc test where noted. A value of p < 0.05 considered as statistically significant.The number of neuroblasts was counted independently by two investigators blinded to the treatment using a 20x objective (Leica DM6000 microscope) by identifying dcx-positive cells with Hoechst-labeled nuclei in the most populated dorsal quadrant of the SVZ. Cells were counted at the same area (35 μm × 15 μm rectangular box) overlaying the entire width of the SVZ using 4 sections per brain. Statistical analyses were performed with Student’s CNTF: Ciliary Neurotrophic factor; JNK: c-Jun N-terminal kinase; FAK: Focal Adhesion Kinase; ERK: Extracellular regulated kinase; MAPK: Mitogen Activated Protein Kinase; SVZ: Subventricular Zone; EAE: Experimental autoimmune encephalomyelitis; ECM: Extracellular matrix; pFAK: Phosphorylated FAK; FAKi: FAK antagonist; pJNK: Phosphorylated JNK; STAT3: Signal Transducer and Activator of Transcription 3; EGFR: Epidermal growth factor receptor; IL-6: Interleukin-6; pSTAT3: Phosphorylated STAT3; PPAR-γ: Peroxisome Proliferator-Activated Receptor gamma; CREB: Cyclic AMP Response Element Binding; ATF2: Activating Transcription Factor 2.The authors have no financial or non-financial conflicts of interest.MPK, SSK, CL and TH designed and performed experiments, analyzed data and wrote the manuscript. All authors read and approved the final manuscript.


ATP channel gatings are unknown.Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors) consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular K2+]i, [ATP]i and [glucose]o uptake measurements.Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for -2-deoxy-glucose uptake and an increase in [ATP]i levels. A time (2–30 min)- and concentration (0.1–10 µM)-dependent increase by simvastatin of p-AMPKα-Thr172 and p-PP2A-Tyr307 expression was observed. The enhanced p-AMPKα-Thr172 expression was inhibited by compound C, ryanodine (100 µM) and KN93 (10 µM). Simvastatin-induced p-PP2A-Tyr307 expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM), ouabain (10 µM), and in [glucose]o-free or [Na+]o-free conditions.The cromakalim (10 nM to 10 µM)- and pinacidil (10 nM to 10 µM)-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM). Simvastatin  suppressed (in okadaic acid (10 nM)-sensitive manner) cromakalim (10 µM)- and pinacidil (10 µM)-mediated opening of whole-cell K2+ release which is important for AMPKα-Thr172 phosphorylation via Ca2+/CaMK II. AMPKα-Thr172 phosphorylation causes [glucose]o uptake (and an [ATP]i increase), closure of KATP channels, and phosphorylation of AMPKα-Thr172 and PP2A-Tyr307 resulted. Phosphorylation of PP2A-Tyr307 occurs at a site downstream of AMPKα-Thr172 phosphorylation.Simvastatin causes ryanodine-sensitive Ca Previous studies have demonstrated that statins can modify the activities of different ion channels in blood vessels including L-type CaATP channels are modulated by multiple cell signaling mechanisms, such as phosphorylation by protein kinase A (PKA) ATP channel gatings. It is well-known that AMP-activated protein kinase (AMPK) serves as a ‘metabolic master regulator’ which is sensitive to changes of intracellular AMP/ATP ratio. Activation of AMPK results in suppression of intracellular energy-consuming pathways and generation of ATP i.e. an increase in cellular ATP level. In mouse isolated pancreatic islets, activation of AMPK by AICAR (an AMPK activator) potentiated insulin secretion by inhibiting KATP channel openings ATP channel openings in mouse aortic smooth muscle cells ATP channel gatings in VSMC. Unfortunately, in various ex vivo studies (multi-cellular preparations), there is no consensus on the vascular effects mediated by AMPK activation as both contraction and relaxation were observed ATP channel gatings and vascular reactivity.Similar to other ion channels, the opening and closing of KATP channel gatings and the simvastatin-mediated effects involve activation of AMPK signaling pathway. Thus, in this study, experiments were designed to evaluate the effects of acute simvastatin on vascular KATP channel gatings of pig’s coronary artery, and the participation of AMPK activation.Therefore, in this study we hypothesize that acute simvastatin could modulate vascular KThis investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health . The protocol was approved by the Animal Ethics Committee of the Chinese University of Hong Kong . Permission prior to the collection of fresh pig’s heart for research purposes was obtained from Sheung Shui Slaughterhouse (Hong Kong).Fresh human left internal mammary arteries were the leftover obtained from patients with cardiovascular diseases undergoing coronary artery bypass grafting (CABG) procedures, and the use of human tissues for research purposes was approved by the Human Research Ethics Committee of the Chinese University of Hong Kong (CREC Ref. No. 2006.313). Written consents were obtained, prior to surgery, from patients voluntarily involved for the usage of tissues solely for research purposes. Patients had read and understood the patient information document provided, and the aims and methods of this study had been fully explained to them. Patients involved had given written informed consent (as outlined in PLOS consent form) to authors of this manuscript for publication of these data.Fresh hearts were obtained from pigs (∼35 kg) that were slaughtered in the morning of the experiment at a local slaughterhouse. The heart was immediately immersed in an ice-cold physiological salt solution. Segment of the left anterior descending (LAD) coronary artery  was dissected within an hour after the animal was slaughtered.Fresh human left internal mammary arteries were bathed in an ice-cold physiological salt solution before transported to the laboratory from the operation theatre of the Prince of Wales Hospital (Hong Kong) within an hour. Fat and connective tissues were carefully removed under the dissecting stereo-microscope.4 1.2, NaHCO3 25, KH2PO4 1.2, CaCl2 2.5, and glucose 11 . Rings (1 mm in length) were equilibrated under resting tension of 1 g ATP channels in these vascular tissues Arterial rings  (endothelium was removed using a blunted watch-maker forceps) were bathed in a 5-ml thermo-regulated wire myograph contained physiological salt solution with the composition (mM): NaCl 118.3, KCl 4.6, MgSOPorcine left anterior descending coronary artery myocytes and human left internal mammary artery myocytes were dissociated using collagenase and protease, as reported ATP channels for a fair/accurate comparison of KATP channel gatings of different cells  in response to drug challenges. In addition, this mode of recording offers the convenience of a rapid delivery of drugs  into the cytosol of cells.Conventional whole-cell, patch-clamp experiments were performed at room temperature (∼ 22°C) using single-cell, voltage-clamp techniques (Axopatch 200B amplifier and Digidata 1200 A/D interface)  with recording patch pipettes of 2–4 MΩ . Whole-cell recording configurations were used so as to maintain a “pre-determined concentration” of ATP (i.e. 1 mM) inside all the cells used during the recording of K2ATP 1, NaADP 0.5, MgCl2 1 and HEPES 10 (pH = 7.4) The external bath solution contained (mM): KCl 10, potassium gluconate 135, EGTA 5, glucose 5 and HEPES 10 (pH = 7.4). The internal pipette solution contained (mM): KCl 10, potassium gluconate 133, EGTA 5, glucose 5, K2 1, CaCl2 1, glucose 10, and HEPES 10 (pH 7.4). After washing, myocytes were imaged using an Eclipse CL Plus Confocal Microscope System  with an excitation at 488 nm and a band-pass filter at 515/530 nm. Fluorescence changes of myocytes (at room temperature) in response to drugs (simvastatin (10 µM) and AICAR (1 mM), with and without ryanodine (100 µM)) were acquired at 15-s intervals. Images were recorded and analyzed by software EZ-C1 3.5 .Porcine coronary artery myocytes were incubated with Fluo-4/AM (5 µM in 0.05% DMSO)  in HEPES buffer (mM): NaCl 140, KCl 5, MgCl3H]-2-Deoxy-glucose uptake into porcine coronary artery myocytes was determined using previously described protocols with minor modifications 2PO4 3.33; Na2HPO4 0.83; CaCl2 1.0; MgCl2 1.0 and HEPES 5 (pH 7.4). Confluent monolayer of cultured porcine coronary artery myocytes in 24-well plates were washed three times in Ringer’s solution. [3H]-2-Deoxy-glucose  was added to each well and incubated for 30 min (37°C). The plates were then washed three times rapidly with ice-cold phosphate-buffered saline before cells were solubilized in 0.5 ml of Triton X-100 (5% vol./vol.). To determine non-specific uptake of [3H]-2-deoxy-glucose, cells were incubated in buffer containing [3H]-2-deoxy-glucose in the presence of cytochalasin B (50 µM) and phloretin (100 µM). The radioactivity was measured using a β-scintillation counter. The protein content was determined spectrophotometrically using the bicinchoninic acid assay .o and [Ca2+]i in mediating simvastatin-induced responses was therefore evaluated. Simvastatin (10 µM) caused an increase in [Ca2+]i level and contraction of single myocytes (data not shown). In myocytes challenged with ryanodine (100 µM), there was a transient increase in [Ca2+]i, (plus contraction of single myocytes) and the subsequent application of simvastatin (10 µM) (2+]i levels.Ca (10 µM)  failed t2+]i in mediating simvastatin-induced AMPK activation as shown above, effects of ryanodine, [Ca2+]o-free solution, KB R-7953 and nifedipine were examined. Ryanodine  abolished simvastatin (10 µM)-, but not AICAR (1 mM)-, induced AMPK activation , KB R-7953 (10 µM)- or nifedipine (10 µM)-containing solutions (data not shown).Ryanodine pre-treatment (100 µM) abolished simvastatin (10 µM)-, AICAR (1 mM) and caffeine (1 mM)-induced changes of p-PP2A-Tyrtal PP2A . Moreovetal PP2A , but noto, effects of simvastatin and AICAR on [glucose]o uptake was determined. Simvastatin (10 µM) and AICAR (1 mM) caused a significant increase in [3H]-2-deoxy-glucose uptake into coronary artery myocytes, and the “enhanced” [glucose]o uptake was eradicated by Compound C (10 µM) (In order to establish the role of [glucose] (10 µM) .o as mentioned above, identification of the [glucose]o uptake transporter involved was performed. [Glucose]o-free solution , phloridzin  blocker), ouabain , 5-(N-ethyl-N-isopropyl) amiloride  (a Na+/H+ exchanger-1 blocker) and [Na+]o-free (replaced with N-methyl-D-glucamine) solution failed to alter simvastatin (10 µM)- and AICAR (1 mM)-mediated increase of p-AMPKα-Thr172 expression (data not shown). In contrast, simvastatin (10 µM)- and AICAR (1 mM)-mediated increase of p-PP2A-Tyr307/total PP2A (i.e. PP2A inhibition) was eradicated in [glucose]o-free .After the confirmation of the essential role of [glucose]e]o-free  or [Na+]nditions , and witnditions  or ouabanditions . Howeveri after [glucose]o uptake induced by simvastatin, cellular ATP level was estimated in response to drug challenges. AICAR caused a Compound C (10 µM)-sensitive increase in cellular ATP level of the arterial myocytes -mediated drug metabolism in mediating simvastatin-induced responses, effects of CYP450 3A4 inhibitor was examined. The biochemical existence of CYP450 3A4 protein was confirmed in porcine coronary artery and human internal mammary artery . PorcineATP channel openings with no apparent effect on basal KATP channel gatings. Thus, a prerequisite opening of KATP channels  is necessary for simvastatin and AICAR to demonstrate KATP channel blocking properties. However, simvastatin Na+ (membrane impermeable) did not alter the cromakalim−/pinacidil-induced relaxation and the KATP channel openings. Therefore, these results suggest that the lipophilic property of simvastatin is essential In this study, acute simvastatin (membrane permeable) suppressed cromakalim- and pinacidil-induced relaxation of U46619 pre-constricted (endothelium-denuded) arteries with no effects on basal tension. In single myocytes of porcine coronary artery and human left internal mammary artery, simvastatin and AICAR inhibited cromakalim- and pinacidil-evoked KATP channels by statins has been reported ATP channel openings of arterial myocytes of pig coronary artery and human left internal mammary artery. Taken together, these results clearly illustrate that simvastatin could alter vasodilatation via the inhibition of KATP channels.Acute application of HMG-CoA reductase inhibitors  elicited an endothelium-dependent relaxation of pre-constricted rat isolated aorta, and cerivastatin-induced relaxation was attenuated by glibenclamide and ouabain i ratio and a rise in [Ca2+]i which signals an increase in energy demands i level is thus restored ATP openings + (incubation ≤ 30 min) altered the expression of HMG-CoA reductase and p-HMG-CoA reductase-Ser871 (the inactivated isoform of HMG-CoA reductase) suggesting that AICAR and simvastatin are acting through different cellular mechanisms (see below). In rat’s liver, AMPK is associated with HMG-CoA reductase 172 expression (i.e. activation) ATP channel openings. Taken together, our results illustrate that acute simvastatin inhibits vascular KATP channel openings probably via AMPKα-Thr172 phosphorylation (i.e. activation).AMPK (formerly termed HMG-CoA reductase kinase) is activated by an increase in [AMP/ATP]ATP channel openings strengthen our conclusion on the participation of PP2A. In contrast to cultured bovine aortic endothelial cells (BAECs) 307 phosphorylation was abolished by Compound C (an AMPK inhibitor) illustrating that PP2A is phosphorylated (i.e. inactivated) in an “AMPKα-dependent” manner, and PP2A phosphorylation occurs at a site downstream of AMPKα activation/phosphorylation. In addition, AMPK can be activated via the LKB1 (the upstream serine/threonine kinase of AMPK) cascade 172 phosphorylation via LKB1-Ser428 phosphorylation 428 phosphorylation of porcine coronary artery suggesting that simvastatin-induced AMPKα-Thr172 phosphorylation is mediated via a LKB1-Ser428 phosphorylation-independent pathway.Okadaic acid (a potent PP2A inhibitor), but not rotterlin (a PKC-δ inhibitor) or H89 (a PKA inhibitor), reversed the inhibitory effects of simvastatin on cromakalim- and pinacidil-mediated K2+/calmodulin-dependent kinase kinase (CaMKK) 172 phosphorylation was abolished by KN93  inhibitor) illustrating the participation of Ca2+/CaMK II. In contrast, AICAR-induced AMPKα-Thr172 phosphorylation was not affected by KN93 suggesting that AICAR-mediated AMPKα-Thr172 phosphorylation is CaMK II-independent 428 expression suggesting that AICAR phosphorylates AMPKα-Thr172 via the LKB1 pathway Apart from LKB1, AMPK is activated/phosphorylated by Ca2+ release from intracellular stores of smooth muscle cells 172 phosphorylation involved CaMK II activation which is a Ca2+-dependent process. Similar to rat aorta 2+]i increase in porcine coronary artery myocytes. Apart from Ca2+ release from intracellular stores, the [Ca2+]i level is also modified by [Ca2+]o influx 2+]o-free conditions did not modify simvastatin-induced p-AMPKα-Thr172 expression. Neither nifedipine (a L-type Ca2+ channel blocker) nor KB R-7953 (a reverse-mode Na+/Ca2+ exchanger blocker) altered simvastatin-induced AMPKα-Thr172 phosphorylation. Taken together, the increased p-AMPKα-Thr172 expression is solely dependent on ryanodine-sensitive [Ca2+]i release which is probably related to the distinct physiological location of HMG-CoA reductase (i.e. sarco/endoplasmic reticulum) Simvastatin inhibited Cao uptake o-dependent manner 3H]-2-deoxy-glucose uptake into the myocytes, simvastatin- and AICAR-induced p-AMPKα-Thr172 expression was [glucose]o−/[Na+]o-independent. Hence, ryanodine-sensitive Ca2+ release and activation of CaMK II, but not [glucose]o uptake, are essential initial steps necessary for AMPKα-Thr172 phosphorylation upon simvastatin challenge. Nonetheless, our results demonstrate that simvastatin and AICAR caused an increase in [ATP]i levels. The elevated [ATP]i contributed to the closure of vascular KATP channels 307) phosphorylation (see below).Activation of AMPK causes [glucose]307 phosphorylation (i.e. inactivation) o. In addition, Na+/K+-ATPase provides the favorable trans-cellular Na+ gradient for [glucose]o uptake via Na+-glucose co-transporter (SGLT-1) +]o depletion or ouabain suppressed PP2A-Tyr307, but not AMPKα-Thr172, phosphorylation illustrating the obligatory role of [glucose]o uptake via SGLT-1 +/K+-ATPase for PP2A-Tyr307 phosphorylation.AMPK is regulated negatively by serine/threonine phosphatase(s) e.g. PP2A + by CYP450 3A4 172 and p-PP2A-Tyr307 expression refuted the possibility of local bio-transformation of simvastatin. Thus, our results strengthen the conclusion on the involvement of simvastatin, but not simvastatin Na+, in inhibiting vascular KATP channel openings.The biochemical existence of cytochrome P450 (CYP450) 3A4 was demonstrated in porcine coronary artery and human left internal mammary artery, and the possible local enzymatic conversion of simvastatin into simvastatin Na307 and AMPKα-Thr172, but not HMG-CoA reductase-Ser871, of porcine coronary artery. Ryanodine-sensitive Ca2+ stores, but not [Ca2+]o entry, play an obligatory role in simvastatin-elicited [Ca2+]i increase and initiated the activation of Ca2+/CaMK II cascade which are essential for the subsequent AMPKα-Thr172 phosphorylation (activation). Activation of AMPKα leads to [glucose]o uptake (and [ATP]i elevation resulted) with the participation of SGLT-1 and Na+/K+ ATPase. An increase in [ATP]i levels not only closed the KATP openers-induced channels openings but also provided the necessary phosphate groups for protein phosphorylation. Phosphorylation (inactivation) of PP2A-Tyr307 probably occurs at a site downstream of AMPKα-Thr172 phosphorylation (In conclusion, our results demonstrate that acute simvastatin caused phosphorylation of PP2A-Tyrrylation .


Lupin is a decorative flower found in gardens around the world. It is a member of the legume family and in recent years, flour produced from its seeds has been increasingly used in food manufacturing, especially in breads, baked products and confectionery. Within a similar timeframe, lupin allergy has emerged as a major problem in European countries, with more cases also now appearing in the United Kingdom.A 46 year old female with a history of asthma and seasonal allergic rhinitis was referred to our Food Allergy clinic. In the previous 2 years, she suffered two episodes of anaphylaxis without being able to identify the cause. The first anaphylactic episode occurred after eating a crab canapé. The other, after eating duck pâté, beef with vegetables and a fruit tart. After eating this meal, she developed a sensation of throat tightness, nausea and felt unwell. Twenty minutes later, she went on to develop wheeze and stridor, generalised pruritus, erythema, vomiting and diarrhoea. She has had milder symptoms after eating lobster chowder and mussels in Belgium and also with chocolate and toffee. She has been avoiding shellfish since the reaction. She was also avoiding peanuts as she was found to have a positive specific IgE test in the general allergy clinic.Total IgE was 159iu/ml. Specific IgE test to lobster, mussel, shrimp, fish, lipid-transfer protein, tree nuts and various legumes were negative. Specific IgE to peanut was 1.02iu/ml. Ara h 1, 3, 8 and 9 were negative while ara h2 was 1.74 iu/ml. Skin prick testing (SPT) to peanut reagent, raw peanut and roasted peanut were negative. SPT to lupin flour was 25mm and specific IgE 42iu/ml. Soy flour was 4mm. She has been eating soy and tolerates other legumes. SPT to tragacanth gum, a legume that she was using in sugar flower making was negative.The strongly positive SPT and specific IgE tests to lupin, in conjunction with this being the common ingredient across the foods our patient reacted to, suggest lupin as the cause of these reactions. It is important for clinicians to consider lupin as a potential cause of unexplained food-related allergic reactions, as its use has now become widespread throughout Europe. Furthermore, cross-reactivity to peanut should be clarified with challenge testing. Of note, our patient had been using a facial anti-wrinkle cream that contained white lupin seed extract for over 7 years. This may represent an additional route of sensitisation.None declared.


Drosophila encodes a VRAC that is also activated by increases in intracellular Ca2+. The role of Best1 as a VRAC has recently been independently confirmed by the Clapham lab in an unbiased RNAi screen. Although dBest1 is clearly a volume-regulated channel, its mechanisms of regulation remain unknown. Here we investigate Drosophila Best1 (dBest1) regulation using the Drosophila S2 cell model system. Because dBest1 activates slowly after establishing whole-cell recording, we tested the hypothesis that the channel is activated by phosphorylation. Two experiments indicate that phosphorylation is required for dBest1 activation: nonspecific protein kinase inhibitors or intracellular perfusion with the non-hydrolyzable ATP analog AMP-PNP dramatically reduce the amplitude of dBest1 currents. Furthermore, intracellular perfusion with ATP-γ-S augments channel activation. The kinase responsible for dBest1 activation is likely Ca2+/calmodulin dependent kinase II (CaMKII), because specific inhibitors of this kinase dramatically inhibit dBest1 current activation. Neither specific PKA inhibitors nor inactive control inhibitors have effects on dBest1currents. Our results demonstrate that dBest1 currents are regulated by phosphorylation via a CaMKII dependent mechanism.Cell swelling induced by hypo-osmotic stress results in activation of volume-regulated anion channels (VRAC) that drive a compensatory regulatory volume decrease. We have previously shown that the Best1 gene in  Drosophila S2 Volume-regulated anion channels (VRACs) are critical for cell volume homeostasis via a process called regulatory volume decrease (RVD) Drosophila S2 cells encodes an anion channel. The dBest1 current can be activated by increases in intracellular Ca2+ and is abolished by RNAi directed against dBest1 We have previously shown that the dBest1 gene in 2+ may independently regulate dBest1 function, because Ca2+ can activate the current in the absence of cell volume changes and increases in cell volume can activate the current even when intracellular Ca is highly buffered. In general, the mechanisms underlying ion channel regulation by cell volume are very complex, and multiple signaling pathways have been implicated 2+ converge on a common regulatory pathway to activate dBest1, as very little is known about mechanisms that contribute to dBest1 channel regulation. Although human Best1 does not seem to require phosphorylation for activation, it is modulated by phosphorylation Drosophila S2 cells.Cell volume and CaDrosophila S2 cells were obtained from the Drosophila Genomics Resource Center (Indiana University) and cultured at room temperature (22–24°C) in Schneider’s Drosophila Medium (GIBCO BRL) containing 10% heat-inactivated FBS (GIBCO BRL), 50 µg/ml penicillin, and 50 µg/ml streptomycin (GIBCO BRL). HEK293 cells (ATCC) were cultured in DMEM supplemented with 10% FBS and 0.5% penicillin-streptomycin at 37°C.HEK293 cells were transfected using Fugene 6 (Roche) with 1 µg plasmid DNA. For electrophysiological recordings, dAno1 cloned from S2 cells (NM_142563.1) was subcloned into the pIRES2-EGFP vector (Clontech). Transfected cells were plated at a low density and used for electrophysiology 24–48 hrs after transfection. Cells expressing GFP were patched.2, 1 MgCl2, HEPES (pH7.2 with NaOH), and 10 glucose. The standard pipette solution contained 143 CsCl, 10 Ca-EGTA-NMDG, 8 MgCl2, 10 HEPES (pH 7.2 with NMDG), 10 glucose, and 3 ATP. The free Ca2+ concentration for this solution was ∼4.5 µM. Osmolarity of both extracellular and intracellular solutions was adjusted to 320 mOsm with water or sucrose. For HEK293 cell recordings, the intracellular solution contained 146 CsCl, 2 MgCl2, 5 Ca-EGTA (∼24 µM free Ca2+), 10 sucrose, and 8 HEPES, pH 7.3 with NMDG. The extracellular solution contained 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 15 glucose, and 10 HEPES, pH 7.4 with NaOH. Osmolarity was adjusted with sucrose to 303 mOsm. Cells were voltage clamped with ramps from −100 mV to +100 mV run at 10-s intervals, followed by a voltage step protocol from −100 to +100 mV.Whole cell patch-clamp was performed at room temperature (22–24°C). Patch pipettes were fire polished to resistances of 2–3 MΩ. The standard extracellular solution used for patch clamping S2 cells contained (in mM) 150 NaCl, 2 CaClATP-γ-S, AMP-PNP, H7-dihydrochloride, calyculin A, K252a, KN-62, KN-93, and staurosporine (STP) (Sigma-Aldrich) were made as stock solutions and diluted to their final concentrations in extracellular and intracellular recording solutions. ATP analogs were included in the patch pipette solution. Kinase inhibitors were included in both the bath and patch pipette for whole-cell recordings. S2 cells were exposed to extracellular solution containing drug for at least 6 minutes prior to recording. Cells were incubated with myristoylated-AIP for 20 min prior to recording due to its relative impermeability to the membrane.Data were analyzed using pClamp 8.2 software and Origin 7.0. A Student’s t-test was performed to determine significant differences in maximum current between control and drug treated cells (p<0.05). Data is expressed as mean +/− SEM.www.hprd.org/PhosphoMotif_finder) or GPS2.1 (http://gps.biocuckoo.org/). Accession numbers for the dBest1 and hBest1 sequences analyzed are AAL29094 and AAH41664.1, respectively.Phosphorylation sites were predicted using either Phosphomotif Finder  for which we used the polypeptide sequence for human Ano1 as the input (NP_060513.5). Several Drosophila anoctamin orthologs were identified: CG16718, CG6938, CG10353, CG15270, and Axs. Of the genes identified, CG16718 had the highest sequence similarity to hAno1. Because several Ano1 orthologs have been clearly shown to function as CaCCs, we chose to more closely examine the channel function of CG16718, which will be referred to as dAno1 for the purpose of this study.Previous studies demonstrate that endogenous CaCC currents in dAno1 (NM_142563.1) cloned from S2 cells was expressed in HEK293 cells to examine its channel function. Expression of dAno1 generated currents with biophysical properties distinct from those of the endogenous dBest1 current. Unlike dBest1, dAno1 currents strongly outwardly rectify and exhibit tail currents upon repolarization to −100 mV from positive voltages . In addiDrosophila Best1 currents are activated by increases in intracellular Ca2+. However, the mechanisms underlying Ca2+ regulation of dBest1 are unknown. Two basic mechanisms could explain activation of dBest1 by Ca2+. Ca2+ could bind directly to dBest1 (or an accessory subunit) to induce a conformational change which gates the channel open. Alternatively, Ca2+ could bind to and activate Ca2+-dependent protein kinases or protein phosphatases, which regulate dBest1 activation via phosphorylation. Because dBest1 activates slowly over a period of minutes after establishing whole cell recording with high Ca2+ in the internal solution 2+ (4.5 µM) and 3 mM ATP, dBest1 currents activate slowly over a period of 4–5 min before reaching a plateau , the C-terminus of hBest1is sufficiently divergent from dBest1 that it is not possible to predict whether these amino acids have any homology. Furthermore, it is unknown if CaMKII directly phosphorylates dBest1, or if there are intermediate signaling events involved in dBest1 activation by CaMKII.Given the importance of the C-terminus in the regulation of hBest by phosphorylation, one wonders whether a CaMKII phosphorylation site may be conserved between human and 2+ binding is also important for activation of dBest1. We previously reported that hBest1 is activated by direct Ca2+ binding to an EF hand at position 312–323, located within the C-terminus of hBest1 2+ binding in dBest1 activation. For hBest1, activation by Ca2+ binding to the EF hand also requires an adjacent acidic amino acid rich regulatory region at position 350–390, which also plays a role in channel rundown. However, several of the acidic residues in this region are not conserved in dBest1. Interestingly, many of the predicted CaMKII phosphorylation sites fall near this regulatory region in a stretch of amino acids that are unique to dBest1. Although speculative, it is possible that the phosphorylation state of this region is important for regulation/activation of dBest1 by Ca2+. Taken together, these findings support the model of channel activation presented in 2+ binding and Ca2+-dependent phosphorylation by CaMKII are critical for channel activation.Although CaMKII is critical for dBest1 activation, it is unknown if direct CaIn the literature, there are many examples of endogenous CaCC currents (whose molecular identities remain uncertain) that are regulated by CaMKII These preliminary studies provide future directions for examining the regulatory mechanisms of Best1. The foremost question posed by this study is: can dBest1 be directly phosphorylated, and if so, is phosphorylation directly mediated by CaMKII? Alternatively, if dBest1 is not directly phosphorylated by CaMKII, CaMKII may act on a regulatory subunit of dBest1. There are several possible mechanisms for how phosphorylation could regulate dBest1 activity. First, the phosphorylation state of dBest1 may directly regulate its channel function, as has been shown for hBest1. Phosphorylation of dBest1 could also alter its trafficking to the plasma membrane. Or, phosphorylation of dBest1 could affect its association with regulatory subunits, and vice versa.2+. It will be interesting to see if CaMKII is also critical for volume dependent activation of dBest1, and if this regulatory mechanism is conserved in hBest1. Inhibition of hBest1 by hypertonic stress was previously shown to be dependent on its phosphorylation state Although the mechanisms underlying dBest1 regulation by phosphorylation are uncertain, it is clear that CaMKII is important for activation of dBest1 by intracellular Ca


The SKF83959-induced ERK1/2 phosphorylation was found to be Ca2+-independent based on the following observations: i. chelating intracellular Ca2+ did not inhibit ERK1/2 phosphorylation and astrocyte migration; ii. blockage of the release of intracellular Ca2+ from the endoplasmic reticulum by an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor did not attenuate ERK1/2 phosphorylation. However, inhibition of phospholipase C (PLC), the upstream molecule of internal Ca2+ release, disabled SKF83959’s ability to elevate the level of ERK1/2 phosphorylation. Both non-selective protein kinase C (PKC) inhibitor and PKCδ selective inhibitor prevented ERK1/2 phosphorylation increase and astrocyte migration, but PKCα inhibitor did not. This suggests that Ca2+-independent and diacylglycerol-dependent PKCδ acts downstream of putative phosphatidylinositol-linked D1-like receptor activation and mediates SKF83959-induced elevation of ERK1/2 phosphorylation in order to modulate astrocyte migration. In conclusion, our results demonstrate that SKF83959-induced increases in ERK1/2 phosphorylation and astrocyte migration are dependent on PLC-PKCδ signals. This might help us to further understand the functions of the putative phosphatidylinositol-linked D1-like receptors in the nervous system.Extracellular signal-regulated kinase 1/2 (ERK1/2) is a member of the mitogen-activated protein kinase family. It can mediate cell migration. Classical dopamine receptor-mediated ERK1/2 phosphorylation is widely studied in neurons. Here, we report that ERK1/2 phosphorylation is also modulated by putative phosphatidylinositol-linked D Among these receptors, classical cyclase-coupled D1 receptors are linked to Gs protein that can stimulate cyclic AMP (cAMP) formation 1-like receptors are connected to Gq protein to promote phospholipase C (PLC) activation and the subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) 1-like receptor was named phosphatidylinositol (PI)-linked D1-like receptor because of its ability to activate Gq/PLC/inositol 1,4,5-triphosphate (IP3) signals 1-like receptor can be used to identify new roles of atypical DA receptors in the nervous system 2+ currents, which was proven to be dependent on PLC/IP3/Ca2+/calcineurin signals in vivo models 2+/calcium-calmodulin-dependent protein kinase II (CaMKII) and PLC/IP3/Ca2+/cyclin-dependent kinase 5 (CDK5) signals, respectively Dopamine (DA) can regulate emotion, cognition, locomotion, and endocrine function 1-like receptors have also been found to modulate astrocyte function. For instance, activation of the putative PI-linked D1-like receptors by SKF83959 up-regulates astrocyte-derived fibroblast growth factor-2 (FGF-2) expression via PLC/IP3/Ca2+/CaMKII signals, which potentially protects dopaminergic neurons from 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity 2+. Data from our previous study also showed a release of internal Ca2+ from endoplasmic reticulum (ER) in cultured astrocytes after SKF83959 treatment 2+-related kinases such as CaMKII in response to SKF83959 application. However, due to the complexity of DA signal transduction pathways, we focused on whether other signal molecules could mediate SKF83959’s effects on astrocytes.Astrocytes, regarded as supporting structures in the nervous system, are generally thought to act as a syncytium of interconnected cells, rather than as individual bodies 1-like receptors can mediate ERK1/2 activation in cultured astrocytes. Our present study demonstrates that SKF83959 promotes ERK1/2 phosphorylation by augmenting PLC-protein kinase Cδ (PKCδ) signaling in cultured rat astrocytes. Both ERK1/2 and PKCδ inhibition functionally inhibit SKF83959-induced astrocyte migration. Our observations regarding SKF83959-induced ERK1/2 activation in astrocytes might provide new perspectives on the roles of the putative PI-linked D1-like receptors in the nervous system.Extracellular signal-regulated kinase 1/2 (ERK1/2) may be involved in this process. ERK1/2 is a member of the mitogen-activated protein kinase family, whose activation in response to stimuli is involved in cell migration and proliferation Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) was obtained from Gibco Invitrogen Corporation . Heat-inactivated fetal bovine serum was purchased from Hyclone . Poly-L-lysine, PD98059, BAPTA-AM, GF109203X, Gö6976, prazosin, hydroxyurea, rottlerin, and Hoechst 33258 were purchased from Sigma . SKF83959, 2-APB, SCH23390, and U-73122 were purchased from Tocris Bioscience . Antibodies against phospho-ERK1/2 and total-ERK1/2 were obtained from Santa Cruz Biotechnology  and Abcam , respectively. Antibodies against total-CaMKII, phospho-CaMKII (Thr286), and β-actin were purchased from Cell Signaling Technology . Antibody against GFAP was the product of NeoMarkers . Both goat anti-mouse IgG polyclonal secondary antibody for Western blot and rabbit anti-mouse tetramethylrhodamine isothiocyanate-conjugated secondary antibody for immunofluorescence were purchased from Thermo Scientific . Other related agents were purchased from commercial suppliers. All drugs were prepared as stock solutions, and stock solutions were stored at −20°C. The final concentration of DMSO was < 0.05%. No detectable effects of DMSO were found in our experiments.g for 5 min, cells were re-suspended and plated on poly-L-lysine (1 mg/mL)-coated culture flasks. The single-cell suspension was then cultured in DMEM/F12 supplement with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin (100 U/mL). For astrocytes, the medium was changed to fresh DMEM/F12 medium after 24 h and replaced every 3 days. After 12 days, mixed cells were shaken gently overnight (18 h), and then the supernatant was removed. Next the remaining cells were digested with 0.125% trypsin for 5 min and plated on new poly-L-lysine-coated culture flasks. All cultures were maintained in a 37°C incubator containing 95% air and 5% CO2. Immunofluorescence with glial fibrillary acidic protein (GFAP) antibody was used to identify the astrocyte. The purity of the astrocyte culture exceeded 98%.The use of rats was approved by the University Animal Ethics Committee of Nantong University (Permit Number: 2110836). Rat primary cultured cells were prepared as described previously with some modifications Cultured astrocytes were fixed with 4% paraformaldehyde in 0.01 M phosphate-buffered saline  for 30 min, and then rinsed three times with PBS for 10 min each. Immunofluorescence study was performed to determine the expression of GFAP in astrocytes. First, cells were permeabilized with PBS containing 0.3% (v/v) Triton X-100 for 30 min and blocked with 1% bovine serum albumin in PBS for 1 h, and then incubated with 1∶100 anti-GFAP antibody in PBS containing 0.3% Triton X-100, 1% bovine serum albumin, and 2% goat serum overnight at 4°C. After rinsing in PBS three times, the cells were incubated with rabbit anti-mouse tetramethylrhodamine isothiocyanate-conjugated secondary antibody (1∶50) for 1 h at room temperature. Then, the cells were incubated with Hoechst 33258 (20 min) for nucleus staining. Eventually, the samples were mounted on glass slides with 30% glycerin and imaged using a confocal laser scanning microscope . The purity of astrocyte culture was calculated by the ratio of the GFAP and Hoechst 33258 co-stained cell numbers to the Hoechst 33258-stained cell numbers.3VO4, 1 mM PMSF, with 1% (v/v) Nonidet P-40, and protease inhibitor cocktail). The lysates were recovered by centrifugation at 12000 g for 15 min. After denaturation, the proteins (30 µg) were separated on 10% SDS/PAGE gels and then transferred to nitrocellulose membranes . After blocking with 5% nonfat dried milk powder/Tris-buffered saline Tween-20 (TBST) for 1 h, membranes were probed with 1∶500 primary antibodies against total ERK1/2 (42/44 kD) and phospho-ERK1/2 (42/44 kD) or 1∶5000 primary antibody against β-actin (43 kD) overnight at 4°C. Primary antibodies were then removed by washing the membranes 3 times in TBST, and incubated for further 1 h at room temperature with goat anti-mouse IgG polyclonal secondary antibody (1∶3000-1∶5000) that are conjugated with polymers of horseradish peroxidase. Following 3 times of washing in TBST, immunoblots were developed on films using the enhanced chemiluminescence (ECL) technique . The band density was quantified using Image J software. All assays were performed at least three times.To extract the total proteins, purified rat astrocytes were lysed on ice for 30 min in lyses buffer -coated coverslips were cultured to conﬂuence in 35 mm dishes. The monolayer cells were scratched manually with a pipette tip to create extended and definite scratches in the center of the coverslips with a bright and clear field, and then the detached cells were immediately removed by washing the cells with fresh medium. Growth medium with or without SKF83959 was added to each dish after pretreatment with inhibitors for 30 min. Hydroxyurea, an inhibitor of DNA synthesis t test and corrected by Bonferroni. Comparisons for other data were made using unpaired t test. Differences were considered statistically significant at P < 0.05 or P < 0.01.Data were expressed as mean ± SE. Comparisons for dose- and time-dependent data were made using a two-tailed Student' paired 1-like receptors SKF83959 on astrocytes, we first confirmed the purity of cultured cortical astrocytes. As shown in Dopamine receptors have been reported to mediate ERK1/2 phosphorylation and activation in the nervous system. To evaluate the effects of the agonist of the putative PI-linked D1-like receptors are the functional receptors for SKF83959. We next determined whether this receptor could mediate SKF83959-induced ERK1/2 phosphorylation. As expected, pretreatment of astrocytes with the selective antagonist of D1-like receptor SCH23390  prior to SKF83959 (30 µM) incubation prevented increases in ERK1/2 phosphorylation    or an an (10 µM)  did not 1-like receptors increases intracellular Ca2+ level through mobilization of ER 2+ can trigger ERK1/2 phosphorylation, we investigated whether an increase in internal Ca2+ could mediate SKF83959-induced ERK1/2 phosphorylation. Astrocytes were pretreated with a Ca2+ chelator BAPTA-AM , followed by application of SKF83959 (30 µM) for 10 min. BAPTA-AM failed to prevent the SKF83959-induced increase in ERK1/2 phosphorylation level, demonstrating that increases in intracellular Ca2+ are not necessary for the elevation in ERK1/2 phosphorylation level  ability to elevate the level of ERK1/2 phosphorylation , a specific inhibitor of PKCδ, significantly reduced the SKF83959-induced increase in ERK1/2 phosphorylation, and the PKCα inhibitor Gö6976 (1 µM) did not  is also present in the cytosol after PLC activation. DAG couples to PKC and positively regulates PKC activity in various disorders such as diabetes and cancer In conclusion, our current data clearly suggest that the PLC-DAG-PKCδ signaling pathway mediates the SKF83959-induced ERK1/2 activation in cultured rat astrocytes. This might help us to understand the new pharmacological roles of SKF83959 in the nervous system .


Acer-dominated forest stands spanning a 500-km transect, community-scale expression of the cellulolytic gene cbhI under elevated N deposition did not differ significantly from that under ambient levels of N deposition. In contrast, expression of the ligninolytic gene lcc was significantly down-regulated by a factor of 2–4 fold relative to its expression under ambient N deposition. Fungal community composition was examined at the most southerly of the four sites, in which consistently lower levels of cbhI and lcc gene expression were observed over a two-year period. We recovered 19 basidiomycete and 28 ascomycete rDNA 28S operational taxonomic units; Athelia, Sistotrema, Ceratobasidium and Ceratosebacina taxa dominated the basidiomycete assemblage, and Leotiomycetes dominated the ascomycetes. Simulated N deposition increased the proportion of basidiomycete sequences recovered from forest floor, whereas the proportion of ascomycetes in the community was significantly lower under elevated N deposition. Our results suggest that chronic atmospheric N deposition may lower decomposition rates through a combination of reduced expression of ligninolytic genes such as lcc, and compositional changes in the fungal community.High levels of atmospheric nitrogen (N) deposition may result in greater terrestrial carbon (C) storage. In a northern hardwood ecosystem, exposure to over a decade of simulated N deposition increased C storage in soil by slowing litter decay rates, rather than increasing detrital inputs. To understand the mechanisms underlying this response, we focused on the saprotrophic fungal community residing in the forest floor and employed molecular genetic approaches to determine if the slower decomposition rates resulted from down-regulation of the transcription of key lignocellulolytic genes, by a change in fungal community composition, or by a combination of the two mechanisms. Our results indicate that across four  In this study, we sampled forest floor from sugar-maple (Acer saccharum Marsh.) dominated forest stands in which chronic simulated N deposition (30 kg NO3- -N ha−1 yr−1since 1994) has been associated with lower levels of lignocellulolytic enzyme activity and increased levels of C sequestration. We tested three alternate hypotheses: 1) simulated N deposition represses lignocellulolytic gene transcription, 2) simulated N deposition alters fungal community composition, 3) declines in decay rates result from a combination of bothHowever, higher N availability may also directly affect the transcription of the functional genes that encode for lignocellulolytic enzymes cbhI and lcc in the forest floor of experimental plots receiving elevated levels of N was determined relative to that of forest floor in plots receiving ambient levels of N deposition at one sugar-maple dominated site . The expression of the cellobiohydrolase cbhI gene relative to β-tubulin (ΔCT cbhβtubI−) varied between sites and showed no clear relationship to N deposition (cbhI expression (2−ΔΔCT) ranged from 0.06- to 6.85-fold across the four sites in 2009, and was not significantly affected by N deposition . Laccase gene expression also showed considerable variation, although mean ΔCT values were generally lower under simulated N deposition (lcc gene expression (2−ΔΔCT) ranged from 0.02-fold to 11.5-fold across the four forest sites in 2009, and tended to be lower under simulated N deposition , providing evidence supporting one of our hypotheses. Laccase gene expression was also significantly lower under simulated N deposition at Site D in the pooled two-year dataset .The expression of the lignocellulolytic genes  Site D,  in 2007  Site D,  in 2009.position . Normaliposition . Normaliposition . Despite−Whitney U = 46, nambient  =  nsimulated N deposition  = 12, Ptwo-tailed  = 0.14). Cellobiohydrolase activity was significantly different between years at Site D (mean (± SD) 2007, 360 (±111) nmol h−1 g−1; mean (± SD) 2009, 920 (± 165) nmol h−1 g−1; P = 0.01). Despite the temporal difference in magnitude, cellobiohydrolase activity tended to be lower under simulated N deposition than under the ambient condition .Cellobiohydrolase enzyme activity across the four sites in 2009 was on average 22% lower under simulated N deposition than under ambient N deposition , althougondition , and thiambient =  nsimulated N deposition  = 12, Ptwo-tailed  = 0.14). Laccase enzyme activity was higher in 2009 than in 2007 at Site D . In each year, laccase activity tended to be lower under simulated N deposition than under ambient N deposition, but this was not statistically significant .Laccase enzyme activity varied considerably across the four sites in 2009 ; it was t Site D , althougvs. 19 Basidiomycota), yet they composed only 24% of the fungal sequences. This discrepancy was driven by the abundance of two basidiomycete OTUs (Ceratobasidium sp. and Athelia sp.) which were widespread (each recovered from five of six plots within the site) and whose combined abundance accounted for 46% of the fungal sequences. Phylogenetic analyses indicated that Pezizomycotina dominated the Ascomyctona, with Leotiomycetes, Dothideomycetes, Sordariomycetes and Geoglossomycetes taxa representing a combined 82% of the library .The response of fungal community composition to chronic N deposition was examined at Site D. We obtained high quality sequence data from 209 of 288 rDNA 28S clones (72.5%), and 203 of these sequences were of fungal origin (97%). Phylogenetic analyses divided the fungal sequences into 47 Operational Taxonomic Units (OTUs). Ascomycotina dominated the OTUs  accounted for 23% of the variability in species relative abundance; the second axis (unconstrained) for a further 37% .Of the 47 fungal OTUs, only 12  were recovered from more than one of the six field plots, and only 6 occurred under both ambient and simulated N deposition. We used Canonical Correspondence Analysis (CCA) to determine if the relative abundance of these 12 most widespread taxa was affected by Nther 37% . There wLitter decomposition is an enzymatically complex process mediated by a species-rich community of saprotrophic fungi cbhI) and laccase (lcc) enzymes. The cbhI gene is unique to fungi, broadly distributed between the Ascomycotina, Basidiomycotina and possibly Chytridiomycotina in forest soils cbhI showed no consistent response to simulated N deposition across four study sites, although it was significantly lower under simulated N deposition at one of these sites over time; cellobiohydrolase enzyme activity likewise was not significantly affected by elevated N deposition. Nitrogen deposition has been associated with higher cellulolytic enzyme activities and higher initial rates of mass loss in cellulose-rich litters such as the predominantly sugar maple leaf litter of these sites We examined the community-scale transcriptional response of lignocellulolytic genes to simulated N deposition by focusing on the relative transcription levels of genes that encode for fungal cellobiohydrolase , saprotrophic ascomycetes isolated from forest soils appear to be primarily cellulolytic and chitinolytic organisms, rather than agents of lignin degradation lcc that we observed may then be the result of basidiomycetes expressing less lcc as a physiological response to higher N availability; however, the extent of laccase distribution among ascomycetes is poorly understood Botryotinia fukeliana) are known Mycosphaerella, Neonectria and the Cryphonectriaceae. Species within these groups may use laccase as an “attack” enzyme during infection, and subsequently to break down senescent plant cells lcc gene expression and laccase activity cannot be discounted. Although ligninolytic basidiomycetes are often considered the primary agents of late-stage litter decomposition We examined the effect of simulated N deposition on fungal community composition at the most southerly of our sites site D, , in whichis site , 3. Althhis site . Lignin itopilus , as welllcc) involved in lignin decomposition is lower under elevated N deposition, suggesting that the physiological response of saprotrophic fungi to higher N availability may be an important link between environmental change and ecosystem function. At the same time, our results indicate that while the relative abundances of the more common fungi were unaffected by elevated N deposition, fewer ascomycete species were recovered in the latter stages of decomposition. Our results emphasize the ongoing need to clarify the functional potential and ecological niche of fungal species, as well as the need to understand how that potential is realized under varying environmental conditions. Overall, our results indicate that chronic N deposition can elicit both compositional and functional changes in fungal communities. These changes may mechanistically underlie the slowing of decay and increased soil C storage associated with N deposition in this ecosystem.Despite their important function in plant litter decay and nutrient recycling Acer saccharum Marsh.) dominated stands distributed across lower and upper Michigan  typic Haplothods of the Kalkaska series. All four sites are floristically and edaphically similar, but differ in mean annual temperature, ambient atmospheric N deposition, and soil N availability −1 y−1) was initiated in 1994 and consists of 6 equal applications of NaNO3 delivered as dry pellets to the forest floor over the growing season; NO3− composes ∼60% of wet plus dry atmospheric N deposition in this region. Soil sampling was performed in November 2007 (site D only) and October 2009  after leaf senescence. In each of the six plots at each site, 10 random 0.1-m×0.1-m forest floor samples (Oe horizon) were collected, composited, and homogenized in order to ensure plot coverage and representation of all overstory tree species. Parts of the homogenized samples were immediately flash-frozen in liquid N2 to prevent RNA degradation, and the remainder, subject to enzyme measurements, was transported on ice and later stored at -20 °C.The influence of chronic atmospheric N deposition was investigated in four sugar maple (Michigan . OverstoIn both 2007 (at Site D) and 2009 , total RNA was extracted using a previously published protocol cbhI), laccase (lcc), and β-tubulin gene sequences using previously described primer sets and PCR conditions lcc genes, the lcc primer pair was modified and adjusted to reduce the degree of degeneracy from 32- and 64-fold to 8-fold. Hence, in the quantitative PCR (qPCR) approach, the new primer pair Cu1Fmod1 (5′- ACG GTY CAY TGG CAY GG -3′) and Cu2Rmod1 (5′- GRC TGT GGT ACC AGA AIG TNC -3′) was used. However, the both original lcc primer pairs were also used and provided the same relative results, but with a slightly lower sensitivity.We initially screened all cDNA for cellobiohydrolase  were independent of starting cDNA template concentration Quantitative PCR (qPCR) was performed using a Mx3000P (Stratagene) real-time PCR system and Brilliant SYBR Green qPCR Master Mix (Stratagene). QPCR-cycling parameters were a 10 min denaturation at 95 °C followed by 40 cycles of 30 s at 94 °C, 1 min at 50 °C, and 2 min at 72 °C with fluorescence measurement during the extension step. A melting curve was obtained by heating to 95 °C for 1 min, cooling to 55 °C for 30 s, and then ramping up the temperature to 95 °C at 0.5 °C min−ΔΔCT for cbhI and lcc in each cDNA with β-tubulin as the housekeeping gene T value for each gene within the ambient N deposition plots at each site, and then used these as the standard from which to calculate 2−ΔΔCT for each sample at that site. Samples were ranked by their relative expression levels, and the significance of the difference between ambient N and simulated N deposition was determined using the non-parametric Wilcoxon-Mann U test.To test the hypothesis that simulated N deposition suppresses ligno-cellulolytic gene expression, we calculated 2470  = 27.5 mM−1 cm−1) as the substrate . Cellobiohydrolase activity was determined using a fluorometric assay, in which methylumbelliferone-linked cellobiose was used as the substrate Potential cellobiohydrolase and phenol-oxidase enzyme activities were determined at Site D in 2007, and at all four sites in 2009. Extracellular enzymes were extracted from 1 g of litter suspended in 125 mL 50 mM acetate buffer (pH 5) using a Bio Homogenizer M133 . The suspension was continuously stirred and 50 µL aliquots were dispensed in 96-well microplates, with 8–16 analytical replicates per sample. Laccase activity was measured spectrophotometrically via 3 min interval-kinetic in a EL800 platereader  using 3 mM ABTS , 0.5 µM of each primer, and 50 µg of BSA. After an initial denaturation step of 3 min at 94°C, 20 cycles of 94°C for 30 s, 55°C for 45 s and 72°C for 90 s and a final extension step of 72°C for 15 min were carried out using Stratagene PCR cyclers . PCR products were gel-purified using the QIAquick Gel Extraction Kit (Qiagen), and cloned into the pCR 2.1-TOPO vector using the TOPO TA Cloning kit  according manufacturer protocol. Altogether, 48 clones from each of the 6 cDNA were randomly picked, cultured overnight in liquid Luria broth and bi-directionally sequenced at the Laboratory for Genomics and Bioinformatics at the University of Georgia using standard M13 primers. Contigs were constructed using Geneious 5.3 .In 2007, fungal 28S rRNA gene fragments  were amplified from cDNA obtained from Site D using the primers LR0R and LR3. Primer sequences were obtained from The rDNA 28S sequences were aligned using the FFT-NSi x1000 algorithm in MAFFT −1 2007). For CCA, we restricted the analysis to the 12 OTU that were recovered from at least two of the six plots at Site D, and tested the strength of the correlation between nitrogen availability and fungal community similarity through Monte Carlo simulation with 500 replications in Canoco Vs. 4.54 . Because variation in soil moisture content can influence the distribution of fungal species The relationship between simulated N deposition and the relative abundances of fungal OTU was assessed for the community active at site D in 2007 using a Canonical Correspondence Analysis CCA; . The rel


Egg is leading the top 8 allergenic foods in infants. During gastrointestinal digestion, various factors can affect the proteolysis of food allergens, such as interaction with lipids or bile salts. Therefore, the amount of immunologically active protein reaching the intestinal mucosa can be substantially modified. Here we investigated egg white digestion under physiological conditions, including presence of bile salts and phosphatydilcholine. We compared it to whole egg digestion and sought resistant proteins or fragments that react with IgE antibodies in allergic children's sera.Whole hen's egg and egg white were subjected to an in vitro gastroduodenal digestion consisting on a gastric pepsinolysis at pH 2, followed by a duodenal proteolysis at pH 7 with trypsin and chymotrypsin. Digestion patterns were analysed by RP-HPLC and 1D/2D SDS-PAGE . The binding of the digests to IgE present in a pooled serum from egg allergic children was investigated by western blotting.Among the major egg white allergens, ovalbumin (OVA) and lysozyme (LZ) were the most resistant to gastroduodenal digestion, whereas ovotransferrin (OVT) and ovomucoid (OM) were rapidly hydrolyzed during pepsinolysis. Three peptides were found to be the most resistant in the presence of phosphatidylcholine during duodenal digestion. Regarding the whole egg, the most abundant egg yolk protein, lipovitellin I, was partially hydrolyzed by pepsin and completely digested by duodenal enzymes in the presence of physiological amounts of bile salts. Coupling 2D electrophoresis and immunoblotting, we detected five major proteins highly immunoreactive. Besides the already known allergens OVA, OM, OVT and LZ, we also found ovoinhibitor strongly reacting with serum IgE. This minor protein has rarely been referred as an allergen. After 60 minutes of egg white duodenal digestion, there remained several resistant peptides of intermediate molecular weight (10-40 kDa) capable of binding IgE.


S938AFLS2, while the acidic phosphomimic mutants FLS2S938D and FLS2S938E conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2S938A, demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2S938A and FLS2S938D mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing S938AFLS2 or D997AFLS2 , but was normally induced in FLS2S938D plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.FLAGELLIN-SENSING 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing  As a Pattern-Recognition Receptor (PRR) that detects conserved microbial molecules, FLS2 mediates the plant innate immunity responses triggered by bacterial flagellin or the flagellin epitope flg22. Even though several protein kinases including BAK1, BKK1, BIK1 are known to play early roles in FLS2-mediated signaling, the specific FLS2 phosphorylation and kinase activities during receptor activation after ligand binding remained unclear. We used in vitro and in vivo methods to identify Ser-938 as a potential phosphorylation site of FLS2. Immune responses are blocked when Ser-938 is mutated to alanine but remain normal when mutated to the phosphorylation-mimic residues aspartate or glutamate, suggesting that phosphorylation of Ser-938 is critical for defense activation after flagellin is detected. Phosphorylation of BIK1 and BIK1-homologous proteins is dependent on FLS2 Ser-938, but the widely noted release of BIK1 from FLS2 and binding of BAK1 to FLS2 after flg22 exposure proceed even when Ser-938 is mutated and defense signaling is blocked, suggesting that phosphorylation of Ser-938 and BIK1 phosphorylation prior to release are key steps controlling defense activation after the FLS2 receptor initially detects flagellin. Arabidopsis thaliana FLAGELLIN-SENSING 2 (FLS2) Xanthomonas pathogens Pattern-Recognition Receptors (PRRs) can initiate innate immunity by perception of conserved Pathogen- (or Microbe-) Associated Molecular Patterns (PAMPs or MAMPs) Prior to ligand exposure, FLS2 is present in FLS2-FLS2 and FLS2-BIK1 complexes that are detectable by co-immunoprecipitation Protein phosphorylation, a reversible switching between phosphorylated and unphosphorylated forms, is a common mechanism by which cell signaling pathways are regulated −fls2 mutants expressing FLS2S938A protein, as is the phosphorylation of FLS2, while plants expressing the phosphomimic proteins FLS2S938D/E exhibit defense responses similar to those of wild-type plants. The data suggest that phosphorylation of Ser-938 is critical for FLS2-mediated immune responses. flg22-stimulated FLS2-BIK1 dissociation and FLS2-BAK1 association still occur with FLS2S938A protein, but phosphorylation of BIK1 and homologs is absent, suggesting revisions to mechanistic models of FLS2 function.In the present study, mass spectrometry (MS) was used to detect apparent autophosphorylation sites of FLS2 kinase purified from a cell-free translation system. Among these sites, we show that Ser-938 is particularly important. FLS2-mediated immune responses including seedling growth inhibition, ROS burst, callose deposition, and MPK phosphorylation are shut down in Arabidopsis To characterize the phosphorylation activity of FLS2, the inferred intracellular portion of FLS2  was expressed and purified from a wheat germ cell-free expression system and then subjected to MS analysis. A majority of the protein had a mass 80 Da greater than the predicated molecular weight, while a smaller fraction had a mass 159 Da greater than the predicated molecular weight, equivalent to the mass increases expected for the addition of either one or two phosphate groups, respectively . AlthougTo identify the detected phosphorylation sites, the in vitro-expressed intracellular domain of FLS2 was digested with trypsin and subjected to further mass spectrometry. As shown in FLS2 promoter, and then transformed into the Arabidopsis fls2 null mutant line Col-0 fls2-101. Wild-type FLS2-HA in a similar configuration has previously been shown to function normally S938DFLS2 and S938EFLS2 transgenic plants produced an ROS burst comparable to WTFLS2 transgenic Arabidopsis after flg22 treatment, while S938AFLS2 failed to mediate an ROS response to flg22. This suggests that Ser-938 is a functionally significant site on FLS2.To allow in vivo studies of protein function the above three serines were then converted by site-directed mutagenesis to phosphorylation-blocking alanine (Ala or A), a substitution that typically does not disrupt overall protein structure S938A, were present in transgenic Arabidopsis .For the site Ser-1084, transgenic Arabidopsis plants expressing ransgene . This suT867V could bind flg22 but lost responsiveness to flg22 treatment T867VFLS2, T867DFLS2 and WTFLS2 alleles under control of the CaMV 35S promoter and transformed them into the Arabidopsis fls2-101 line. Expression of the mutated proteins was confirmed by immunoblot analysis, and responsiveness to flg22 was tested using the seedling growth inhibition assay  and aspartate (D) by site-directed mutagenesis and transformed into Col-0 −efr mutant plants under control of the EFR native promoter. Multiple independent T1 transgenic plants were tested for EFR function using the seedling growth inhibition assay. As shown in S777A, EFRS778A, and EFRS781A) and phosphomimic  variants still conferred responsiveness to elf18, suggesting that unlike FLS2 Ser-938, these EFR serine residues are not essential for EFR function.Unlike the sites that are conserved across many or all non-RD kinases , the functionally important Ser-938 residue and nearby residues (FLS2 residues 933–942) are not well conserved among other non-RD kinases in the LRR-RLK subfamily XII S938D was observed . As with FLS2-BIK1 dissociation and FLS2-BAK1 association, no alteration in FLS2-FLS2 associations was detected when Ser-938 was mutated into a non-phosphorylatable alanine.Wild-type FLS2 is present in FLS2-FLS2 associations both before and after exposure to flg22 fls2-101 cells that carry a wild-type (non-epitope tagged) BAK1 and BIK1 but lack any FLS2 not supplied transgenically, kinase-dead FLS2D997A still associated with kinase-dead BAK1D416A after cells were exposed to flg22  translation was suggested to have autophosphorylating protein kinase activity. MS analysis identified Ser-909, Ser-938 and Ser-1084 as in vitro autophosphorylation sites or sites phosphorylatable by the wheat germ in vitro translation extract, in either case making them candidate in vivo autophosphorylation sites. Published phosphoproteomic screens of plant extracts have not detected phosphorylation of FLS2 The small ‘non-RD kinase’ sub-group of protein kinases was identified based on their lack of the highly conserved Arg-Asp (R-D) residue pair in kinase subdomain VIb fls2-101 Arabidopsis plants transgenically expressing a phosphomimic FLS2T867D protein or the previously studied non-phosphorylatable FLS2T867V both lose flg22-responsiveness. We also did not detect in vitro autophosphorylation of Thr-867 in mass spectrometry experiments. There are several possible explanations for these results, including: (i) Thr-867 is not a phosphorylation site, but mutations to Asp or Val cause function-blocking disruptions to FLS2 structure; (ii) Thr-867 is not an autophosphorylation site but it is a phosphorylation site, and both phosphorylated and non-phosphorylated status are necessary at some juncture for FLS2 signaling; (iii) Thr-867 is a phosphorylation site but only in vivo, and for this site the T867D mutation does not correctly mimic phosphorylation, or (iv) Thr-867 is a phosphorylation site but only in vivo, and both phosphorylated and non-phosphorylated status are necessary at some juncture for FLS2 signaling. Similar logic may apply to Ser-909 and Ser-1084, the other candidate autophosphorylation sites identified and tested in this study. Integrity of Ser-909 is needed for full FLS2 signaling, but the loss of function of S909D and S909E phosphomimic proteins suggests that phosphorylation of this residue is not needed to activate FLS2 function. Neither phosphorylation nor lack of phosphomimic status at Ser-1084 is required for substantial signaling by FLS2.Some potential FLS2 autophosphorylation or phosphorylation sites received lower priority in the present study, but merit brief discussion. Arabidopsis FLS2 Thr-867 was previously reported as a potential autophosphorylation site S938A), flg22 induction of downstream responses including seedling growth inhibition, ROS burst, callose deposition, MPK phosphorylation and restriction of P. syringae growth were all blocked. This was not true of FLS2S938D or FLS2S938E. Detectable FLS2 autophosphorylation activity and phosphorylation of BIK1, PBL1, PBS1 and PBL2 were also lost with FLS2S938A. However, the ligand-dependent release of BIK1 from FLS2 and the ligand-dependent association of BAK1 with FLS2 were not detectably altered by mutation of Ser-938. As noted in Results, the above yields two important conclusions: (i) Ser-938 controls essential elements of FLS2 activity other than FLS2-BIK1 and FLS2-BAK1 dissociation/association, and (ii) flg22-elicited FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2 activity.It is well established in mammalian systems that autophosphorylation of protein kinase-containing receptors plays a critical role in regulating signal transduction to downstream components after perception of ligand D997A), and could occur at sites other than Ser-938 (see FLS2S938D and FLS2S938A), and did not strictly require that Ser-938 be phosphorylatable or in the phosphomic state (see FLS2S938A). In future studies, it will be of interest not only to directly confirm phosphorylation of Ser-938, but also to learn about the in vivo stoichiometry, timing and target sites of FLS2 phosphorylation by BIK1.In vitro, a quantitative contribution of Ser-938 phosphorylation to BIK1 phosphorylation of FLS2 was suggested. However, some phosphorylation of FLS2 by BIK1 still occurred in the absence of FLS2 kinase activity , they would need to exhibit substantial flexibility if one kinase catalytic site is to phosphorylate more than one substrate in that complex. Such flexibility has been proposed for human growth hormone receptors, albeit with only partial supporting data Spatial models for FLS2 function must also accommodate the fact that the above inter-protein events are physically launched by extracellular interaction of the FLS2 LRR with flagellin or flg22. This FLS2-ligand binding event drives BIK1 release and BAK1 association, and can do so independent of FLS2 Ser-938 and plant defense activation. With each infusion of new experimental data, refined experimental questions arise. In the case of transmembrane LRR-RLK receptors, the demand is increasingly present for methods that can track the activity of single protein molecules over time, or that supply spatially defined protein complex data.The standard Center for Eukaryotic Structural Genomics (CESG) platform for cloning 4HCO3, 2.5 µl of 25 mM DTT, 2.5 µl ACN, 32 µl 25 mM NH4HCO3 and 3 µl trypsin solution (100 ng/µl Trypsin Gold from PROMEGA Corp. in 25 mM NH4HCO3). Digestion was for 2 hours at 42°C followed by the addition of 3 µl more of trypsin solution  and o/n incubation at 32°C. The reaction was terminated by acidification with 2.5% TFA (Trifluoroacetic Acid) to 0.3% final. 4 µl was loaded on the instrument.“In Liquid” digestion and mass spectrometric analysis was done at the Mass Spectrometry Facility . In short, 50 µg of purified protein was concentrated via acetone precipitation then re-solubilized under denaturing conditions for tryptic digestion with 10 µl of 8 M urea/100 mM NHPeptides were analyzed by nanoLC-MS/MS using the Agilent 1100 nanoflow system  connected to a hybrid linear ion trap-orbitrap mass spectrometer  equipped with a nanoelectrospray ion source. Capillary HPLC was performed using an in-house fabricated column with integrated electrospray emitter using 360 µm×75 µm fused silica tubing packed with Jupiter 4 µm C12 particles . Sample loading and desalting were achieved using a trapping column inline with the autosampler . HPLC solvents were as follows: for isocratic loading, 1% (v/v) ACN, 0.1% Formic acid; for gradient elution, Buffer A (0.1% formic acid in water) and Buffer B (95% (v/v) acetonitrile, 0.1% formic acid in water). Gradient elution was performed at 200 nL/min and increasing %B from A of 1 to 40% in 135 minutes, 40 to 60% in 20 minutes, and 60 to 100% in 5 minutes. The LTQ-Orbitrap was set to acquire MS/MS spectra in data-dependent mode with MS survey scans from m/z 300 to 2000 collected in centroid mode at a resolving power of 100,000. MS/MS spectra were collected on the 5 most-abundant signals in each survey scan. Dynamic exclusion of 40 seconds intervals with 1.05 m/z above and 0.55 m/z below previously selected precursors was employed. Singly-charged ions and ions for which the charge state could not be assigned were rejected for MS/MS. Raw MS/MS data was compared to a user-defined amino acid sequence database  using an in-house Mascot search engine  with methionine oxidation, asparagine and glutamine deamidation, and serine, tyrosine and threonine phosphorylation as variable modifications. Peptide mass tolerance was set at 20 ppm and fragment mass at 0.6 Da.Pfu Ultra II polymerase and cloned into the pCR8 vector (Invitrogen), resulting in pCR8-Prom-gFLS2 Pfu Ultra II polymerase. After digestion with Dpn I, PCR products were transformed into E. Coli DH5α. The resultant plasmids were verified by sequencing. The wild type and point-mutated versions of FLS2 under its native promoter were recombined into pGWB13 binary vector All oligonucleotide primers used for PCR are listed in BAK1, BIK1, PBS1, PBL1, and PBL2 were amplified by PCR with Pfu Ultra II polymerase and inserted into pCR8 vector. The DNA fragment flanking 35S promoter and NOS terminator was amplified from pGWB17 and ligated into pre-linearized plasmid pUC19 by Sma I, resulting pUC-GW17 BAK1, BIK1, PBS1, PBL1, and PBL2 were recombined into pUC-GW17 and verified by sequencing.Genomic DNA sequences for Agrobacterium tumefaciens GV3101 (pMP90) and transformed by the floral dip method fls2 T-DNA insertion mutant Col-0 fls2-101All the binary vectors were electroporated into Transgenic seeds (T1) were screened on 0.5× MS agar plates containing 25 mg/L kanamycin and 25 mg/L hygromycin in a growth chamber for 7 days. Positive transgenic seedlings were transferred into 24-well plates containing liquid 0.5× MS with or without 30 nM flg22 for 10 days of additional growth. Seedling growth inhibition was determined by dividing the weight of flg22-treated seedlings by the mean weight of untreated seedlings from the same experiment. For MPK phosphorylation, T2 transgenic seedlings from 0.5× MS liquid were treated with 2 µM flg22 for 15 minutes, and crude protein was extracted with 2× SDS buffer and separated on 12% SDS-PAGE gel. Phosphorylated MPK3 and MPK6 were detected by anti-P44/P42 antibody .Callose deposition analysis was performed as described by Adams-Phillips, et al. ROS burst experiments performed as described by Sun et al. P. syringae pv. tomato strain DC3000 at OD600 = 0.0001 in 10 mM MgCl2 solution. After 3 days, leaf discs were taken from four inoculated rosette leaves and ground in 10 mM MgCl2. The samples were then diluted serially, plated on NYGA plates with 25 mM rifampicin, and colony counts were recorded two days after incubation at 28°C.Six-week-old Arabidopsis seedlings were inoculated with FLS2 intracellular domain (encoding amino acids 840–1172) and full length BIK1 were amplified by PCR and inserted into pGEX 4T-2 at BamHI and HindIII sites. The resultant plasmids were transformed into BL21 for expression of recombinant protein. BL21 strains harboring plasmid were induced at OD600 0.6 by adding 0.2 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 12°C for 24 hours. The cells were collected at 4°C and resuspended in 10 ml 1× PBS (Sigma) supplemented with 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1% Triton X-100, 1 mg/ml lysozyme and put on ice for 30 minutes with slow shaking. After lysis using a French pressure cell , the lysate was centrifuged at 14,000 rpm for 30 minutes at 4°C. The resultant supernatant was applied to a column containing glutathione-agarose beads for protein purification. The column was washed with at least 20 bed volumes of 1× PBS. For thrombin digestion to separate FLS2 or BIK1 protein from GST tag, the beads were added to 2 µg/ml thrombin and incubated at 4°C overnight. GST-fusion proteins were eluted with reduced glutathione. GST-free proteins were eluted with 1×PBS. Both GST-fusion proteins and proteins without GST were dialyzed with exchange buffer (50 mM Tris (PH 7.6), 50 mM KCl, 2 mM DTT, and 10% glycerol).2, 5 mM MgCl2, 10 µM ATP and 10 µCi γ-32P-ATP at 30°C for 30 minutes. The reaction was stopped by adding EDTA to 1 mM final concentration. Samples were separated on 10% SDS-PAGE gel, and stained with Coomassie blue prior to autoradiography using storage phosphor screens and a Storm 840 phosphorimager (Amersham Biosciences).About 1 µg of purified FLS2 and/or BIK1 intracellular domain as described above were used for in vitro phosphorylation assays. Typically, assays were performed in buffer containing 50 mM Tris (PH 7.6), 50 mM KCl, 2 mM DTT, 5 mM MnCl6 cells) were transfected with 100 µg plasmid Arabidopsis mesophyll protoplasts were isolated from 6-week-old plants according to the method described by Yoo et al Protoplasts with expression of different proteins as described were lysed with 0.6 ml protein extraction buffer (50 mM Tris (PH 7.6), 150 mM NaCl, 0.5% Triton X-100, and 1× Cocktail protease inhibitor (Sigma)) for 30 minutes on ice. After centrifugation at 14,000 rpm for 15 minutes at 4°C, anti-cMyc was added to the supernatant and incubated at 4°C for 3 hours or overnight, and then 20 µl pre-equilibrated protein A agarose was added for 2 hours with end-to-end shaking at 4°C. Beads were collected by centrifugation at 500 g at 4°C and washed with 1 ml wash buffer (50 mM Tris (PH 7.6), 150 mM NaCl) at least 4 times. After washing, beads were eluted with 50 µl 1× SDS loading buffer by heating at 100°C for 10 min. Eluted proteins were separated on 10% SDS-PAGE gels and transferred to PVDF membrane by electroblotting. Immunoblot detection using the designated horseradish peroxidase-conjugated antibodies was performed using SuperSignal West Pico chemiluminescent substrate .fls2-101 plant transfected with plasmids of FLS2-cMyc, FLS2S938A-cMyc, and FLS2S938D-cMyc were incubated in W5 solution 32P-phosphate for 10 hours, and the subjected to immunoprecipitation as above using anti-cMyc. After washing 6 times with 1 ml protein extraction buffer, samples were separated on 10% SDS-PAGE gels and detected by autoradiography using a storage phosphor screen and Storm 840 phosphorimager (Amersham Biosciences).Protoplasts from Figure S1Mass spectrometry analysis of in vitro phosphorylation sites of FLS2.(PDF)Click here for additional data file.Figure S2Expression level of FLS2 in transgenic Arabidopsis.(PDF)Click here for additional data file.Figure S3Oxidative burst under unstimulated conditions.(PDF)Click here for additional data file.Figure S4Thr-867 of FLS2 is not likely to be an autophosphorylation site of FLS2.(PDF)Click here for additional data file.Figure S5Homology model of 3D structure of FLS2 kinase domain, generated using SWISS-MODEL, and annotated amino acid sequence of full-length FLS2 (At5g46330).(PDF)Click here for additional data file.Figure S6Lack of conservation of FLS2 Ser-938 in related non-RD kinases.(PDF)Click here for additional data file.Figure S7Functional test of the response to elf18 peptide mediated by EFR Ser-777, Ser-778, and Ser-781 mutants.(PDF)Click here for additional data file.Figure S8Ser-938 may be important for phosphorylation of FLS2 in vitro and in vivo.(PDF)Click here for additional data file.Figure S9Second example see also  of trans(PDF)Click here for additional data file.Table S1Primer sequences used in this study.(PDF)Click here for additional data file.


To rapidly quantify total immunoglobulin E levels in human serum, we developed a novel quantum-dot-based immunochromatographic assay that employs digital recording of fluorescence. It can detect IgE levels of 5–1000 kU/L, with a coefficient of variation ranging from 2.0 to 9.5%. The assay can be processed in 10 min. The developed assay was tested on 95 serum samples. The correlation coefficient between the IgE values obtained by the proposed assay and those obtained by a commercial ELISA kit was 0.9884. Our assay thus shows promise as a new diagnostic tool for IgE detection. More than a quarter of the world’s population suffers from allergic diseases such as bronchial asthma, allergic rhinitis, and dermatitis. Immunoglobulins of type E (IgE) play a major role in allergen recognition and the initiation of allergic reactions. IgE are located on the surface of mast cells and basophils; upon interactions with allergens they cause cell degranulation, leading to the release of mediators that induce allergic reactions Normally, the content of IgE in human blood is less than 0.001% of the total amount of all immunoglobulins Clinical diagnostic laboratories mainly use the enzyme-linked immunosorbent assay (ELISA) to determine the total IgE Immunochromatographic assays are attractive alternatives to ELISA because they are faster and less labor-intensive Currently available immunochromatographic tests for human serum can detect total IgE with qualitative precision. For example, the ALFA Total IgE test from Dr. Fooke Laboratorien  The use of other labels, such as fluorescent markers, in immunochromatographic assay may help decrease the detection limit and decrease the influence of the matrix. Several studies have reported low detection limits for assays based on fluorescent nanoparticles The purpose of our study was to develop an immunochromatographic test, using QDs as a label, for the determination of total IgE in human serum. Levels of IgE were assessed with a portable fluorescence detector REFLEKOM-UV, containing a UV light source Water-soluble quantum dots were obtained from Invitrogen . The QDs were composed of CdSe/ZnS and coated with carboxylated polymer, and their emission maximum was 625 nm. The non-ionic detergent Tween 20 and bovine serum albumin (BSA) were obtained from Sigma . Clone 4F4 of anti-human IgE monoclonal antibodies was obtained from Polygnost  and clone ME-113 was obtained from GeneTex . Goat anti-mouse IgG polyclonal antibodies were obtained from IMTEK . N-(4-dimethylaminopropyl)-N’-ethyl-carbodiimide hydrochloride (EDC) and sodium N-hydroxysulfosuccinimide (sulfo-NHS) were obtained from Fluka . Sephadex G-100 was obtained from MP Biomedicals . Standard solutions of IgE with concentrations of 5, 20, 50, 100, 200 and 1000 kU/L were obtained from Dr. Fooke Laboratorien . All other reagents were obtained from Chimmed . Deionized water, with a resistance of 18.2 MΩ • cm at 25°C, was obtained using a Simplicity system from Millipore  and used to prepare all aqueous solutions.CNPC-backed nitrocellulose membranes and pretreated TYPE-GBF-R7L sample pads were obtained from Advanced Microdevices . The following materials were purchased from Millipore : CFSP223000 adsorption pads, Amicon Ultra 100 kDa centrifugal filter units, and fiberglass macroporous CFCP203000 conjugate pads for application of quantum dot conjugates.We used the following buffers: 50 mmol/L phosphate buffer supplemented with 100 mmol/L NaCl, pH 7.4 (PBS 50), 10 mmol/L phosphate buffer supplemented with 100 mmol/L NaCl, pH 7.4 (PBS 10), and 50 mmol/L borate buffer, pH 8.5 (BB).Blood samples were collected from subjects  during allergy consultations as part of a routine health check-up at the Institute of Immunology , Moscow, Russia. Before sampling, all subjects had fasted for at least 12 h. Blood samples were collected into glass tubes without anticoagulant. Serum samples were divided into 200 µL aliquots and kept frozen at −20°C until analysis. All subjects gave written informed consent to participate in this study. All samples were provided anonymously. This study was approved by the Local Ethics Committee at the Institute of Immunology and the investigation was performed according to the ethical guidelines in Document No. 79-11/12, 11 October 2012.−3 mol/L. 50 µL of each activator solution was added into the reaction mixture. The molar ratio of QDs to activator was 1∶400. The reaction was performed with constant mixing on an Intelli-Mixer RM-2 shaker  for 90 min at room temperature in the dark. After the reaction, the mixture was concentrated and simultaneously dialyzed against BB to remove excess activator. The antibody-labeled QDs were isolated by centrifugal filtration five times in Amicon Ultracel 100 K centrifugal filter units at 10,000 g for 15 min each time. The collected conjugate was concentrated 10 times.The procedure is based on the technique presented in reference Gel filtration, on columns packed with Sephadex G-100 and equilibrated with PBS 10 was performed to further purify the conjugate from free low-molecular-weight substances and unconjugated antibodies The conjugate was diluted 20 times with PBS 10 and placed in the upper part of the column, followed by separation using the same buffer solution as an eluent. All fractions exhibiting color and/or fluorescence were collected. All fluorescent fractions were tested for binding by an immunochromatographic method, and those that showed maximum fluorescence intensity upon binding to the test zone were pooled.Fractions obtained after gel filtration were again concentrated and simultaneously dialyzed against BB by 10 centrifugations at 10,000 g for 15 minutes each time in Amicon Ultracel 100 K centrifugal filter units. After the last centrifugation, the 10-fold concentrated conjugate was transferred into a microcentrifuge tube and stored at 4°C.A solution of monoclonal anti-human IgE antibodies, dissolved in PBS 50 to a concentration of 3 g/L, was used to prepare the test zone. A solution of goat anti-mouse IgG polyclonal antibodies, dissolved in PBS 50 to a concentration of 1 g/L, was used to prepare the control zone. Aliquots of the antibody solutions were applied to a CNPC12 nitrocellulose membrane via an IsoFlow dispenser  The complete test structures were then assembled, and the CNPC12 nitrocellulose membrane with immobilized antibodies, the fiberglass macroporous membrane (CFCP203000) coated with the antibody-QD conjugates, a sample pad (R7L) and an adsorption pad (CFSP223000) were comprised. The multimembrane composite was cut into individual 3.5-mm-wide test strips by a guillotine cutter. These test strips were vacuum packed, with silica gel as a desiccant, in bags of laminated aluminum foil by using an FR 900 solder with a mini conveyor . Cutting and packing were conducted at 20–22°C in a climate-controlled room with relative air humidity not exceeding 30% The total level of IgE in the human serum samples was determined via ELISA using the Total IgE HRP EIA  according to the manufacturer’s instructions. A 100 µL of the dilution buffer was added to each well containing adsorbed mouse antibodies against human IgE. A 20 µL of either the serum sample or the total IgE positive/negative control sera was then added, and the plates were incubated for 30 min at room temperature. This step was followed by further incubation with100 µL/well anti-IgE antibodies conjugated to horseradish peroxidase. The plate was washed 3 times with washing buffer between each step. TMB substrate (100 µL/well) was then added, and the plates were incubated for 15 min at room temperature in the dark until the color developed, at which point, TMB peroxidase stop solution was added (50 µL/well). The optical density at 450 nm was measured by a Zenyth 3100 microplate multimode detector from Anthos .Samples and test strips were brought to room temperature (20–25°C) prior to analysis. A 20 µL of 5% Tween 20 was added to a 100-µL sample, and the solution was mixed. A test strip was immersed into the sample for 10 min, removed from the sample, and analyzed while wet. Fluorescence intensity in the test area was measured using a portable detector REFLEKOM-UV containing a UV light source equipped with Videotest software .Calibration curves were obtained using the standard solutions, by plotting the relationship between the known IgE concentration and the fluorescence intensity in the test zone. Based on the calibration curves, IgE concentration in the samples was estimated using the four-parameter sigmoidal and exponential equations in Origin 7.5  software.Anti-human IgE (clones 4F4 and ME-113) antibodies were conjugated with quantum dots by the activated ester technique We checked the differences of unconjugated and conjugated QDs by transmission electron microscopy, dynamic light scattering and immunochromatography. Transmission electron microscopy confirmed the absence of nanoparticles aggregation as well as the increase of particle sizes after the conjugation compared to unconjugated QDs. The same increase was found for data of dynamic light scattering; size values for this technique were higher due to impact of hydration shell. Immunochromatography results indicate the appearance of specific antigen-binding capacity of conjugated QDs –S4.The proposed method for IgE immunochromatographic assay is based on the “sandwich” immunoassay format. Anti-human IgE antibodies labeled with QDs bind to IgE contained in the sample. This complex in turn reacts with anti-human IgE antibodies applied to the working membrane.Buffer compositions  were selected based on previous results obtained for the immunochromatographic test system for determination of specific human serum IgE for the diagnosis of allergy to pollen, timothy meadow, with colloidal gold Anti-human IgE antibodies, clones 4F4 and ME-113, and were immobilized on the working membrane from a solution with the concentration of 1 g/L. Upon UV irradiation of the test strip, maximum fluorescence intensity in the test zone was achieved by a combination of the clone 4F4, immobilized on a nitrocellulose membrane, and the clone ME-113 labeled with quantum dots . All othWe then optimized the concentration of anti-IgE-QD conjugate. Changing the concentration of the conjugate from 4 to 20 nmol/L showed that the fluorescence intensity in the test zone increases with conjugate concentration and reaches a maximum at 12 nmol/L . A conjuNext, we investigated the relationship between the fluorescence intensity and the duration of the analysis. Using the optimal conditions determined in Section 2, and IgE standard solutions , we constructed a calibration curve for immunochromatographic analysis using our test system . The calTo determine the accuracy of the analysis, and to confirm differences between negative and positive samples, we also analyzed samples containing low (2 and 5 kU/L) levels of total IgE and samples without IgE (0 kU/L). Ten replicates of each sample were analyzed. When the calibration curve for the immunochromatographic analysis was plotted in the linear coordinates, it could be described by an exponential model  [36].(1)The calibration curve of ELISA  was obta2 = 0.9989). Thus the immunochromatographic data can be analyzed using the same model as data of ELISA.The given four-parameter sigmoidal model was used for further work.Next, we investigated the influence of the sample matrix on the immunochromatography results. IgE samples were prepared from a serum sample containing 100 kU/L of total IgE, which was used undiluted and also diluted by 2- to 10-fold with PBS 50. 2+ is measured. The limit of IgE detection was 5 pg/mL, but this assay necessitates about 2 days for providing. Liu and coworkers As indicated above, there are several publications describing application of nanocrystals for the immunodetection of human IgE by using such techniques as ELISA However, the both earlier described methods require long time for the analysis (20 hours or more), and due to this are less suitable for medical practice as compared with the developed immunochromatographic assay .We used 95 serum samples obtained from adult peoples to test the developed method. All samples were characterized by ELISA using the Total IgE HRP EIA kit . The 95 sera included 43 samples from healthy donors and 52 samples from the patients with allergic diseases. We have compared data for these two groups for further evaluation of the applicability of the developed immunochromatographic method. The variations in the fluorescent signal for serum samples from healthy donors and from patients with allergic diseases are shown on sigmoidal/Cexp ranged from 0.97 to 1.09, thus indicating good correlation between the sigmoidal and exponential functions for interpreting the immunochromatographic results.The approximations in 2 of 0.9884.Because nearly identical results were obtained by both methods of calculation, we chose the sigmoidal equation because it unifies processing of data acquired by both ELISA and immunochromatography. This equation was used to calculate the IgE content in 95 serum samples. As 2 = 0.9884) with the data obtained by a commercial ELISA kit.We have developed a quantum-dot-based immunochromatographic analysis method for the rapid quantitative determination of total IgE in human serum over a wide range of concentrations, 5–1000 kU/L and the coefficient of variation was less than 10%. Fluorescence of the bound label was measured using a portable handheld detector. The obtained results were well correlated  and quantum dots conjugated with anti-human IgE antibodies (B).(EPS)Click here for additional data file.Figure S2Size distribution of unconjugated quantum dots  and quantum dots conjugated with anti-human IgE antibodies  determined by transmission electron microscopy.(EPS)Click here for additional data file.Figure S3Size distribution of unconjugated quantum dots  and quantum dots conjugated with anti-human IgE antibodies  determined by dynamic light scattering.(EPS)Click here for additional data file.Figure S4Images of the test strips indicating specific conjugate binding. A – positive reaction in test and control lines owing to the interaction of anti-human IgE antibodies labeled by quantum dots with antibodies immobilized on the membrane. B – unconjugated quantum dots do not interact with the same antibodies immobilized.(EPS)Click here for additional data file.Methods S1Transmission electron microscopy; Dynamic light scattering; Immunochromatographic testing of conjugates binding.(DOC)Click here for additional data file.