In this study, we investigated the effects of the addition of ARA on cellulase hydrolytic action.α-We found that approximately 15% of the xylan was converted into AXOS during the hydrolysis of aqueous ammonia-pretreated corn stover and that this AXOS fraction was approximately 12% substituted with arabinose. The addition of ARA removes a portion of the arabinose decoration, but the resulting less-substituted AXOS inhibited cellulase action much more effectively; showing an increase of 45.7%. Kinetic experiments revealed that AXOS with a lower degree of arabinose substitution showed stronger affinity for the active site of cellobiohydrolase, which could be the mechanism of increased inhibition.Our findings strongly suggest that the ratio of ARA and other xylanases should be carefully selected to avoid the strong inhibition caused by the less-substituted AXOS during the hydrolysis of arabinoxylan-containing biomass. This study advances our understanding of the inhibitory mechanism of xylooligomers and provides critical new insights into the relationship of ARA addition and cellulose digestibility.The online version of this article (10.1186/s13068-019-1412-0) contains supplementary material, which is available to authorized users. Biofuels from lignocellulosic biomass have been considered a promising alternative to traditional fossil fuels –3. Howevl-arabinofuranosyl unit on either the C2- or C3-position of xylose, or both the C2- and C3-positions of xylose [O-methyl-glucuronopyranosyl groups [l-arabinofuranosidase (ARA), and acetyl esterase to remove the corresponding side groups; and β-xylosidases which hydrolyze xylooligomers to xylose from its non-reducing end. Single arabinofuranosyl substitutions that decorate β-D-xylopyranose residues can be removed by ARA of Glycoside Hydrolase Family 51, 54 or 62 ; arabinofuranose of di-substituted β-D-xylopyranose residues can be removed by GH 43 ARA [As previously reported, there was a nearly linear relationship between the increases in xylose and glucose yields during the hydrolysis of biomass after pretreatment by many technologies, such as ammonia fiber expansion, ammonia recycle, and hydrothermal , 12. Thif xylose , 14. Furl groups , 16. TheH 43 ARA , 18. HowH 43 ARA –21. TherH 43 ARA , 20, 22.The addition of ARA has shown increase in xylose yields, but whether ARA can significantly increase the hydrolysis of cellulose in lignocellulosic biomass remains controversial. Alvira et al. reported that the addition of ARA to the mixture of cellulase and endoxylanase resulted in increased conversion of cellulose in steam explosion-pretreated wheat straw into glucose . HoweverCompared to xylan, xylooligomers show a much stronger inhibitory effect on cellulase activity and present a major cause of the loss in cellulase activity . It was In this study, we investigated the role of additional ARA on the conversion of cellulose and xylan in ammonia aqueous-pretreated corn stover into fermentable monosaccharides. The formation and composition of xylooligomers during the hydrolysis process were tested. The inhibitory effects of xylooligomers on the commercial cellulase, CTec2, and the thermostable individual cellulases, including endoglucanase, cellobiohydrolase, and β-glucosidase, were compared before and after debranching by ARA. The kinetics of the inhibition of cellobiohydrolase by xylooligomers with or without substitution was analyzed to elucidate the mechanism behind the different inhibitory abilities.To assess whether substitution affects the inhibition of AXOS on cellulase, AXOS with or without debranching enzyme was added to the hydrolysis of Avicel. Two different types of cellulase, namely, commercial cellulase (CTec2) and a mixture of thermostable cellulases (CEL), were used in this study to compare their effectiveness. The amount of monosaccharides in the AXOS preparation was measured by HPLC. No xylose or arabinose was observed, indicating that all of the arabinose substituents were linked to the AXOS. To determine the content of the arabinose substituent, the AXOS fraction was treated with 4% sulfuric acid as described by the NREL . The resThe results in Fig. l-arabinofuranose from β-D-xylopyranose residues and that most of the residual arabinofuranoses released were doubly substituted to β-D-xylopyranose backbone sugars.In addition, some xylose (1.13 mg/mL by CTec2 and 0.34 mg/mL by CEL) was detected, showing that a considerable amount of endoxylanase and/or β-xylosidase activity is present in CTec2, while only small amounts of endoxylanase and/or β-xylosidase activity are present in the thermostable CEL mixture, which agrees well with previous results on the composition of these cellulase preparations , 32. AftIn this work, the addition of ARA to AXOS resulted in the release of xylose , arabinose , and less-substituted AXOS in the hydrolysate. Our previous results have confirmed that such a low amount of xylose showed no inhibitory effect on Cel5A, Cel7A, and Cel3A . The resThe pretreatment of corn stover with aqueous ammonia, a commonly used pretreatment technology that can retain most of the xylan, was used to investigate the effect of ARA during the hydrolysis of the lignocellulosic biomass Fig. a, b. As To mimic this possible inhibitory effect, an AXOS preparation  was added to the hydrolytic system of AA-CS Fig. c. As expThe effects of ARA addition on the inhibition of AXOS on the individual cellulases, Cel5A, Cel7A, and Cel3A were evaluated to thoroughly understand the role of ARA addition on cellulase activity Fig. . The dat2-α-l-Arabinofuranosyl-xylobiose (A3X), 23-α-l-arabinofuranosyl-xylotriose (A2XX), and 23,33-di-α-l-arabinofuranosyl-xylotriose (A2+3XX), were used in these experiments as comparisons , xylotriose (XXX), and the corresponding xylooligomers with arabinose groups, 3ons Fig. . All theKm and Ki values were calculated to quantify the degree of inhibition  (P < 0.05) and A2+3XX (7.57 mM) (P < 0.01). These results further confirmed that xylooligomers without an arabinose group tended to be more easily and firmly bound to the active site of Cel7A, and thus showed a stronger inhibitory effect on Cel7A compared with those that were fully substituted.The on Table . It was O-1,4-structure of xylooligomers and cellobiose was the reason why xylooligomers could occupy the active site of Cel7A, and thus prevent the cellulose chain from moving into the tunnel of cellobiohydrolase [Ki, of xylobiose on Cel7A was found to be 0.98 mM, which was lower than that of xylotriose (3.22 mM) (P < 0.01) , 23-α-l-arabinofuranosyl-xylotriose (A2XX), and 23,33-di-α-l-arabinofuranosyl-xylotriose (A2+3XX) were purchased from Megazyme . All other chemicals used in this work were of analytical grade and purchased from Sigma.Microcrystalline cellulose (Avicel PH-101) was purchased from Sigma Chemical Co. . Arabinoxylan (P-WAXYRS) was purchased from Megazyme , and its detailed structure is shown in Fig. Corn stover was collected from a local farm in Yangling, China. The material was milled, sieved through a 60-mesh screen scale, and pretreated with 25% (w/v) aqueous ammonia with a solid to liquid ratio of 1:10 at 50 °C for 24 h. The composition of these pretreated materials was analyzed as described by the National Renewable Energy Laboratory (NREL) , and theThermoascus aurantiacus (Cel5A), thermostable GH 7 family cellobiohydrolase from T. aurantiacus (Cel7A), and thermostable GH 3 family β-glucosidase from Acremonium thermophilum (Cel3A) [Trichoderma reesei strain in which the genes cbh1, cbh2, egl1, and egl2, encoding Tr Cel7A, Tr Cel6A, Tr Cel7B and Tr Cel5A, respectively, had been deleted as previously described [T. reesei enzymes. Because of the high expression level of native T. reesei xylanase genes, it is difficult to totally inactivate the xylanase activity. Hence, there are still some xylanolytic enzymes present in the heat-treated enzymes [Thermostable glycosyl hydrolase (GH) 5 family endoglucanase from  (Cel3A)  were proescribed –45. The  enzymes . The the enzymes  with bov® CTec2  was used as a reference commercial cellulases. The activity of the enzyme was 176 FPU/mL (284.8 mg protein/mL) as measured by the IUPAC standard assay. GH 11 endo-xylanase from Trichoderma viride  and GH51 α-l-arabinofuranosidase from Aspergillus niger  were purchased from Megazyme .The commercial enzyme preparation Cellicg, 4 °C for 15 min.The AXOS used in this study was prepared by the hydrolysis of arabinoxylan (5%) using endoxylanase (2 mg/g DM) for 1 h. A sample was then withdrawn and boiled for 10 min to stop the enzymatic hydrolysis. Protein and unhydrolyzed arabinoxylan in the hydrolysate were removed using Amicon Ultra centrifuge tubes (Millipore) by centrifugation at 16,280×3 at 50 °C [g for 10 min, and the supernatants were analyzed for glucose and xylose.The hydrolysis of Avicel and corn stover after pretreatment with aqueous ammonia (AA-CS) using cellulase preparations was performed in tubes with a working volume of 3 mL in 50 mM sodium citrate buffer (pH 5.0) containing 0.02% NaNat 50 °C . The hydp-nitrophenol-D-cellobioside (pNPC) in 50 mM sodium citrate buffer at 50 °C for 30 min as described by Deshpande et al. [2CO3. The formation of pNP was measured at an absorbance at 410 nm to determine the activity of Cel7A. A kinetic inhibitory analysis of Cel7A was carried out with different concentrations of XX, XXX, A3X, A2XX, and A2+3XX. Lineweaver–Burk plots were applied to monitor the mechanism of inhibition. The kinetic parameters were determined by fitting the data to the appropriate Michaelis–Menten equations and plotting the double reciprocal of the reaction rates and the substrate concentrations as Lineweaver–Burk plots. In addition, Michaelis maximum velocity (Vmax), constant values (Km), and inhibitory constant (Ki) were evaluated.Cel7A activity was assayed by the hydrolysis of 1 mg/mL e et al. . The rea2SO4. After incubation at 121 °C for 1 h in an autoclave, the samples were withdrawn and neutralized with CaCO3 to a pH of 5–6. The samples were then filtered into HPLC vials to determinate the amount of monosaccharide as described below. The amount of oligomer sugars was calculated by subtracting the monosaccharide concentration of the non-hydrolyzed samples from the total sugar concentration of the acid-hydrolyzed samples.The concentrations of the oligomer sugars in the supernatants were analyzed using the procedure published by the NREL . The fil2SO4 as the eluent at a flow rate of 0.5 mL/min. Before injection, the samples were filtered through 0.22-µm MicroPES filters, and a volume of 10 µL was injected. The peaks were detected by refractive index and were identified and quantified by comparison to the retention times of authentic standards .The amounts of glucose, xylose, and arabinose were determined using an HPLC system . The system was equipped with a refractive index detector and an autosampler. An ion-moderated partition chromatography column (Aminex column HPX-87H) with a Cation H micro-guard cartridge was used. The column was maintained at 45 °C with 5 mM HThe glucose, xylose, and arabinose yields in the hydrolysis of the biomass material were calculated as described in NREL LAP-009 .P values of 0.05 or less were considered significant.Analysis of variance (ANOVA) was performed at 95% confidence level to compare group means of experimental data in triplicate using Microsoft Excel 07 and GraphPad Prism. Differences with Additional file 1: Figure S1. Effect of arabinose on hydrolytic action of cellulases. Hydrolysis of 2% Avicel by CTec2 (10 FPU/g DM) and CEL  with the addition of arabinose  at 50 °C for 48 h. The error bars represent the standard error of three independent experiments.Additional file 2: Figure S2. Hydrolysis of 10% corn stover after pretreatment with aqueous ammonia (AA-CS) by CEL  (B), and XYL (4 mg/g DM), and/or ARA (2 mg/g DM) at 50 °C for 24 and 72 h. The error bars represent the standard error of three independent experiments.


Antibody Fc-driven engagement of macrophages is critical for evoking cellular activation and effector functions and influencing tumour-associated macrophage (TAM) recruitment. We previously reported that IgE class antibodies promote restriction of cancer growth in rodent models associated with significant TAM infiltration. However, the human macrophage-associated IgE-Fc Receptor (FcεR) axis remains unexplored. We investigated the effects of anti-tumour IgE stimulation on human macrophage activation.Human blood monocyte-differentiated quiescent (M0), classically-(M1) and alternatively-(M2) activated macrophages were crosslinked with IgE and polyclonal antibodies to mimic immune complex formation. We examined surface marker expression, cytokine secretion, protein kinase phosphorylation and gene expression in IgE-stimulated macrophages and IgE antibody-dependent macrophage-mediated cytotoxicity (ADCC) against tumour cells.Lyn gene expression by M1 and M2 macrophages. Anti-tumour IgE engendered ADCC of cancer cells by all macrophage subsets.A proportion (40%) of M2 and (<20%) M0 and M1 macrophages expressed the high-affinity IgE receptor FcεRI. IgE crosslinking triggered upregulation of co-stimulatory CD80, increased TNFα, IFNγ, IL-1β, IL-12, IL-10, IL-13, CXCL9, CXCL11 and RANTES secretion by M0 and M2 and additionally enhanced MCP-1 by M2 macrophages. IgE-stimulated M1 macrophages retained secretion of pro-inflammatory cytokines. IgE crosslinking enhanced the FcεRI-dependent signalling pathway, including phosphorylation of the Lyn kinase, ERK1/2 and p38 in M2 macrophages and upregulated IgE can engage and re-educate alternatively-activated macrophages towards pro-inflammatory phenotypes and prime all subsets to mediate anti-tumour functions. This points to IgE-mediated cascades with potential to activate immune stroma and may be significant in the clinical development of strategies targeting tumour-resident macrophages. Tumour-associated macrophages (TAMs) with alternatively-activated pro-inflammatory features represent a significant component of the immune infiltrate in the tumour microenvironment (TME). Monoclonal antibodies may influence macrophage activatory states and functions, however the impact of antibody isotype on engagement of different macrophage subsets remains unclear. We have previously reported that IgE class antibodies recognising tumour-associated antigens elicit anti-tumour immune responses in rodent models. IgE-mediated anti-tumour effects were associated with an increased influx of macrophages into tumour lesions likely engendered through IgE Fc-mediated cascades.In this study we demonstrate that human IgE is responsible for potentiating Fc-mediated activation of alternatively-activated macrophages that leads to enhanced FcεRI-dependent signalling and a cytokine signature that bears characteristics normally associated with classically-activated, pro-inflammatory macrophages. Moreover, we show for the first time that IgE can prime quiescent, classically-activated and alternatively-activated macrophages to mediate anti-tumour cell cytotoxic effects.This cross-talk of IgE class antibodies with human macrophages may be significant for the clinical development of cancer immunotherapies that exploit the plasticity of these often tissue- and tumour-resident cells to restore their antitumor properties. Our findings thus point to IgE class antibodies as mediators of a cascade with potential to activate and re-educate immune stroma against cancer.Alt-text: Unlabelled Box1Tumour-associated macrophages (TAMs) form a significant proportion of the immune cell infiltrate and are thought to play important roles in defining the pro- or anti-tumour status of the tumour microenvironment (TME). The mechanisms by which macrophages can influence tumorigenesis, though, has been controversial: these cells represent a multi-faceted immune compartment and their contributions in shaping a pro- or anti-tumour environment heavily depend on their polarisation status . While tSeveral anti-cancer approaches targeting macrophages have been explored aiming to either inhibit their tumour-supporting functions or to promote their anti-tumour effects. More specifically, macrophage-centred anti-cancer strategies can include macrophage depletion, modification of macrophage recruitment and macrophage re-polarisation or re-programming. Macrophage reprogramming involves the suppression of alternatively-activated (M2) macrophages or, possibly, the stimulation of an M1-biased phenotype in the TME . The devThe demonstration that re-polarisation of macrophages can be achieved through monoclonal antibody treatment opened the door to the development of new therapeutics targeting macrophage surface proteins . We previously demonstrated that treatment with MOv18 I.E. a monoclonal human/chimeric antibody engineered with Fc regions of the IgE class and specific for a tumour-associated antigen (TAA) , triggered an immune-activatory TNFα/MCP-1 axis in tumours, which resulted in recruitment of macrophages into tumour lesions and was associated with significantly-reduced tumour growth , in a syIn our study we investigated how a monoclonal antibody  from healthy donors. To isolate PBMCs, equal volumes of blood and 2% FCS/2 mM EDTA were gently mixed to a final volume of 30 ml and gently pipetted on top of 15 ml of Ficoll-Paque™ PLUS density gradient in a 50 ml conical tube. The tube was then centrifuged at 1200 x g with slow acceleration and no brake at room temperature (RT) for 20 min. The plasma interface was collected and washed with PBS at 600 x g at 4 °C for 10 min. The erythrocytes present in the sample were lysed with RBC lysis buffer for 5 min at RT, followed by washing with PBS + 2% FCS/2 mM EDTA. Human samples were collected with informed written consent, in accordance with the Helsinki Declaration. Study design was approved by the Guy's Research Ethics Committee, Guy's and St. Thomas' NHS Foundation Trust. Peripheral blood was also obtained through the UK National Health System (NHS) Blood and Transplant system from anonymous donor leukocyte cones.K2.2Isolated PBMCs were passed through a 40 μm cell strainer to remove cell clumps. Next, PBMCs were transferred in a new vial and washed with PBS at 600 x g for 5 min. After discarding the supernatant, cells were resuspended in 400 μl of MACS buffer (PBS + 0.5% FCS/2 mM EDTA). Human monocytes were isolate by negative selection, following the Pan Monocyte Isolation Kit protocol (Miltenyi Biotec). The purity of monocytes was evaluated by flow cytometry staining with a BV786-conjugated monoclonal antibody anti-CD14 and BV711-conjugated anti-CD16. Acquisitions were performed using a BD FACS Canto™ II at the Biomedical Research Centre Flow Cytometry Core, King's College London.2.3ex vivo. Thereafter, every 3 days, half of the volume in each well fresh were replaced with media containing 25 ng/ml GM-CSF (for M1) or 40 ng/ml M-CSF (for M2). On Day 7, 20 ng/ml of Interferon-Gamma (IFNγ) and 100 ng/ml of Lipopolysaccharide (LPS) were added to GM-CSF cells for 72 h. M-CSF cells were instead added with 20 ng/ml of Interleukin-4 (IL-4) for 72 h.Isolated monocytes were resuspended in RPMI 1640 2% FBS to promote adhesion and cells were seeded in 6-well plates at a density of 1–1.5 × 10 [2.45 cells per sample. Cells were detached with PBS + 5 mM EDTA, resuspended in PBS + 2% FBS (FACS buffer) and incubated with 5 μg/ml of primary antibody for 30 min at 4 °C, followed by a wash with 3 ml of FACS buffer (spinning at 500 g for 5 min). Cells were incubated with the secondary antibody goat anti-human IgE–fluorescein isothiocyanate  or goat anti-human IgG1–fluorescein isothiocyanate  at 30 μg/ml in FACS buffer for 30 min at 4 °C, followed by one wash as above. Samples were resuspended in 100 μl of FACS buffer and analysed with a BD FACS Canto™ II.Binding of SF-25 IgE and IgG1 to FcεR- and FcγR-expressing cells or antigen-expressing cells was evaluated by using 102.55 cells were aliquoted in FACS tubes in RPMI1640 medium supplemented with 2% FBS. Cells were incubated with 5 μg/ml of IgE antibody for 30 min at 4 °C. Macrophages were then washed with PBS to remove any unbound antibody. Cells were then treated with 1 μg/ml of goat polyclonal anti-human IgE–fluorescein isothiocyanate  for 30 additional mins at 4 °C. Cells were washed again with PBS by centrifugation for 5 mins at 400 ×g and resuspended in FACS buffer, before analysing them on a BD FACS Canto™ II.M0, M1 and M2 monocyte-derived (MDM) macrophages were detached with Accutase and 1 × 102.6M0, M1 and M2 (MDM) macrophages were incubated with 5 μg/ml of IgE antibody for 30 min at 37 °C by adding the antibody solution directly onto the cell culture plate. Cells wells were washed with PBS to remove any unbound antibody. Cells were then treated with 1 μg/ml of goat polyclonal anti-human IgE–fluorescein isothiocyanate  for 30 more mins at 37 °C. Cells were harvested 4 h after stimulation to examine soluble mediator release and 24 h after stimulation to investigate surface marker expression.2.7Macrophage supernatants were collected 4 h post-stimulation, centrifuged at 600 x g to remove floating cells and diluted 1:1 with sterile PBS. The multiplex assay was performed with the Magnetic Luminex Kit following the protocol instructions. The plate was sealed and left on the shaker up to 90 min until ready to be analysed on a Luminex™ analyzer.2.86 cells/ml. 5 ml poly-propylene tubes were used for staining and flow cytometry acquisition. To each sample, 1 μl of each of the staining antibody solution was added, and cells were incubated for staining at 4 °C for 40 min, before being washed with 2 ml of FACS buffer and centrifuged as above. Cells were resuspended in 100 μl of PBS only and stained for live/dead cell discrimination with LIVE/DEAD® Fixable Dead Cell Stain Near-IR and incubated at room temperature for 30 min in the dark. Tubes were added with 2 ml of PBS and washed by centrifugation as above, before being resuspended in 200 μl of FACS buffer and acquired at BD FACS Canto™ II.The day after the treatments, cells were detached with Accutase (Sigma). Plates were incubated for 10 min at 37 °C to facilitate detachment. After the incubation, 1 ml of RPMI 1640 supplemented with 10% FCS and penicillin/streptomycin was added to each well. Cells were counted, washed with PBS by centrifugation at 400 x g for 5 min and resuspended at a concentration of 1 × 102.9Tim-3 or Lyn kinase) and 6 μl RNAse free water. The qPCR master mix was added to every well containing DNA. Negative qPCR controls included cDNA-free wells. 40 cycles of RT-qPCR were performed.cDNA samples were diluted 1:4 with RNAse/DNAse free water and 2 μl were distributed in triplicate on a fast-optical 96-well reaction plate. A qPCR master mix was prepared by mixing 10 μl of TaqMan gene expression master mix, 1 μl of GAPDH Probe, 1 μl of the GOI Probe (T cell (or transmembrane) immunoglobulin and mucin domain protein 3 2.106 cells. Cells were incubated at 37 °C for 10 min and the reaction was stopped with one wash in ice-cold RPMI 1640 standard medium. Normal culture conditions were resumed overnight. The following day, CFSE-labelled tumour cells were detached using PBS + 5 mM EDTA, washed in RPMI 1640 standard medium and re-suspended to 1 × 105 cells/100 μl culture medium. Macrophages were detached with Accutase (Sigma), re-suspended in culture medium and mixed with the tumour cells at a 10:1 ratio. Cells were placed in FACS tubes and co-incubated at 37 °C for 3 h in the presence of either PBS or 5 μg/ml of either SF-25 IgE or control antibody. After incubation, cells were washed in FACS buffer and macrophages were identified by staining mixed cell populations with 5 μg/ml of anti-human CD206-APC/Cy7-conjugated antibody to detect M1 or anti-human CD163-PE/Cy7-conjugated for M2 for 30 min at 4 °C. Cells were washed once and re-suspended in 200 μl FACS buffer for immediate acquisition using a dual laser FACSCanto™ (Becton Dickinson). Every sample was treated with 100 μl of DAPI (100 ng/ml), mixed using a vortex and acquired immediately. Tumour cell killing was calculated on the percentage of DAPI+ tumour cells and the total number of tumour cells in SF-25 antibody-treated samples compared to negative controls. Analysis of flow cytometry data was performed by FlowJo (TreeStar Inc) software.ADCC/ADCP assays were performed using a protocol adjusted from a previously-described method . Briefly2.11Proteome Profiler Human Phospho-Kinase Array (R&D Systems) were incubated with 400 μg lysate overnight at 4 °C. The following day, chemiluminescent detection was performed according to manufacturer's indications. Densitometry analysis was conducted using ImageJ.2.12t-test p-value performed on the means of each condition (baseline/IgE crosslinked), resulted in a P value of <0.05. A False Discovery Rate of <0.05 and a minimum gene set size of 4 was used to filter the results. Overly general pathways and processes were manually removed. Version 67 of the Reactome database was used. The KEGG database was used to depict the structure of the FcεRI pathway.Pathway analysis was performed using the Reactome Pathway Analysis tool .The set 2.13in vitro and in ex vivo evaluations. Clinical associations of gene expression were assessed using publicly-available data in Kaplan-Meier Plotter . One way-ANOVA was performed in combination with Bonferroni's or Tukey's post-test to determine statistical significance of the data.All statistical analyses were performed using GraphPad™ Prism Software (version 6.0). Error bars represent SD and SEM in 33.1++, CD16−), intermediate  and alternative  subsets . Non-adherent, non-monocytic cells were removed after two hours of seeding in serum-free medium, consistently yielding >80% monocyte purity based on CD14 and CD16 expression and physical parameters . Macrophages were differentiated over 7 days  and alternatively-activated M2) subsets  subsets ,17 comprWe ascertained expression of key markers of macrophage differentiation , and the mannose receptor CD206 for M2) B. Consisn = 4), while CD23 expression was expressed by 15–20% of all macrophage phenotypes .Very little information is available regarding IgE receptor expression for distinct macrophage subpopulations . We founex vivo in the absence of antigen engagement and immune complex formation [Based on the high affinity of IgE for cognate Fc receptors and its slow dissociation rates most notably following binding to FcεRI on immune cells, we hypothesised that some endogenous IgE may be retained by effector cells ormation ,25. An aormation . Over 20Together, as previously demonstrated with primary human monocytes ,30, thes3.2We previously reported that systemic administration of rat anti-tumour IgE was associated with recruitment of CD80+ macrophages into syngeneic tumours of immunocompetent rats . Here weBased on previous evidence that IgE can influence macrophage recruitment and activation in rodent models , we invePro-inflammatory cytokines TNFα, IFNγ, IL-12, IL-1β: Crosslinking of IgE led to a drastic increase (from 100 to approximately 2000 pg/ml) in TNFα secretion in both M0 and M2 macrophages, while IgE did not affect the low TNFα levels produced by M1 cells , which remained unaffected upon IgE treatment. Baseline concentration of IL-1β were very low in all three cell subsets. Crosslinking of cell-bound IgE triggered increased IL-1β secretion in M0 and M2 macrophages, while stimulation did not influence production by M1 macrophages.M1 cells B. IgE crAnti-inflammatory cytokines IL-4, IL-6, IL-10, IL-13: IL-4 production slightly increased upon IgE crosslinking in both M0 and M2 subsets and remained unaffected in M1 cells. Baseline levels of IL-10 and IL-13 were similar between macrophage subsets, however IgE crosslinking upregulated IL-10 and IL-13 release by M0 and M2 subsets but not by M1 macrophages. IL-6 secretion remained unchanged in M1 macrophages but increased upon treatment with IgE only and with IgE/anti-IgE crosslinking in both M0 and M2 subsets ( subsets C.Interferon Gamma-induced chemokines MIG (CXCL9) and I-TAC (CXCL11): In M1 macrophages, secretion of MIG (CXCL9) and I-TAC (CXCL11) remained unchanged (ca. 400 pg/ml) with IgE stimulation, while crosslinking of IgE in M0 and M2 macrophages led to modest increases. Levels of I-TAC (CXCL11) were 4-fold greater in M1 macrophages (ca. 400 pg/ml) compared to M0 and M2 at baseline, however, IgE crosslinking increased secretion of I-TAC (CXCL11) by M0 and M2 cultures from ca. 80 to ca. 160 pg/ml in M0 cells and from 50 to ca. 200 pg/ml in M2 macrophages (rophages D.Macrophage chemoattractant chemokines MCP-1 (CCL2) and RANTES (CCL5): In a rat model of cancer and in human monocytes in vitro, Josephs and colleagues demonstrated, that IgE crosslinking triggered a TNFα/MCP-1 axis, which resulted in recruitment of macrophages into tumours [ tumours . Here, cTogether these data demonstrate that crosslinking of cell-bound IgE triggered enhanced expression of cytokines TNFα, IFNγ, IL-12, IL-1β, IL-6, IL-10, IL-13 and of the chemokines MIG (CXCL9), I-TAC (CXCL11) and RANTES (CCL5) by M2 and M0 macrophages. IgE crosslinking on M2 macrophages but not on M0 or M1 subsets, triggered enhanced secretion of the chemoattractant MCP-1 (CCL2). On the other hand, M1 macrophages appeared to retain their cytokine/chemokine expression upon IgE activation including expression of pro-inflammatory mediators such as IFNγ and IL-12 .Table 1S3.3via a phosphorylation array   A. Furtheatabase) B. These 3.4Lyn and the checkpoint molecule Tim-3 (T cell or transmembrane immunoglobulin and mucin domain protein 3), both known to participate in the FcεRI signalling pathway in mast cells [Lyn mRNA expression increased to 2-, 7- and 70-fold in M0, M1 and M2 macrophages, respectively , lung (P = 6.7e-05) and ovarian (P = .036) cancers. These findings suggest that enhanced Lyn gene expression - one of the downstream signalling effects of IgE crosslinking its FcεR on alternatively-activated and classically-activated macrophages - may be associated with better prognosis in cancer patients. On the other hand, the association of Tim-3 gene expression with clinical outcomes appeared dependent on the tumour type: specifically, higher levels of Tim-3 expression in lung and ovarian cancers correlated with improved patient prognosis, while it was associated with less favourable overall survival in patients with gastric cancer. This suggests that Tim-3 may exert different functions in different malignancies. These findings may be significant in future IgE class-mediated immune cell activation in different solid tumour types.To gain insights into a potential clinical relevance of IgE/FcεR signalling through  origins B. We fou3.5ex vivo can be activated by IgE to kill cancer cells by applying a three-colour flow cytometric assay to detect cytotoxicity and phagocytosis (14)  A. The ansis (14) B. SF-25 In summary, crosslinking IgE antibodies on the surface of human macrophages influenced their polarisation status , leading4Macrophages represent a major component of the tumour microenvironment in many solid cancers. Their phenotypes and activation states can be influenced in the TME in response to environmental stimuli, so that different macrophage subsets could orchestrate different and often opposite aspects of the immune response, striking a balance between anti-tumour immunity and wound healing or inflammation. The potential of antibodies to influence the dynamics and polarisation states of these cells against cancer may offer opportunities to tip the balance in favour of host immunity. However, the mechanisms of human macrophage-associated immune complex formation engendered by different antibody isotypes, and especially through the IgE/FcεR (IgE FcR) axis, remain insufficiently understood.in vivo models of cancer, associated with significant recruitment of macrophages towards tumours [We have previously reported that systemic treatment with anti-tumour IgE reduced tumour growth in different  tumours ,29. Howe tumours . We therAround 40% of M2 macrophages expressed the high affinity IgE receptor FcεRI, while approximately 20% of cells in each subset expressed the low affinity IgE receptor CD23. Macrophage cell surface-bound IgE crosslinked on alternatively-activated macrophages triggered increased expression of the co-stimulatory molecule CD80, a typical M1 macrophage marker. Upregulation of the co-stimulatory molecule CD80 on the surface of professional antigen-presenting cells (APC) including macrophages, may signify cell maturation and also confer capacity for effective antigen presentation and may point to the potential of IgE class antibodies to promote macrophage maturation, polarisation and APC functions. These findings are consistent with previously described evidence of: a) infiltration of immune cells, including macrophages, in human ovarian cancer patient-derived xenograft residual tumours in immunocompromised mice treated with an anti-tumour IgE and human PBMCs as effector cells; and b) higher recruitment of monocyte/macrophages into tumours and enhanced expression of CD80+ rat macrophages into syngeneic tumours in an immunocompetent rat model following systemic treatment with rat MOv18 IgE . FurtherWe furthermore explored whether IgE stimulation may promote differential activation of specific macrophage subsets. In M1 macrophages, IgE crosslinking retained the production of key pro-inflammatory cytokines including IFNγ and IL-12. In concordance, in Josephs et al., we recently demonstrated upregulation of pro-inflammatory immune-associated pathways including IL-12 and NK-cell immune activation signatures in the lungs of tumour-bearing rats treated with MOv18 IgE . ConsideImportantly, secretion of IL-1β, IL-6, IL-10, IL-12, IL-13, IFNγ, RANTES, MIG and I-TAC was triggered by crosslinking of cell-bound IgE on quiescent M0 and on M2 macrophages. This may suggest that the two subsets might feature similar molecular mechanisms in their ability to regulate activation of the FcεRI pathway. Furthermore, production of all soluble mediators tested was either retained at the same level or increased with IgE-crosslinking. Although the cytokine panel secreted by M0 and M2 after crosslinking with IgE did not correspond to a defined macrophage subtype, the mediator signature displayed by IgE-stimulated cells may represent a newly polarized macrophage subset secreting both pro- and anti-inflammatory mediators, as well as chemoattractant factors . An addiin vivo suggested that tumour antigen-specific IgE treatment influences the spatial distribution and activation of macrophages around tumour lesions. Systemic IgE treatment in this model was also associated with a specific cytokine signature, featuring elevated levels of TNFα, MCP-1 and IL-10 in the tumour microenvironment and upregulated expression of TNFα by tumour-associated macrophages in rat lungs. Furthermore, ex vivo crosslinking of MOv18 I.E. triggered TNFα upregulation by human monocytes. TNFα stimulation triggered production of MCP-1 by macrophages and also by tumour cells of different origins. MCP-1 triggered monocyte and macrophage infiltration in the tumour microenvironment. These studies demonstrated the significance of the TNFα/MCP-1 axis in the modulation of cellular populations and functions in the TME [We also report that IgE crosslinking on both M0 and M2 macrophages triggered enhanced levels of the pro-inflammatory M1 cytokine TNFα, which this had different effects in M0 and M2 macrophages in the relation to the production of the macrophage chemoattractant MCP-1. M2 macrophages, upon IgE treatment, enhanced the production and the release of MCP-1. In line with the findings presented in this work, upregulation and the potential roles of TNFα and MCP-1 have been described by Josephs and colleagues. Our previous findings with the anti-tumour IgE antibody MOv18 in a rat tumour model  the TME ,35. Our  the TME ,37.Lyn and Tim-3 both part of the FcεRI signalling pathway and known to regulate effector cell activation [Lyn gene expression in M0, M1 and M2 macrophage subtypes. This may be significant in the cancer context since in vivo studies demonstrated that a loss-of-function of Lyn may predispose to tumorigenesis [Lyn were characterised by defective macrophage populations [Lyn expression on all macrophage subsets may therefore signify the importance of activating macrophages by antibodies of the IgE isotype, which could potentially confer protection from tumour growth. The potential of activating the FcεR downstream pathway, and by inference anti-tumour IgE engagement of immune effector cells may have clinical significance, since higher intra-tumoural Lyn expression in gastric, lung and ovarian cancer is associated with more favourable prognosis. On the other hand, engagement and crosslinking of IgE induced a reduction in Tim-3 gene expression in M2 macrophages, while Tim-3 was upregulated in M0 macrophages. Tim-3, known to participate in multiple suppressor pathways of anti-tumour responses, can be expressed by cancer cells and may support alternatively-activated macrophage polarisation. Simultaneous blocking of Tim-3 and PD-1 in infectious diseases was shown to restore lymphocyte functions and it was recently hypothesised that blocking Tim-3 may support the anti-tumour functions by macrophages [Tim-3 gene expression in M2 macrophages may induce immune cell activation, in line with what was observed with T lymphocytes upon treatment with anti-PD-1 and anti-Tim-3 antibodies. Therefore, reduction of Tim-3 expression on M2 macrophages upon IgE treatment may be further explored in the context of cancer immunotherapy and macrophage functions in the TME.Analysis of gene expression of the protein kinase tivation , showed igenesis , and traulations . The retrophages . IgE croex vivo upon IgE crosslinking may provide a new insight on this antibody class that may have relevance in tissues and perhaps in TME. Quiescent M0 macrophages, when crosslinked with IgE, featured: upregulation of the classic M1 surface marker CD80, increased production of pro-inflammatory cytokines and chemoattractant mediators, increased expression of the kinase Lyn and other downstream signalling molecules including Erk1/2 and p38. M1 macrophages retained their pro-inflammatory status upon activation via IgE antibodies, which led to increased expression of Lyn downstream of the FcεRI pathway. The crosslinking of IgE antibodies on M2 macrophages was characterised by a significant increase in the production of the chemoattractant, pro-inflammatory molecule MCP-1 and the enhanced phosphorylation and gene expression of Lyn. Therefore, crosslinking of IgE on human macrophages confers M0 and M2 subsets features more likely associated with classically-activated macrophages  shown to engage and activate canine macrophages more effectively than the equivalent IgG1 to target and kill tumour cells in vitro [. Human adipose tissue-derived mast cells (ADMC) can be specifically activated to display tumoricidal activity against breast cancer cells when engaged with tumour antigen-specific IgEs [NCT02546921, www.clinicaltrials.gov).The anti-cancer potential of IgE class antibodies has been reported in multiple studies in the emerging field of AllergoOncology, concerned with the links between Th2 and IgE responses with cancer ,43. Repoin vitro . Human afic IgEs . Our ownTaken together, our findings point to the potential of anti-tumour IgE antibodies not only to mediate immune effector cell-mediated killing of cancer cells, but also to impact on immune stroma, influencing macrophage phenotype and maturation states. Our findings provide new insights into the interactions between human macrophages and IgE class antibodies. This may be highly significant in the clinical development of new immunotherapy strategies targeting these often tissue- and tumour-resident cells and may point to previously-unappreciated cascades with potential to activate and re-educate immune stroma.


Implants provide support, stability and retention for restorations used in fully and partially edentulous patients. This clinical report describes prosthetic treatment of a 30-year-old man suffering from a dentoalveolar deficiency due to an old gunshot trauma to the left side of the mandible. The patient was rehabilitated with implant-supported Toronto prosthesis following surgical placement of three implants with undesirable location and angulations due to lack of sufficient bone. This prosthetic option offers advantages of both screw-retained and cement-retained prostheses and provides acceptable aesthetic and functional results. Screw-retained restorations provide ease of retrievability of restoration for repair, hygiene measurements or retightening of abutment screw.15 However, they have some disadvantages such as difficulty in removing the restoration and remaining of cement excess around the implant.16On the other hand, cement-retained restorations present several advantages over screw-retained restorations. These benefits include ease of fabrication, being aesthetically pleasing, simpler placement in posterior regions of the oral cavity and higher potential for achieving passive fit in the superstructure.17 or abutment-hybrid overdenture18 has been suggested in order to overcome problems of both types of aforementioned restorations and yet benefiting from their merits. In this technique the substructure is a screw-retained framework on which individual crowns would be cemented and pink or gingiva-colored porcelain or laboratory composite is used for mimicking the soft tissues.20 Rajan and Gunaseelan21 first described fabricating retrievable cement-retained and screw-retained implant prosthesis for single-tooth implant-supported restorations. This prosthesis offers the advantages of both cement-retained and screw-retained restorations such as retrievability of the abutment and the prosthesis, and easy removal of the prosthesis for cleaning the excess cement. The aim of this article was to present a case treated with Toronto prosthesis for restoration of partial edentulism in a post-traumatic mandible.Toronto bridge techniqueA 30-year-old partially edentulous patient was referred to the Implant Department of Tehran University, School of Dentistry, for prosthetic reconstruction . The pat22 Also, an irreversible hydrocolloid impression  was made of the upper jaw. After attaching implant analogs to the impression copings in the impression, a silicone material  was injected around the coping analogs to serve as gingival tissues around the implants. Then, type IV dental stone  was used for pouring the impressions.Using auto-polymerizing acrylic resin  square impression copings were splinted, and an open-tray implant-level impression was made with polyvinyl siloxane impression material .To record the jaw relationship, a screw-retained record base was made on the master cast. Before making the jaw relationship record, the occlusal surface of maxillary second premolar was reduced about 1 mm using a football-shaped diamond finishing bur  to address its minimal supraeruption. After recording the centric position of the mandible with lower record base and occlusion rim opposing the upper teeth, using an inter-occlusal registration material , the casts were mounted in a semi-adjustable articulator  using a facebow transfer record. Then, acrylic denture teeth  were set up on the record base and tried in the patient mouth for evaluation of tooth arrangement, position, length, gingival level and contour. Subsequently, a silicone  index was fabricated of the approved tooth set-up on the record base.6After connecting plastic abutments (burn-out sleeve)  to the implant analogs on the master cast with 10 N/cm torque, the acrylic resin  pattern of infrastructure (meso-structure) was made using previously made silicone index as the guide . The patSubsequently, gingiva-colored porcelain was baked on the gingival part of the framework by the aid of silicone index for controlling the gingival contour. After evaluating gingival adaptation of the framework intraorally , the copFinally after screwing the meso-structure with 30 N/cm torque according to manufacturer’s instructions, Teflon tape  was placed at the orifice of screw holes and covered with light-cured composite resin. Finally, the crowns were cemented with temporary cement  on the metal framework .16 This kind of prosthesis could be used for restoring a single tooth,21 or partial19 and even a full20 edentulous arch. The main advantage of this kind of restoration is that not only implant position or angulation would not affect the design of the substructure, but also implant emergences could be corrected.18 Considering severe misalignment of the middle implant due to insufficient bone volume in the canine site as a result of an old gunshot injury in this case, this prosthetic option was selected. Also, regardless of location of screw access holes in the meso-structure, each crown can be fabricated in a position which is esthetically and functionally desirable.,17 Furthermore, when a great amount of porcelain is fired on the metal framework in cases of increased inter-occlusal space, distortion of the framework and loss of passive fit is expected. However, Toronto bridge technique prevents such distortion and also enables the possibility of replacing a damaged crown without jeopardizing the fit of the framework by re-firing it.20 Another advantage of this technique is that the esthetic and occlusion concerns usually seen in screw-retained restorations due to screw access hole are overcome.18 Moreover, retrievability of crowns cemented with temporary cement makes their maintenance relatively easy.20The patient presented in this report was treated with a hybrid cement-retained and screw-retained implant restoration. This kind of restoration is mainly indicated for demanding esthetic situations, excessive inter-occlusal space, and when vertical bone augmentation is not feasible.19However, the disadvantages entitled to this technique are difficulty of adjusting the crown's contact points, probability of crown dislodgement due to the use of a temporary cement, difficulty of achieving passive fit of screw-retained framework and high cost.21 zirconia19 and CAD-CAM milled titanium20 frameworks. Also, gingiva-colored composite resin16 could be used for covering the gingival part of the framework.This technique offers some varieties in terms of material used for framework, including casting,,None.All authors contributed to the case selection, and treatment planning. FN, MA, and EB contributed to the treatment of the patient. FN and EB together drafted the manuscript. All authors have contributed to the critical revision of the manuscript, and have read and approved the final manuscript.Not applicable.The authors declare no competing interests with regards to the authorship and/or publication of this article.The individual whose information is included in the report has given written informed consent for the publication of this paper.


Although DAPK1 is tightly regulated under physiological conditions, DAPK1 deregulation in the brain contributes to the development of neurological disorders. In this review, we describe the molecular mechanisms of DAPK1 regulation in neurons under various stresses. We also discuss the role of DAPK1 signaling in the phosphorylation-dependent and phosphorylation-independent regulation of its downstream targets in neuronal cell death. Moreover, we focus on the major impact of DAPK1 deregulation on the progression of neurodegenerative diseases and the development of drugs targeting DAPK1 for the treatment of diseases. Therefore, this review summarizes the DAPK1 phosphorylation signaling pathways in various neurodegenerative diseases.Regulated neuronal cell death plays an essential role in biological processes in normal physiology, including the development of the nervous system. However, the deregulation of neuronal apoptosis by various factors leads to neurodegenerative diseases such as ischemic stroke and Alzheimer’s disease (AD). Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin (Ca In the central nervous system, neuronal cell death is a crucial process in nerve damage and development. The death of neurons under normal physiological conditions in the adult brain is limited and adequately controlled, even in the elderly. In general, mature neurons are more resistant than immature neurons to cell death . HoweverDeath-associated protein kinase 1 (DAPK1), as a serine/threonine (Ser/Thr) kinase, plays a critical role in the regulation of stress-induced cell death ,7. DAPK1This review focuses on the role of DAPK1 in neuronal cell death and neurodegenerative diseases. Furthermore, we discuss the currently understood mechanisms of neuronal cell death associated with DAPK1 phosphorylation in the damaged brain.2+/CaM)-regulated Ser/Thr kinase that was originally identified by an unbiased genetic screen of an antisense cDNA expression library from HeLa cells that underwent γ-interferon (IFN-γ)-mediated cell death [2+/CaM autoregulatory domain and a 40-amino-acid C-terminus [2+/CaM domain but instead has a nuclear localization signal and a leucine zipper structure at the C-terminus [2+/CaM domain and are exclusively localized in the nucleus [DAPK1 is a calcium/calmodulin  domain, a C-terminal of ROC (COR), a death domain and a Ser-rich C-terminal tail  ,24. The echanism . Calmoduic cleft . In addine sites . DAPK1 cne sites ,29,30.Although the function of the two putative P-loops at residues 639-646 and 695-702 of DAPK1 is not well known, the second of the P-loops (residues 695-720) partially overlaps a cytoskeleton binding region, and DAPK1 binds to GTP through the second P-loop . GTP binThe serine-rich C-terminal tail of DAPK1 acts an autoinhibitory module that negatively regulates the putative function of the protein residues of one of the various extra-catalytic domains . The deaPhosphorylation plays a very important role in the regulation of DAPK1 activity . In partAnother important DAPK1 phosphorylation site is Ser735 in the ROC-COR domain . DAPK1 iIn addition to Ser308, another phosphorylation site, Ser289, is present in the CaM autoregulatory domain. p90 ribosomal S6 kinases (RSK1/2) are located downstream of ERK in the mitogen-activated protein kinase (MAPK) pathway . ERK proPhosphorylation sites that regulate DAPK1 activity include Tyr491 and Tyr492, which are present in ankyrin repeats ,58. AlthNeuronal cell death occurs extensively during the development of the central nervous system as well as in pathologies associated with neuronal injury. The death of neuron results in many chronic neurodegenerative diseases due to the limited growth and replacement of adult neurons . The pheDAPK1 has an important role in various types of neuronal cell death mechanisms. DAPK1 expression in the brains of AD patients is significantly increased compared with age-matched normal subjects . In addiL through their BH3 domains [L [L [Furthermore, DAPK1 mediates death mechanisms through TGF-β stimulation . TGF-β i domains . Howevermains [L . DAPK1 pns [L [L . Moreovens [L [L . DAPK1 ans [L [L ,78.DAPK1 phosphorylates p53 at Ser23 by the direct binding of the DAPK1 death domain to the DNA binding motif of p53 (residues 270–281) . In primDAPK1 regulates the c-Jun N-terminal kinase (JNK) signaling pathway through the binding and activation of protein kinase D (PKD) in response to oxidative stress . DAPK1 a2+-permeable ionotropic glutamate receptors that are known to be responsible for the neurotoxic effect of glutamate, which stimulates fast synaptic transmission in the majority of excitatory synapses in the human brain [2+ influx via GluN1/GluN2B receptor channel conductance [DAPK1 causes cell death under pathological conditions via N-methyl-D-aspartate (NMDA) receptors in neurons . NMDA rean brain ,89. Natian brain ,91. GluNan brain . DAPK1 dan brain . Activatductance . Howeverductance .3Rs), is a novel membrane-associated protein predicted to contain a partial MAB-21 domain [3Rs to improve the Ca2+-mediated inhibition of IP3R-dependent Ca2+ release and regulates neuronal death without affecting IP3 ligand binding [2+, have identified DAPK1 as an interacting protein [DANGER, which was first identified on the basis of binding to inositol 1,4,5-trisphosphate receptors , is membrane-permeable, specifically blocks the phosphorylation of GluN2B by DAPK1 following transient focal ischemia, and dramatically decreases the infarct size [Activated DAPK1 directly interacts with the NMDA receptor GluN2B protein complex and phosphorylates the GluN2B subunit at Ser1303 in the cortex of ischemic stroke mice . A variarct size . Thus, trct size . Interesrct size . The micrct size ,105,106.rct size . Howeverrct size . Moreoverct size . ConsequAD is a progressive neurodegenerative disease associated with cognitive impairment and is the most common type of dementia in the elderly, with approximately 44 million patients worldwide . AD is cInterestingly, DAPK1 expression is highly up-regulated in the human AD brain . DAPK1 oDAPK1 increases the phosphorylation of specific sites of tau, namely Thr231, Ser262, and Ser396, in neurons . The phoDAPK1 is responsible for the phosphorylation of Pin1 ,117,118.40 secretion; however, DAPK1 K42A mutations do not affect Aβ40 secretion compared with that of WT DAPK1 in a neuronal culture model [42 in cells stably overexpressing the Swedish mutant form of APP (APPswe), thus shifting APP processing toward the β-secretase-mediated pathway [DAPK1 increases the phosphorylation and amyloidogenic processing of APP . Interesre model . Moreove pathway . However pathway . DAPK1 i pathway . The pho pathway . Further pathway . The kno pathway . In cont pathway .Through phospho-peptide library screening, N-myc downstream-regulated gene 2 (NDRG2) has been identified as a novel substrate for DAPK1 . NDRG2 iDAPK1 is a potential molecular target for neuronal cell death, and certain inhibitors of DAPK1 have indicated the potential of new therapeutic strategies for treating neurodegenerative diseases such as ischemic stroke and AD .50 = 13 μM) is an alkylated 3-amino-6-phenylpyridazine that is known to significantly attenuate brain damage after ischemic stroke [50 = 69 nM) was identified via a structure-based virtual screening approach [40 and Aβ42 secretion via the down-regulation of APP phosphorylation [A specific small-molecule inhibitor of DAPK1  in an ATP-competitive manner [50 value of morin against DAPK1 is 11 μM, and morin moderately inhibits the catalytic activity of DAPK1. Although its role in neurons has not been elucidated, morin is a strong candidate for drug development [Peptide-based methods of DAPK1 inhibition rapidly and reversibly knockdown endogenous proteins in situ . The DAPe manner . It was e manner . Althouge manner . Morin ie manner . The IC5elopment .50 = 0.247 μM) [50 = 0.03 μM) and is highly selective for the ATP binding sites and substrate recognition motifs, including Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN) [N-(4-(4-methoxyphenoxy)-2-((6-morpholinopyridin-3-yl)amino)pyrimidin-5-yl)benzamide), is a unique and selective inhibitor of both DAPK1 (IC50 = 1.25 μM) and CSF1R (IC50 = 0.15 μM) [50 = 5.0 μM) in vitro [In addition, a novel small-molecule imidazo-pyramidazine inhibitor was identified by a GluN2B peptide-based method using a caliper microfluidics capillary electrophoresis system, and this molecule was found to have a potent inhibitory effect on DAPK1 (IC.247 μM) . Recentl.247 μM) . This insn (PEN) . More resn (PEN) . CSF1R psn (PEN) . The dua0.15 μM) . More imin vitro .Interestingly, there is a conformational-specific tau antibody that influences DAPK1 activity. As mentioned above, DAPK1 specifically inhibits Pin1 activity and subsequently induces of cis p-tau, particularly at Thr231 ,118,123.In a variety of neurodegenerative diseases, DAPK1 is the major key protein kinase that regulates cell death in the brain. DAPK1 is a well-known protein that plays a crucial role in neuronal cell death, including apoptosis and autophagy, through the death signaling pathway. The most important mechanism that regulates DAPK1 catalytic activity is phosphorylation. DAPK1 consists of multiple domains with special phosphorylation sites, such as Ser308, that are important for catalytic activation related to pathology in neurons. Therefore, DAPK1 inhibitors are likely to be very potent therapies because DAPK1 activity is markedly elevated in ischemic stroke and AD brains. Most of the DAPK1 inhibitors that have been developed have been shown to improve neurological function. However, there are still many open questions about the development of DAPK1 inhibitors as therapies. Further research is needed to clinically validate these DAPK1 inhibitors. To date, there is no potent treatment for neurological disorders in humans, but the treatment of neurodegenerative diseases with DAPK1 inhibitors should be strongly considered. Thus, animal experiments with DAPK1 inhibitors should be given priority. Since in vivo studies of most identified inhibitors have been limited, it is necessary to demonstrate the efficacy of these inhibitors in animals. Furthermore, the clinical utility of these inhibitors should be tested. The development of DAPK1 inhibitors is still at an early stage, and the development of new drugs with therapeutic effects on humans may be challenging. In addition, it is important to determine the underlying mechanism and function of DAPK1 to provide knowledge for novel drug therapies. DAPK1 is a key regulator that binds to numerous substrates involved in cell death. A better understanding of the mechanism of DAPK1 and its regulation under different pathological conditions, should help to define the main substrates for therapy. A further approach is to demonstrate the function of various PTMs of DAPK1 other than phosphorylation. The function of DAPK1 occurs mainly through phosphorylation, but PTMs such as acetylation and methylation may also play a role. Therefore, subsequent studies are also needed to clarify how signals are transmitted and how DAPK1 interacts with its binding partners. This study provides an opportunity for novel drug development based on the role of DAPK1, which is not yet fully understood.


During polar springtime, active bromine drives ozone, a greenhouse gas, to near-zero levels. Bromine production and emission in the polar regions have so far been assumed to require sunlight. Here, we report measurements of bromocarbons in sea ice, snow, and air during the Antarctic winter that reveal an unexpected new source of organic bromine to the atmosphere during periods of no sunlight. The results show that Antarctic winter sea ice provides 10 times more bromocarbons to the atmosphere than Southern Ocean waters, and substantially more than summer sea ice. The inclusion of these measurements in a global climate model indicates that the emitted bromocarbons will disperse throughout the troposphere in the southern hemisphere and through photochemical degradation to bromine atoms, contribute ~ 10% to the tropospheric reactive bromine budget. Combined together, our results suggest that winter sea ice could potentially be an important source of atmospheric bromine with implications for atmospheric chemistry and climate at a hemispheric scale. Short-lived natural bromocarbons, which contribute to ozone depletion in the atmosphere, are believed to be produced through light-driven processes, mainly in oceans. Here the authors present bromocarbon measurements in snow, sea ice, and air during polar winter that show an unexpected source of bromine to the polar atmosphere during periods of no sunlight. Of these naturally produced substances, bromoform (CHBr3) is the best known, since it is the most abundant brominated organic. It is formed in enzymatic processes developed to protect algal cells of reactive oxygen species, such as hydrogen peroxide, formed during photosynthesis2. In addition, several other bromocarbons are formed, such as dibromomethane (CH2Br2), dibromochloromethane (CHBr2Cl), and bromodichloromethane (CHBrCl2), either directly through the same enzymatic pathway, or by nucleophilic substitution of CHBr3. In the atmosphere, bromocarbons are photochemically degraded to reactive bromine which initiate the depletion of ozone and mercury, a contaminant of global concern5. BrO, formed by the reaction of atmospheric Br atoms with O3, has an impact on the sulfur cycle through the oxidation of dimethyl sulfide (DMS), leading to a 18% reduction in the DMS burden and lifetime6. The Br atom is the dominant oxidizer in mercury depletion events in the polar atmosphere7. Reactive bromine from the decomposition of bromocarbons also contributes to the depletion of ozone in the lower stratosphere8. The interplay between the oceanic sources of bromocarbons and their atmospheric impacts has led to the suggestion that these compounds are a link between climate change and atmospheric ozone9.Bromine-containing organic compounds (bromocarbons) are ubiquitous in the oceans, and they are mainly formed by macro- and microalgae10. Reactive bromine is anti-correlated with ozone, which suggests that the efficiency of halogen-driven polar tropospheric ozone depletion is related to sea ice dynamics and biogeochemical processes. Research on the role of sea ice in the formation of reactive halogens has in recent years mainly focused on the inorganic chemistry of saline surfaces, where sea-salt halides are oxidized to more reactive forms12. Similarly, inorganic bromine has also been shown to form in saline snow13. Arctic and Antarctic sea ice algae have been estimated to emit organo-bromine with ~ 70–80 × 108 g yr−1, suggesting that the produced compounds are released into seawater and then reach the atmosphere14. Other investigations have pointed out the possibility of sea ice as a source of atmospheric CHBr3 in the Amundsen Sea (Antarctica) during the sunlit period15. In coastal Antarctica, concentrations of iodo-carbons in sea ice were found to be strongly enhanced relative to seawater, suggesting a significant local source within the sea ice17. Therefore, although halocarbons are thought to be produced within sea ice, two key questions remain unknown: (i) whether this source is significant in supplying bromocarbons to the atmosphere relative to the flux from the surface waters in leads and the open ocean, and (ii) the role of biogeochemical cycles of bromocarbons during polar winter, as their production, and thereby emissions, have so far been assumed to require sunlight.Recently, it has been shown that variability in polar tropospheric bromine oxide (BrO) levels is correlated with first year sea ice concentrations, through the production of halogens in sea ice regionsIn this work, we report profile measurements of bromocarbons in snow, sea ice, and air during Antarctic winter that represent a new source of atmospheric bromine during the polar night. We combine the observations with a state-of-the-art global chemistry-climate model. The results provide strong evidence that Antarctic winter sea ice is a significant source of bromocarbons, which spread and contribute to the burden of atmospheric bromine throughout the southern hemisphere.3, CH2Br2, CHBr2Cl, and CHBrCl2) were measured in snow, sea ice, and air to estimate their source strength of seasonal sea ice in the absence of sunlight , 0.53 pptv (CHBrCl2), 0.91 pptv (CHBr2Cl), and 1.2 pptv for CH2Br2   revealed unexpectedly high concentrations of bromocarbons under conditions of solar zenith angle (SZA) larger than 90° , with average values of 9.5 pptv (CHBr21 and snow22. Note that on one location (Station 500) one of the coring sites was flooded, which led to slightly different profiles, as indicated by the high salinity found close to the sea ice interface  on ice revealed that CHBr3 is mobile at temperatures as low as 85 K, which allows us to infer that the bromocarbons were not adsorbed/desorbed on snow surfaces.Next, the magnitude of the release of individual bromocarbons from the ice surface to the atmosphere was calculated based on the assumption that the bromocarbons were formed on the surface of sea ice and subsequently diffused through the snow to the atmosphere. The interstitial air concentrations were calculated from the concentrations found in melted snow, the fraction of water and air, respectively, and the Henry's law constants (Methods). We found the concentration gradient in snow to decrease linearly Fig. . Adsorpt31. As our results show the presence of concentration gradients that could be assigned to diffusive processes, a simplified diffusion calculation was performed using the molecular diffusivities in air (Methods). These diffusivities are comparable to the effective diffusivities calculated for SF6 in snow at Summit, Greenland32. The median ice–snow–air fluxes of individual halocarbons varied between 1 and 50 nmol m−2 h−1 (Table −2 h−1 (CHBr3); 0.5 nmol m−2 h−1 (CH2Br2); 0.3 nmol m−2 h−1 (CHBrCl2 and 0.5 nmol m−2 h−1 (CHBr2Cl) at a significance level of 0.05. Fluxes from all stations are given in Supplementary Table It has been shown that wind pumping could be important for the flux of gases through the snow. In such cases, concentration gradients are absent and the fluxes are not governed by diffusive processes3 varied between 20 and 100 pptv with the highest values at midnight. These variations were related to wind speed. Around midnight the wind speed dropped from ca 7 m s−1 to ca 1 m s−1 to, later in the night, increase again to ca 9 m s−1. This trend was valid for all bromocarbons .On board, attempts were made to perform an air gradient flux estimate. There were some indications that the air concentration just above the snow had elevated concentrations compared with a height of 2.2 or 20 m . The results show an accumulation of brominated halocarbons in the atmosphere during the polar winter until the austral sunrise in August when photodecomposition starts to reduce the bromocarbon concentrations 36, based on the average emission fluxes of bromocarbons from Table In the austral spring, the arrival of sunlight initiates the photochemical breakdown of bromocarbons, accumulated during the polar night, leading to the formation of reactive inorganic bromine species throughout the troposphere of the southern hemisphere  and divided into 5–10 cm sections, individually packed in gas-tight Tedlar© bags. The snow samples were thawed in darkness at room temperature for ~ 12 h. The halocarbon compounds CHBr3, CH2Br2, CHCl2Br, and CHClBr2 were quantified. They were pre-concentrated using three purge-and-trap systems: Velocity XPT (Teledyne Tekmar) connected to an autosampler , and two custom-made purge-and-trap system, which were coupled to gas chromatographs with electron capture detection (Varian 3800), according to the methods described by Mattsson et al.15. One of the custom-made purge-and-trap instruments , CHBrCl2 , CH2ClI , CHBr2Cl , and CHBr3  diluted from stock solutions in methanol  in seawater to give final concentrations in the picomolar range in the purge chamber. The same standard solutions were used for calibration of air measurements. The detection limits of the systems are given in Supplementary Table Data were collected during the ANTXXIX/6 expedition conducted during austral winter aboard the R.V Polarstern from 8 June to 12 August 2013 in the Weddell Sea  and a high precision RTD thermometer with a Pt100 temperature sensor with an accuracy of ± 0.1 °C (winter expedition). Salinity and conductivity of the melted sea ice were measured using a conductivity meter, WTWCond330i or 3210, with a precision and accuracy of ± 0.1.vb) was derived from ice temperature (Ti) and bulk salinity (Si) according to the equation of Frankenstein and Garner [1967]37 .X] is the individual concentration of bromocarbons, Da the molecular diffusivity and z is the snow depth.Stations 493–506 had a snow cover of 5–40 cm. Vertical profiles in snow (thickness > 10 cm) indicated that the snow was ventilated above this level. The interstitial air concentrations were calculated from the measured concentrations in melted snow, the volume of air and water in the samples and the Henry's law constant at ambient temperature. The linearly decreasing concentrations in snow along with the following air diffusion coefficients were used to calculate fluxes according to Fick's first law Eq.  , as a linear function of first year sea ice fraction. CAM-Chem has been configured in this work with a horizontal resolution of 1.9° latitude by 2.5° longitude and 26 vertical levels, from the surface to ∼ 40 km altitude. All model runs in this study were performed in the specified dynamics mode34 using offline meteorological fields instead of an online calculation, to allow direct comparisons between different simulations. This offline meteorology consists of a high-frequency meteorological input from a previous free running climatic simulation. The MERRA reanalysis data set45 is used in the model to supply the implemented sea ice fields.The model employed in this work is the global 3-D chemistry-climate model CAM-Chem  to assess the impact of the measured bromocarbons emissions upon the distribution of bromocarbons and reactive bromine throughout the southern hemisphere. The model includes a comprehensive chemistry scheme to simulate the evolution of trace gases and aerosols in the troposphere and the stratosphereSupplementary InformationDescription of Additional Supplementary Files


Ptc is a gatekeeper to avoid abnormal Hh signaling activation, but the key regulators involved in Ptc-mediated inhibition remain largely unknown. Here, we identify PKAc as a key regulator required for Ptc inhibitory function. In the absence of Hh, PKAc physically interacts with Ptc and phosphorylates Ptc at Ser-1150 and -1183 residues. The presence of Hh unleashes PKAc from Ptc and activates Hh signaling. By combining both in vitro and in vivo functional assays, we demonstrate that such Ptc–PKAc interaction and Ptc phosphorylation are both important for Ptc inhibitory function. Interestingly, we further demonstrate that PKAc is subjected to palmitoylation, contributing to its kinase activity on plasma membrane. Based on those novel findings, we establish a working model on Ptc inhibitory function: In the absence of Hh, PKAc interacts with and phosphorylates Ptc to ensure its inhibitory function; and Hh presence releases PKAc from Ptc, resulting in Hh signaling activation. Aberrant inactivation or activation of Hh signaling is associated with various human congenital disorders and cancers4. One of the key features which distinguishes Hh signaling with other signaling pathways is the existence of an important inhibitory receptor, a 12-transmembrane protein Patched (Ptc). Physiologically, in the absence of ligands, Ptc inhibits the activation of a downstream seven-transmembrane signal transducer, Smoothened (Smo), and consequently silences the Hh signaling5. Conversely, in the presence of Hh, Ptc binds to Hh and releases Smo from inhibition, resulting in the activation of Hh signaling as evidenced by accumulation of Ci full-length and induction of downstream target genes of Hh signaling including dpp, ptc, and en5. Therefore, the inhibitory function of Ptc is critical in regulating the switch-off and -on of Hh signaling and is inevitably subjected to precise regulation at multiple levels to guarantee the appropriate Hh signaling activity in Hh-responsive cells.As one of the most evolutionarily conserved morphogen pathways, the Hedgehog (Hh) signaling pathway plays vital roles in embryo development, tissue regeneration, and homeostasis8. In those Hh-driven malignancies, loss of the inhibitory function of Ptc is the predominant alteration leading to abnormal Hh activation. For example, over 70% sporadic BCCs possess Ptc mutations9; and mutations inactivating Ptc inhibitory function have been reported in a subset of sporadic MBs10. Moreover, although the loss of both copies of Ptc is embryonic lethal, one copy loss of Ptc is sufficient to induce MBs in mouse models11. Therefore, those findings from both clinical samples and animal models strongly indicate the physiological and pathological importance of Ptc-mediated inhibition on Hh signaling. A long-standing idea believes that Ptc inhibits Hh signaling in a catalytic manner by pumping activating sterols away from Smo12, but whether alterative mechanisms are involved in Ptc inhibitory function remains elusive.Previous studies have demonstrated that Hh signaling is abnormally activated in many types of cancers including basal cell carcinomas (BCCs), medulloblastomas (MBs) and non-small cell lung carcinomas15, Hh signaling is extensively subjected to multiple PTMs16. Among them, phosphorylation is one of most important PTMs in regulating Hh signaling activity. When Hh signaling is off, cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylates Ci and primes it for the subsequent phosphorylation by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in phosphorylation-dependent ubiquitination and partial degradation of Ci full-length (Cifl) to Ci repressor (CiR)18. However, when Hh signaling is on, PKA phosphorylates Smo at its C terminus and triggers the subsequent adjacent phosphorylation of Smo by CK1 and GPCR kinase 2 (Gprk2) in Drosophila, which leads to plasma membrane accumulation and active conformation changes of Smo20. Therefore, PKA is one of the key regulators on Hh signaling activity.Post-translational modifications (PTMs) play a key role in intracellular signal transduction. Like other signaling pathways, including Wnt, Notch, and Hippo22. The kinase activity of PKA is regulated by cAMP, an intracellular second messenger. In the absence of cAMP, PKA exists as an inactive tetramer composed of a regulatory (R) subunit dimer and two catalytic (C) subunits23. In the presence of cAMP, the two catalytic (C) subunits are released from their inactive tetramer and phosphorylate their specific substrates23. It is a long-standing concept that the subcellular distribution of PKA is largely determined by a family of structurally diverse proteins, so called A-kinase anchoring proteins (AKAPs), which confine PKA at specific cellular locations by binding to the regulatory (R) subunits of PKA25. However, some recent studies have indicated that the catalytic (C) subunits of PKA (PKAc) may have their distinct cellular distribution patterns independent on PKA regulatory (R) subunit dimer or AKAPs due to various posttranslational modifications including myristylation and deamidation28. For example, the myristylation at PKAc N terminus helps its plasma membrane localization28, and the deamidation at Asn2 supports PKAc to localize in the nucleus27. Given the importance of PKA in regulating Hh signaling and its potential for plasma membrane localization, we hypothesized that PKA might serve as a key regulator for the inhibitory function of Ptc.PKA ubiquitously expresses in eukaryotic cells and functions as a signal switch in regulating the on and off of various signaling pathways, including Wnt, Hippo, and HhIn the current study, we demonstrate that, in the absence of Hh, PKAc can physically interact with Ptc at its 4th intracellular domain (C4) and phosphorylate the 7th intracellular domain of Ptc (C7). Interestingly, such a Ptc–PKAc interaction is mitigated in the presence of Hh ligands parallel with enhanced Smo-PKA interaction and the subsequent Smo activation. Following functional experiments indicate that both Ptc–PKAc interaction and PKAc-mediated Ptc phosphorylation are required for Ptc inhibitory function on Hh signaling. In addition, we also demonstrate that PKAc can be palmitoylated, and the palmitoylation is required for its plasma membrane localization and its physiological function on Ptc-mediated inhibition on Hh signaling. Therefore, we establish a working model for our newly identified Ptc–PKAc regulatory axis: in the absence of Hh, Ptc–PKAc interaction, and the subsequent PKAc-mediated Ptc phosphorylation are required for the inhibitory function of Ptc; in the presence of Hh, PKAc is released from Ptc, resulting in Hh signaling activation.Drosophila Ptc and PKAc, we have to examine the possible interaction between the PKAc and Ptc by overexpressing V5-tagged Ptc (PtcWT-V5) and HA tagged PKAc (3XHA-PKAc) in S2 cells followed with immunoprecipitation (IP) assays. Reciprocal IP assays by using capture antibodies against V5 or HA clearly showed co-IP between Ptc and PKAc , overexpression of PtcWT can markedly decreased the plasma membrane accumulation of Smo, while PtcWTΔC4 cannot , ptc , whereas PtcWTΔC4 overexpression showed impaired inhibition on dpp-lacZ , ptc-lacZ  and En expression levels . Interestingly, in addition to the inability of PtcWTΔC4 to inhibit Hh signaling, PtcWTΔC4 overexpression induced dpp expression , suggesting the dominant negative role of PtcWTΔC4 and the C4 is important for Ptc inhibitory function. Moreover, loss of inhibition on Hh signaling was also observed in another line of PtcWTΔC4 transgenic flies driven by MS1096 or APGal4 . More importantly, in vivo rescue assays showed that only the PtcWT  could rescue the inhibition loss on En expression caused by endogenous Ptc depletion . These findings suggest that Ptc–PKAc interaction is critical for Ptc inhibitory function on Hh signaling in vivo.To further investigate whether the Ptc–PKAc interaction is critical for Ptc inhibitory function in vivo, we generated transgenic flies overexpressing either PtcWT or PtcWTΔC4 respectively, and examined their functions on Hh signaling activities. Compared with control flies, PtcWT overexpression driven by either APGal4 or AG4 greatly inhibited ptc Fig. ‴, whereaacZ Fig. ‴, ptc-laacZ Fig. ‴ and En els Fig. ‴. Intereion Fig. ‴, sugges33. Since we have demonstrated the physical interaction between Ptc and PKAc, we sought to determine whether Ptc is a novel phosphorylation substrate of PKAc. Compared with the vehicle control  by PKAc in the presence of purified Ptc-C1, -C4 and -C7 intracellular domains Fig. , which aTo precisely determine the PKAc-phosphorylation sites on Ptc, we separately substituted all five Ser residues in phospho-cluster 3 with Ala in the backbone of ‘GST-C7-1 mut’, in which all Ser residues in phospho-cluster 1 are mutated to Ala Fig. . Thiopho‴, Fig. 3g–g‴ and Supplementary Fig. S3d–d‴), whereas PtcWT-S1150/1183D restored the inhibition on En . Given that the Ptc–PKAc interaction was decreased in response to Hh ligands, we then examined whether PKAc-mediated Ptc phosphorylation is also regulated by Hh. MS data indicated that Hh treatment eliminate the phospho signals at Ser-1150 and greatly diminished them at Ser-1183  and Ptc phosphomimetics (PtcWT-S1150/1183D). As shown in Supplementary Fig. 2k–kdpp  expression, PtcWTΔC4-PKA-K75R functions as a dominant negative Ptc mutant to induce the expression of dpp  expression levels. In addition, compared with control fly  and ptc  expression levels. Meanwhile, rescue assays showed that PtcWTΔC4-PKA  but not PtcWTΔC4-PKA-K75R  could restore Ptc-mediated inhibition on Hh signaling. Therefore, these findings indicate that both Ptc–PKAc interaction and PKAc-mediated Ptc phosphorylation are important for Ptc-mediated inhibition on Hh signaling.To further confirm our findings that both Ptc–PKAc interaction and PKAc-mediated Ptc phosphorylation are important for Ptc inhibitory function, we replaced the Ptc domain C4 with wild-type PKAc (PtcWTΔC4-PKA) or its kinase dead form, PKAc-K75R  to mimic Ptc interacting with PKAc or kinase-dead PKAc. A ‘soft linker (GSGGS)’ was included between Ptc and PKAc to avoid possible structure alterations in the Ptc–PKAc fusion protein Fig. . In addi75R Fig. ‴ could r28, the mechanism leading to PKAc membrane localization is still not fully understood. Given that palmitoylation is one of the important PTMs to enhance hydrophobicity and the subsequent membrane localization of many target proteins35, we started to examine whether PKAc can be palmitoylated. Thiopropyl captivation of S-palmitoylated protein assays showed that treatment of hydroxylamine but not control Tris–HCl greatly increased the association of purified PKAc with thiopropyl beads and such association was markedly reduced in the presence of 2-Brp, a palmitoylation inhibitor  markedly inhibited Ci and dpp-lacZ expression, whereas overexpression of membrane-tethered PKAc or PKAc-3C mut has no effect on Ci levels but can greatly increase dpp-lacZ levels . These findings indicate that PKAc is subjected to palmitoylation and such palmitoylation is important for PKAc plasma membrane localization and plasma membrane activity of PKAc on Ptc.To further determine whether PKAc palmitoylation affects Ptc inhibitory function, we determined the plasma membrane localization of both Ptc and Smo by IF staining assays. Compared with vector control, soluble or membrane-tethered PKAc greatly increased plasma membrane accumulation of Ptc and Smo, but PKAc-3C-mut failed to do so  in the absence of Hh, PKAc interacts with and phosphorylates Ptc to ensure Ptc inhibitory function on Hh signaling and (ii) in the presence of Hh, PKAc is released from Ptc, resulting in activation of Hh signaling.10. Unlike Ptc whose inhibitory function is mainly attributed to its inhibition on Smo, PKAc can differentially regulate Hh signaling activity by functionally and/or physically interacting with and phosphorylating multiple key components of Hh signaling including Ci and Smo. In contrast to PKAc-mediated Smo phosphorylation which activates Hh signaling in response to Hh, in present study we demonstrated that Ptc–PKAc interaction and PKAc-mediated Ptc phosphorylations at Ser-1150 and -1183 are both important for Ptc inhibitory function in the absence of Hh ligand . But the fact that the flank amino acid sequences of Ser-1150 and Ser-1183 are not consensus PKA sites raised the concern whether those Ptc phosphorylation is directly mediated by PKAc. To address this concern, we constructed fusion proteins by fusing Ptc with PKAc and kinase-dead PKAc (PKA-K75R) respectively. Like the unphosphorylable Ptc (PtcWT-S1150/1183A), Ptc fused with PKA-K75R showed impaired inhibitory function compared with the Ptc–PKAc fusion protein  showed impaired plasma membrane accumulation in S2 cells , 20 min each time. Third, blocked the larvae with 1%BSA in PBST for 15 min. Forth, incubated the larvae with primary antibody diluted in PBST for overnight at 4 °C. Fifth, washed with PBST three times again and incubated with secondary antibody diluted in PBST for 2 h at room temperature. Finally, washed the larvae for another three times, dissected wing discs and mounted in 40% glycerol. A Leica LAS SP8 confocal microscope was employed to take immunostaining images.Primary antibodies used in this study: rabbit/mouse anti-V5 (Invitrogen), rabbit/mouse anti-Flag (Sigma), rabbit/mouse anti Myc (Sigma), rabbit/mouse HA (Sigma), rabbit/mouse anti lacZ (Sigma), rat anti-Ci , mouse anti-Ptc (DSHB), mouse anti-Smo (DSHB), and mouse anti-En (DSHB).S2 cells were cultured at 25 °C in a Schneider’s Drosophila Medium (Invitrogen) with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin. Ptc-related plasmids transfection and dsRNA transfection were performed using the lipo3000 according to manufacturer’s instructions.For S2 cell immunostaining analysis, cells were fixed in 4% formaldehyde for 10 min and permeabilized in 0.25% Triton X-100 for 2 min . The cells were blocked in 1% BSA in PBS for 30 min and incubated in primary and secondary antibody for 2 and 1 h, respectively. Images were captured by confocal microscopy.6) were transfected with 0.3 μg ub-Gal4, 0.05 μg Ci, 0.025 μg Sufu, 0.05 μg Ptc reporter, 0.005 μg Renilla, 1 μg Ptc-related plasmid, 2 μg dsRNA of ptc 5′UTR, then cells were lysed in indicated buffer and next the luciferase assays were performed as standard procedures.For the dual luciferase reporter assay, 1 ml S2 cells in 24 well (1.0 × 10AP − CFPBP)/CFPAP] × 100. Each data point was calculated using 15–20 individual cells.For the FRET assay, S2 cells were transfected with ub-Gal4, Myr-AKAR3 and related PKAc mutants. Cells were washed with PBS, fixed with 4% formaldehyde for 10 min, and mounted on the slides in 40–50% glycerol. The intensity of CFP emission before (BP) and after (AP) photobleaching of YFP with 514 nm laser at full strength for ten times was acquired through the standard procedure built in Leika TCS SP8 WLL. FRET% = [ for 30 min at 4 °C. After centrifugation, the supernatants were incubated with indicated primary antibodies for 2 h and protein A/G gel for 1 h at 4 °C. After affinity pull-down, gels were washed in lysis buffer for 10 min three times and then boiled in 40 μl 1× SDS loading buffer. Western blot analysis was carried out by SDS-PADE electrophoresis by using standard protocol.For the IP and western blot assay, S2 cells were lysed in NP-40 buffer (50 mM Tris–Cl pH 8.0, 0.1 M NaCl, 10 mM NaF, 1 mM Nat test. P-values are presented in figures, *P < 0.05, **P < 0.01, N.S., not significant.Data were presented as Mean ± SD and statistical significance was calculated using two-tailed Student’s Supplementary figures and data.


Gap junctional, intercellular communication (GJIC) is interrupted in cells transformed by oncogenes such as activated Src. The Src effector, Ras, is required for this effect, so that Ras inhibition restores GJIC in Src-transformed cells. Interestingly, the inhibition of the Src effector phosphatidyl-inositol-3 kinase (PI3k) or Signal Transducer and Activator of Transcription-3 (Stat3) pathways does not restore GJIC. In the contrary, inhibition of PI3k or Stat3 in non-transformed rodent fibroblasts or epithelial cells or certain human lung carcinoma lines with extensive GJIC inhibits communication, while mutational activation of PI3k or Stat3 increases GJIC. Therefore, it appears that oncogenes such as activated Src have a dual role upon GJIC; acting as inhibitors of communication through the Ras pathway, and as activators through activation of PI3k or Stat3. In the presence of high Src activity the inhibitory functions prevail so that the net effect is gap junction closure. PI3k and Stat3 constitute potent survival signals, so that their inhibition in non-transformed cells triggers apoptosis which, in turn, has been independently demonstrated to suppress GJIC. The interruption of gap junctional communication would confine the apoptotic event to single cells and this might be essential for the maintenance of tissue integrity. We hypothesize that the GJIC activation by PI3k or Stat3 may be linked to their survival function. Gap junctions are aqueous channels connecting the cytoplasm of adjacent cells that permit the passage of small molecules and ions ,2. Gap jA number of oncogenes such as vSrc , vRas 1,15,16,17Engagement of cadherins, cell to cell adhesion proteins, was shown to be required for gap junction formation . In addiThe discovery of PI 3-kinases began with the identification of a phosphoinositide kinase activity present in immunoprecipitates of the polyoma virus middle Tumor antigen oncogene (mT). Later results demonstrated that this lipid kinase consists of two subunits: A catalytic, 110kDa (p110α or PIK3C), which phosphorylates phosphoinositides on the 3′ position of the inositol ring  and a re3, known as PIP3] from phosphatidylinositol -bisphosphate . This provides docking sites for signalling molecules with plekstrin-homology (PH) domains such as Akt , PI3k translocates to the membrane by binding through three SH2 (Src-homology-2) domains of p85, onto phosphorylated, pYXXM motifs of RTKs or adaptor proteins. Once on the membrane, PI3k initiates signalling cascades by generating the second messenger phosphatidylinositol -trisphosphate [PIPiewed in ). Akt isiewed in . Akt’s piewed in ,39.Evidence on the effect of PI3k upon GJIC is mainly from two systems: c-Src complex offers the distinct advantage for signalling studies compared to activated Src in that each pathway can be individually inhibited, using mT constructs that are mutated at specific phospho-tyrosine residues which are responsible for activating specific pathways.The study of mT has led to the discovery of important concepts in signal transduction. mT is a membrane-bound, 421-amino-acid protein which is able to transform cultured rodent fibroblasts and cause tumors following transgenic expression in mice . mT tranThree major tyrosine residues of mT provide docking sites for key signal transducers:250 in a NPTpY250SVM motif recognizes the PTB domain of Shc, an adaptor which binds Grb2-sos and activates the Ras/Raf/Erk pathway [a. p-Y pathway ,44.315 in a pY315MPM motif recognizes an SH2 domain of p85, the adaptor subunit of PI3k [b. p-Y of PI3k ,49,50,51322 recognizes the SH2 domain of Phospholipase-Cγ (PLCγ) [c. p-Yγ (PLCγ) .250 phosphorylation (and presumably Ras/Raf/Erk activation) is in fact a key to GJIC suppression by mT [315 is not required for GJIC suppression. Therefore, activation of the Shc/Grb2/Ras pathway (through pY250 which is intact in 315F-mT) appears to be sufficient to drive the dramatic reduction in gap junctional communication [250, Shc binding site, rather than the Y315, in GJIC suppression is further supported by the fact that a mT mutant which activates PI3k/Akt more than wt-mT  mT can effectively suppress GJIC in mouse fibroblasts ,54. To eon by mT . In shars intact ) has acts intact , as longPI3k inhibition with two different inhibitors (wortmanin or LY294002) resulted in a dramatic reduction of GJIC in T51B cells, pointing to a positive role of PI3k upon gap junctional communication in this system . Examination of the effect of hyperactive transforming PI3k was performed through expression of two mutants of the catalytic subunit of PI3k, p110α (E545K and H1047R) found in a number of cancers , and a mXenopus oocytes [In spite of extensive efforts, the effect of Akt upon GJIC of different cellular systems is unclear. Akt-mediated phosphorylation was shown to stabilise membrane-localised Cx43 ,62,63, a oocytes . In this oocytes . Since i oocytes .myc. However, Stat3 is also a potent cell survival signal that acts through a number of pathways: (1) Transcriptional upregulation of genes such as bcl-xL, Mcl-1 and survivin; (2) transcriptional downregulation of the tumor suppressor p53 [Stat3, a member of the STAT family of transcription factors, is normally inactive in the cytoplasm of quiescent cells. Following stimulation of cytokine receptors especially of the IL6 family, certain RTKs, or oncoproteins such as Src, Stat3 is phosphorylated at a critical Y-705 by the associated Jak or Src kinases. Reciprocal SH2-pY interactions follow leading to dimerization, nuclear translocation and homing of the complex towards a specific sequence (TTCNNNGAA) on the promotors of target genes . Stat3 assor p53 ,70,71;  In ssor p53 ,74. Statssor p53 ,75,76. Stat3 is found to be overactive in a number of cancers and to be required for transformation by a number of oncogenes such as Src ,78,79. I705 phosphorylation and activity . The. The705 ctivity . In fact, an inverse relationship between Src activity levels and GJIC was noted; in five lines with high Src activity , GJIC was absent (e.g., A549, Further examination of additional lung cancer lines revealed a greater level of complexity , indicatNeoplastic transformation is invariably accompanied by GJIC reduction ,97. In f250-Ras/Erk pathway can occur in the absence of full neoplastic conversion and the concomitant changes in cell shape in this system.Since neoplastic transformation leads to inhibition of junctional communication, the question of the transforming abilities of the two pathways, Ras and PI3k is next. In fact, both the Ras and PI3k pathways were shown to be required for full neoplastic transformation by mT . Still, PI3k and Stat3, two evolutionarily very different oncogenes, both constitute potent survival signals. The question therefore arises as to whether the effect of PI3k or Stat3 upon GJIC is linked to their survival function.Apoptotic death of adherent cells is associated with dramatic shape changes and a reduction in the area of cell to cell contact, which would lead to a disruption of gap junctions. Previous data demonstrated that apoptosis triggered by treatment with etoposide, cycloheximide or puromycin leads to a loss of GJIC, probably due to caspase-3-mediated degradation of Cx43 . In factDespite the fact that Ras, PI3k and Stat3 are generally growth promoting, required for transformation by a multitude of oncogenes, and in an activated form can act as oncogenes in their own right, PI3k and Stat3 are not transmitting signals leading to GJIC suppression. In the contrary, the available evidence reveals a dramatic difference in the response of GJIC to the three pathways emanating from many oncogenes and jointly required for full neoplastic conversion; PI3k and Stat3 actually promote GJIC. Therefore, it is tempting to speculate that neoplastic transformation requires Ras/Erk as well as PI3k/Akt, Stat3 and survival, while a survival function is actually required for the maintenance, rather than suppression, of GJIC. When all three pathways are activated then the Ras pathway prevails, with GJIC suppression as a net result. Upon PI3k or Stat3 inhibition, the interruption of GJIC would confine the apoptotic event to single cells and this might be essential for the maintenance of tissue integrity. This novel role of PI3k and Stat3 may be an important regulatory step in the progression of tumours that exploit such a pathway. In fact, a correlation was noted between nuclear Stat3 levels and Cx43 and Cx26 in grade 1 and 2 uterine endometrioid adenocarcinomas, while this association was progressively weaker as the tumors dedifferentiated to G3, presumably due to the activation of additional oncogenes .Significance: A large number of oncogenes activate the transcription factor E2F family, which targets genes involved in cell proliferation. Paradoxically however, E2F by default activates genes involved in apoptosis [poptosis . Certain


We compared pediatric populations with primary food allergies (n = 59), insect venom allergies (n = 9), allergic asthma (n = 24), atopic dermatitis (n = 25), food‐sensitized nonallergic children (n = 31), and nonallergic controls (n = 17). Additionally, other sensitized groups and controls (n = 147) were included in the study Table .Serum samples were analyzed by ELISA to detect IgE‐bound and total serum sFcεRI levels Figure . First, Since IgE‐sensitization profiles toward food allergens are generally a poor measure of clinical symptoms, we compared sFcεRI titers in two food‐sensitized nonallergic groups (FS and Ghana) with FA patients Table . The GhaWe then investigated whether serum sFcεRI levels were different in patients diagnosed with atopic dermatitis or asthma, with (Pos sIgE) or without (Neg sIgE) a clinically relevant sIgE profile. sFcεRI titers did not differ based on the patients’ sIgE profile. However, we found significantly higher titers in patients with elevated IgE Figure  in both Recently, we demonstrated that sFcεRI is released from dendritic cells and mast cells after antigen‐specific FcεRI crosslinking.m) were generated. Prior to a commercial IgE ELISA, samples containing human cIgE were incubated with the recombinant proteins . This interference with IgE detection by rsFcεRI was confirmed with human IgE  and a mutated version which cannot bind IgE (rsFcεRIs Figure A‐C. Our E Figure F and humE Figure G. In addE Figure .To the best of our knowledge, this is the first analysis of sFcεRI levels in a pediatric population of well‐classified sensitized and allergic individuals. We show that sFcεRI is correlated with IgE levels, is significantly increased in IgE‐sensitized individuals, and can be modulated by allergen exposure. We collected evidence that sFcεRI can interfere with IgE detection in serum, which might be of importance in regard to interference in sIgE detection and diagnosis. Although further research on the modulation by allergen exposure and interference with sIgE molecules is needed, sFcεRI represents an additional biomarker for IgE‐mediated diseases and its use could be a valuable tool in clinical practice. Click here for additional data file. Click here for additional data file.