Targeting of immunoglobulin E (IgE) represents an interesting approach for the treatment of allergic disorders. A high-affinity monoclonal anti-IgE antibody, ligelizumab, has recently been developed to overcome some of the limitations associated with the clinical use of the therapeutic anti-IgE antibody, omalizumab. Here, we determine the molecular binding profile and functional modes-of-action of ligelizumab. We solve the crystal structure of ligelizumab bound to IgE, and report epitope differences between ligelizumab and omalizumab that contribute to their qualitatively distinct IgE-receptor inhibition profiles. While ligelizumab shows superior inhibition of IgE binding to FcεRI, basophil activation, IgE production by B cells and passive systemic anaphylaxis in an in vivo mouse model, ligelizumab is less potent in inhibiting IgE:CD23 interactions than omalizumab. Our data thus provide a structural and mechanistic foundation for understanding the efficient suppression of FcεRI-dependent allergic reactions by ligelizumab in vitro as well as in vivo. Immunoglobulin E (IgE) plays a central role in allergic responses, yet therapeutic targeting of IgE with antibodies such as omalizumab is met with various limitations. Here the authors characterize the molecular properties and crystal structure of a new anti-IgE antibody, ligelizumab, for mechanistic insights related to its enhanced suppression activity. IgE is known to interact with two major receptors, FcεRI and CD23/FcεRII4, which are involved in different immunological processes5. Binding of allergen-specific IgE to FcεRI expressed on immunological effector cells including basophils and mast cells occurs with high affinity (KD 10−10 M). This interaction occurs via two asymmetric binding sites on the receptor and is stabilized through the induction of a conformational change in IgE8. Exposure to allergens induces cross-linking of IgE-bound FcεRI resulting in immediate activation of allergic effector cells, which culminates in cellular degranulation and the release of vasoactive and pro-inflammatory mediators9.As a key driver in the development and manifestation of hypersensitivity reactions against normally non-hazardous substances, immunoglobulin E (IgE) has become a major target of therapeutic intervention strategies13. Binding of IgE to monomeric CD23 is of low affinity (KD 10−6–10−7 M)14. However, cell surface CD23 is prone to oligomerization leading to enhanced binding of IgE:allergen complexes on antigen presenting cells15. Furthermore, various studies have provided evidence that IgE binding to CD23 on B-cells negatively regulates IgE synthesis16.While this FcεRI-dependent cellular degranulation process accounts for immediate hypersensitivity reactions and the induction of clinical allergy symptoms, the interaction of IgE with CD23 has been reported to be involved in antigen presentation, the transport of antigens across airway and intestinal epithelial barriers and the regulation of IgE synthesis22, DARPin® proteins25 and nanobodies27 have been generated and tested in pre-clinical studies. To date, the monoclonal antibody omalizumab (Xolair®) represents the only licensed anti-IgE compound for clinical use. Omalizumab shows remarkable therapeutic efficacy in allergic asthma and chronic spontaneous urticaria29. Recently, a next-generation high-affinity anti-IgE monoclonal antibody  has been developed with the intention of overcoming some of the limitations associated with omalizumab30.Over the last decades, various anti-IgE inhibitors including antibodies31.Given its current status as a potential anti-IgE therapeutic and successor to omalizumab, we sought to investigate the IgE binding characteristics of ligelizumab and its modes-of-action. Here, we report the crystal structure of ligelizumab bound to IgE, revealing that it recognizes a distinct IgE epitope only partially overlapping with that of omalizumab. Ligelizumab interacts across the IgE-Fc dimer and favors the recognition of IgE in an open conformation different from its FcεRI- or CD23-bound conformations. Moreover, it binds IgE with significantly higher affinity than omalizumab and shows a correspondingly enhanced inhibition of IgE binding to FcεRI and basophil activation. In contrast and despite its higher affinity for IgE, ligelizumab is inferior to omalizumab in preventing IgE binding to CD23. Structural analysis indicates that differences in the ligelizumab epitope and spatial orientation on IgE contribute to this differential inhibition. We further observe that ligelizumab features the ability to reduce IgE production in peripheral blood mononuclear cell (PBMC) cultures, a process which may be mediated by its ability to bind IgE:CD23 complexes at the surface of B-cells. Together, our data provide a structural and mechanistic foundation for understanding why ligelizumab exerts a qualitatively and functionally distinct inhibition profile from omalizumab and is superior in suppressing FcεRI-dependent allergic reactions in vitro, in a passive systemic anaphylaxis mouse model in vivo and in clinical studies with chronic spontaneous urticaria patients32). Different concentrations of ligelizumab IgG as well as its F(ab′)2 and Fab fragments were measured in consecutive cycles on surface displayed IgE  and dissociation (kd) constants were calculated using a 1:1 binding langmuir curve fitting model 2 or Fab fragments on surface displayed IgE in a titration ELISA  measurements. Human monoclonal Sus11-IgE was displayed on the chip surface via the non-competitive anti-IgE capture antibody Le27  that is trapped in a closed FcεRIα incompatible conformation36, while the wild-type IgE-Fc3-4 (C328) can adopt an open FcεRIα-binding state  construct of ligelizumab to a resolution of 3.65 Å (Supplementary Table 2. This primary contact is formed by ~15 residues of the ligelizumab VH domain with heavy chain complementary determining region 1 (HCDR1) residues W31, Y32 and W33 forming key contacts at the center of the interface  at the midpoint of its binding titration with FcεRIα, which falls within physiological IgE concentrations  constitutively express surface FcεRI resulting in rapid binding and endocytosis of serum IgE37. The mechanism of FcεRI-mediated IgE:antigen complex uptake and presentation by DCs has further been proposed to promote Th2 immune responses39. To assess the effect of ligelizumab and omalizumab on IgE-binding and IgE:antigen complex internalization with DCs, we isolated human BDCA1+ DCs from whole-blood donations . The mice were pre-treated with 10 µg of anti-IgE antibody or PBS prior to sensitization with 20 µg of NIP-specific JW8-IgE IgE Fig. . The folion Fig. . These dtry Fig. . Thus, ttry Fig. , with th18. To assess whether this effect might additionally be related to ligelizumab-mediated inhibition of IgE production by B-cells, we performed IgE ELISpot assays. In vitro IgE synthesis is induced through incubation of isolated human PBMCs with IL-4 and anti-CD40 antibody46. Following this stimulation, we determined the number of IgE-producing B-cells in the cell culture by ELISpot assays and determined soluble IgE in culture supernatants. Incubation of the PBMCs with ligelizumab resulted in a significant reduction of IgE-producing B-cells in PBMC cultures, whereas the inhibition with omalizumab was less pronounced 2 had no effect on IgE production, strongly suggesting that its suppression is mediated via Fcγ-receptors. Interestingly, blocking of CD32b (FcɣRIIb) in PBMC cultures completely abrogated omalizumab-mediated suppression of IgE production, whereas ligelizumab-mediated suppression remained unaltered. Together our results demonstrate that the distinct IgE binding profiles and inhibition properties of ligelizumab and omalizumab significantly impact their functional activity.A recent study has provided strong evidence that ligelizumab suppresses free serum IgE levels in humans for a significantly longer period-of-time than omalizumab after single dose injectionced Fig. . In line48. This balance of modulating IgE interactions with its two receptors, along with their associated functions, might have future consequences for the design of anti-IgE therapeutics.Here, we compare structural and functional studies of two anti-IgE antibodies, ligelizumab and omalizumab, to better understand how their interactions with IgE impacts inhibitory mechanisms and predicts their potential therapeutic benefit. Ligelizumab and omalizumab recognize distinct binding epitopes in the IgE Cε3 domain with some overlap and show different sensitivities to IgE conformation. Therefore, the two anti-IgE antibodies display different abilities to inhibit IgE interactions with FcεRI and CD23 and feature a qualitatively distinct inhibition profile. Consequently, they greatly differ in their functional activities in blocking effector cell activation and IgE synthesis. While the increased affinity of ligelizumab for IgE explains superiority over omalizumab regarding neutralization of free serum IgE, we have identified an additional mode of action for ligelizumab through the inhibition of IgE production, which may provide additional therapeutic benefit. We observe that ligelizumab is more efficient in suppressing FcεRI-dependent allergic reactions in an in vivo model, while omalizumab may have advantages in blocking antigen presentation and transport processes that are dependent on IgE:CD23 interactions3-4 fragments from FcεRIα, while omalizumab binds such complexes without disrupting them41. Furthermore, we could show that the interaction of omalizumab with such FcεRI-bound IgE-Fc3-4 fragments on primary human basophils results in a dose-dependent degranulation of the cells. Whether such IgE fragments exist in vivo in certain situations remains to be investigated as well as whether this could potentially explain rare systemic anaphylactic reactions in roughly 0.2% of omalizumab-treated patients49. Ligelizumab lacks the ability to cross-link FcεRIα-bound IgE-Fc3-4 fragments and it will be interesting to see whether it shows a reduced risk of anaphylaxis and an altered safety profile as compared to omalizumab.Our data confirm that ligelizumab does not recognize FcεRIα-bound IgE, which represents the critical safety requirement for therapeutic anti-IgE antibodies. Interestingly, we observed that ligelizumab efficiently removes IgE-Fc50. It has been shown that allergen-IgE complex-mediated eosinophilic lung inflammation in mice was CD23 dependent48. These findings might explain why ligelizumab treatment lacked superior efficacy in phase 2 clinical trials with severe asthma patients compared to placebo and omalizumab (NCT02075008). On the other hand, ligelizumab has the ability to recognize CD23-bound IgE on B-cells and decreases IgE production in PBMC cultures. This effect was not mediated by ADCC as F(ab′)2 fragments revealed a similar inhibition of IgE production. Our data indicating that ligelizumab downregulates IgE production is in line with the finding that single dose injection of ligelizumab resulted in a significantly longer suppression of free serum IgE compared to omalizumab18. Further, our results strongly suggest that the mechanism of ligelizumab suppression of IgE synthesis is independent of Fcγ-receptor engagement but might rather be due to its ability to bind and aggregate CD23:IgE complexes on the surface of B-cells, which has not been observed for omalizumab.We further observe that omalizumab inhibits IgE-binding to CD23 more potently than ligelizumab, which could potentially play an important role in the inhibition of IgE-mediated antigen presentation and IgE-mediated transport across epithelial barriers31.In summary, the structural and mechanistic differences that we have found between ligelizumab and omalizumab may have different impacts on FcεRI- and CD23-mediated pathways in patients. Presently, it is concluded that ligelizumab has the potential to be particularly efficacious in diseases driven predominantly by FcεRI-dependent reactions, as observed in CSU3-4 and the mutated C335 IgE-Fc3-4 were produced in our laboratory36. Recombinant human CD23 was purchased . Recombinant human IL-3 and IL-4 were purchased from Peprotec . Cells were cultured in RPMI+/+ medium composed of RPMI 1640 medium  complemented with 10% Hyclone FCS , penicillin 100 U/ml, 100 µg/ml streptomycin  and 10 mM HEPES buffer . For flow cytometry, we used the following antibodies: anti-human IgE FITC , monoclonal mouse anti-human FcεRIα APC  and the appropriate isotype controls monoclonal mouse IgG1, κ Isotype control FITC  and mouse IgG2b Isotype control APC , monoclonal rat anti-mouse CD200R FITC , monoclonal rat anti-mouse CD117 PE , monoclonal mouse anti-human CD19 APC , monoclonal mouse anti-human CD23 FITC and PE , monoclonal mouse anti-human Ig kappa light chain PE . For basophil activation testing, the anti-human CCR3 and anti-human CD63 antibody staining mix from the Flow CAST® kit was used .Ligelizumab as well as omalizumab antibodies and fragments were produced and kindly provided by Novartis Pharma AG . Sus11-IgE, JW8-IgE and the anaphylactogenic monoclonal anti-IgE antibody Le27 were purchased from NBS-C BioScience . Monoclonal anti-human CD40 antibody was purchased . Recombinant extracellular part of human FcεRIα as well as the wild-type C328 IgE-Fc3AS linker, the ligelizumab VL domain, a TEV cleavage site and linker to a His8 tag (MNFLLSWVHWSLA LLLYLHHAKWSQAAPMAEGGGQNQVQLVQSGAEVMKPGSSVKVSCKASGYTFSWYWLEWVRQAPGHGLEWMGEIDPGTFTTNYNEKFKARVTFTADTSTSTAYMELSSLRSEDTAVYYCARFSHFSGSNYDYFDYWGQGTLVTVSSGTGGSGGGGSGGGGSGGGASEIVMTQSPATLSVSPGERATLSCRASQSIGTNIHWYQQKPGQAPRLLIYYASESISGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQSWSWPTTFGGGTKVEIKENLYFQSGGSGHHHHHHHH). The wild-type IgE-Fc3-4 with a VGEF signal sequence derived from the pTTVH8G vector was cloned into pYD7 vector .A scFv codon-optimized construct of ligelizumab was synthesized (Genscript) and cloned into EcoRI/BamHI sites in the pTTVH8G vector for expression in mammalian HEK 293-6E cells . The scFv construct consists of the VEGF signal sequence, the ligelizumab VH domain, a GTG(GSGGG)3-4 by co-transfection in mammalian HEK 293-6E cells. A 1:1 mixture of plasmids was transfected using linear polyethylenimine (PEI), 25 KD (Polysciences) in a DNA:PEI ratio of 1:3. The proteins were expressed for 100–120 h. After harvesting, the cell supernatant was filtered through a 0.45-μm filter (Millipore) and incubated overnight at 4 °C with Ni-NTA resin (Qiagen), washed with wash buffer  and eluted with elution buffer . Eluted protein complex was concentrated using an Amicon Ultra-15 (Millipore) and purified with gel filtration on a Superdex 200 10/300 GL column (GE) using a running buffer consisting of 20 mM Tris pH 8.0, 150 mM NaCl. His-tags were removed by cleavage with TEV protease in the presence of 2.5 mM BME.The ligelizumab scFv was co-expressed with the human IgE-Fc3-4 complex was concentrated to a final concentration of 7.3 mg/ml in 20 mM Tris pH 8.0. Crystallization was carried out using the hanging drop method, with a precipitant composed of 0.2 M Na thiocyanate pH 6.9, 20% PEG3350 and 10 mM spermidine as an additive. Crystals were obtained in 2–5 days at 14 °C. Crystals were harvested and frozen in 0.2 M Na thiocyanate, 23% PEG 3350, 25% glycerol.Purified scFv:IgE-Fc51. The crystals belong to space group P212121 (Supplementary Table 52 using the structure of the IgE-Fc2-4 (Protein Data Bank code: 2Y7Q53) as a search model for IgE and the scFv structure of the anti-CD277 antibody 103.2 (Protein Data Bank code: 4F9P54) as a search model for ligelizumab. Manual model building was carried out in Coot55 and refinement was done in Phenix Refine52 using data from 3.65 to 20.0 Å. Model quality was analyzed by Coot, and structure figures were generated using PyMol . The final model has Ramachandran statistics of 97.3% of residues in the preferred region, 2.7% of the residues in the additionally allowed region and no residues in the disallowed region. Additional refinement statistics are collected in Supplementary Table A complete diffraction dataset for the complex crystal was collected at 100° K at the Advanced Photon Source at Argonne National Laboratories LS-CAT beamline using an X-ray wavelength of 0.979 Å. The diffraction data were processed with X-Ray Detector Software (XDS)+ was used as running buffer at a flowrate of 10 μl/min. The target proteins were immobilized on flow cell 2 (Fc2) of a CM5 sensor chip by standard amine coupling. The sensorgrams reflect binding responses on Fc2 minus binding responses on the reference Fc1. To determine binding kinetics, we used the BIAevaluation software. Affinity constants were calculated using a 1:1 langmuir curve fitting model.All SPR measurements were carried out on a GE Healthcare Biacore X100 device . HBS-EPTo determine binding kinetics of ligelizumab and omalizumab antibodies/fragments for human full-length IgE, 3000 RU of the anti-IgE Le27 were immobilized on flow cell 2 at pH 4.0. A concentration of 30 nM Sus11-IgE was subsequently captured for 120 s to reach a response of ~100 RU. Various concentrations (1.56–25 nM) of ligelizumab and omalizumab antibodies/fragments were injected for 120 s and the dissociation was measured for 240 s under constant buffer flow. After each run, the chip surface was regenerated with 50 mM NaOH and reloaded with Sus11-IgE.3-4 variants, 100 RU of ligelizumab or omalizumab IgG were immobilized on individual chips . A blank immobilization was performed on flow cell 1. Different concentrations (0.3–5 nM) of C328 and C335 IgE-Fc3-4 were injected for 120 s and the dissociation was measured for 180 s under constant buffer flow. After each run, the chip surface was regenerated with 10 mM glycine-HCl pH 2.0.To determine binding kinetics of ligelizumab and omalizumab IgG for wild-type C328 and mutated C335 IgE-FcTo assess whether ligelizumab or omalizumab IgG recognize FcεRIα:IgE complexes, 1000 RU of recombinant human FcεRIα was immobilized on flow cell 2 at pH 4.0. A blank immobilization was performed on flow cell 1. A concentration of 20 nM Sus11-IgE was subsequently captured for 120 s to reach a response of >100 RU. Various concentrations (3.13–100 nM) of ligelizumab, omalizumab antibodies/fragments and Le27 were injected for 120 s and the dissociation was measured for 180 s under constant buffer flow. After each run, the chip surface was regenerated with 50 mM NaOH and reloaded with Sus11-IgE.To assess whether ligelizumab or omalizumab IgG may disrupt FcεRIα:IgE complexes, 1000 RU of recombinant human FcεRIα was immobilized on flow cell 2 at pH 4.0. A blank immobilization was performed on flow cell 1. A concentration of 20 nM Sus11-IgE was subsequently captured for 120 s to reach a response > 100 RU. Three concentrations  of ligelizumab or omalizumab antibodies/fragments were injected for 42 times 540 s with a dissociation time of 180 s between each injection under constant buffer flow. At the end of each run the chip surface was regenerated with 50 mM NaOH and reloaded with Sus11-IgE.3-4 complexes, 1000 RU of recombinant human FcεRIα was immobilized on flow cell 2 at pH 4.0. A blank immobilization was performed on flow cell 1. A concentration of 20 nM IgE-Fc3-4 was subsequently captured for 120 s to reach a response > 100 RU. Various concentrations (3.13–100 nM) of ligelizumab, omalizumab antibodies/fragments and Le27 were injected for 120 s and the dissociation was measured for 180 s under constant buffer flow. After each run, the chip surface was regenerated with 50 mM NaOH and reloaded with IgE-Fc3-4.To assess whether ligelizumab or omalizumab IgG may disrupt FcεRIα:IgE-FcTo assess whether ligelizumab or omalizumab IgG interact with CD23:IgE complexes, 3000 RU of recombinant human CD23 was immobilized on flow cell 2 at pH 4.0. A blank immobilization was performed on flow cell 1. A concentration of 125 nM JW8-IgE was captured for 120 s to reach a response > 100 RU. Various concentrations (31.25–500 nM) of ligelizumab, omalizumab antibodies were injected for 120 s and the dissociation was measured for 180 s under constant buffer flow. After each run, the chip surface was regenerated with 50 mM NaOH and reloaded with JW8-IgE. Ligelizumab and omalizumab IgG binding to preformed CD23:IgE complexes was assessed using a polyclonal sheep anti-human IgG antibody at 125 nM concentration .To measure the binding interaction of ligelizumab or omalizumab with human IgE, the anti-IgE antibodies were immobilized on plastic surface of a 96-half-well plate  at a concentration of 30 nM by overnight incubation in PBS at 4 °C. The next day, the plate was blocked with PBS/0.15% casein for 2 h at room temperature (RT) and washed with PBS/0.05% Tween. Human Sus11-IgE was incubated at a serial dilution (0.03–7.5 nM) for 1 h at RT. The plate was washed two times with PBS/0.05% Tween and incubated for 1 h with the same wash buffer followed by three times washing with PBS only. IgE was detected with monoclonal non-competitive anti-IgE Le27 coupled to horseradish-peroxidase (HRP). TMB  was used as a substrate for HRP and the reaction was stopped with 1 M sulfuric acid. Absorbance was measured at 450 nm wavelength using the standard ELISA reader SpectraMax M5 from Molecular Device LLC .3-4 variants, the anti-IgE antibodies were immobilized on plastic surface of a 96-half-well plate  at a concentration of 10 nM by overnight incubation in PBS at 4 °C. The next day, the plate was blocked with PBS/0.15% casein for 2 h at RT and washed with PBS/0.05% Tween. Wild-type C328 and mutated C335 IgE-Fc3-4 variants were incubated at a serial dilution (0.0001–10 nM) for 1 h at RT and subsequently washed with PBS/0.05% Tween and PBS. Development of the assay was performed as mentioned above.To measure the binding interaction of ligelizumab or omalizumab with wild-type C328 and mutated C335 IgE-FcTo measure IgE-binding to FcεRI, we immobilized recombinant human FcεRIα on a plastic surface of a 96-half-well plate  at 30 nM by overnight incubation in PBS at 4 °C. The next day, the plate was blocked with PBS/0.15% casein for 2 h at RT. Biotinylated JW8-IgE was incubatd at a 1:3 serial dilution (60–0.001 nM) for 1 h at RT. IgE-binding was detected with poly-HRP-conjugated streptavidin  and was followed by development with TMB.To assess the inhibition of IgE-binding to FcεRIα by ligelizumab and omalizumab, we immobilized recombinant human FcεRIα on plastic surface of a 96-half-well plate  at a concentration of 30 nM by overnight incubation in PBS at 4 °C. The next day, the plate was blocked with PBS/0.15% casein. Anti-IgE antibodies (0.0078–600 nM) were pre-incubated with biotinylated JW8-IgE (0.78 nM) for 30 min at RT and were added for 1 h to the plate. The plate was washed with PBS/0.05% Tween and PBS. IgE binding was detected with poly-HRP-conjugated streptavidin and was followed by development with TMB.7-BSA  in PBS/0.15% casein at a 1:2 serial dilution (280–2.1875 nM) and incubated for 30 min at RT. The IgE-antigen complexes were then incubated on the plate for 1 h at RT. IgE binding was detected with biotinylated anti-IgE antibody Le27 (2.66 μg/ml) and poly-HRP-conjugated streptavidin followed by development with TMB. In the inhibition ELISA for CD23-binding IgE-antigen complexes (70 nM:70 nM) were incubated with a 1:2 serial dilution of the anti-IgE antibodies (280–0.5469 nM) for 30 min at RT before incubation on the ELISA plate.For the CD23-binding ELISA, we immobilized 60 nM recombinant human CD23  on a 96-half-well plate  by overnight incubation at 4 °C. The following day, the plate was blocked with PBS/0.15% casein. The JW8-IgE was mixed 1:1 with NIP+ DCs were isolated from whole-blood donations. Human peripheral whole-blood was obtained from volunteering donors, who provided informed consent in accordance with the Helsinki Declaration. The study was approved by the local ethics committee (KEK 2018-00204). Basophils and BDCA1+ DCs were enriched by Percoll density centrifugation of dextran-sedimented supernatants. Furthermore, basophils were purified with negative selection using the Milteny basophil isolation kit II . BDCA1+ DCs were isolated with positive selection using the Milteny human CD1s ). Cells were analyzed for purity via flow cytometry.Primary human basophils and BDCA16 cells/ml per well in a 96-well round-bottom plate . First, we removed endogenous IgE from the cell surface by addition of 5 µM of the disruptive anti-IgE DARPin® protein bi53_79 in RPMI+/+ supplemented with 10 ng/ml recombinant human IL-3 overnight for basophils. The next day, the cells were washed and incubated with different concentrations of JW8-IgE (0.006–100 nM) to assess dose-dependent binding. The concentration of 2 nM was further used for inhibition experiments with anti-IgE antibodies. JW8-IgE was pre-incubated with increasing concentrations (0.032–100 nM) of ligelizumab or omalizumab for 30 min at RT. Surface IgE was stained and quantified by flow cytometry 1 h after adding this mix to the cells. To assess the inhibition profile of ligelizumab and omalizumab IgG to inhibit IgE binding to FcεRIα BDCA-1+ DCs without the presence of any additionally cytokines or growth factors, we modified the protocol above and removed the endogenouse IgE from the cells by the disruptive anti-IgE DARPin® protein bi53_79 only in RPMI+/+ medium during an incubation of 4 h. Subsequently, the cells were washed and incubated with different concentrations of JW8-IgE (100–0.005 nM) to assess dose-dependent binding. The concentration of 25 nM JW8–IgE was further used for inhibition experiments with anti-IgE antibodies. JW8-IgE was pre-incubated with increasing concentrations (0.0012–312.5 nM) of ligelizumab or omalizumab for 30 min at RT. Surface IgE was stained and quantified by flow cytometry 1 h after adding this mix to the cells. Surface IgE was stained and quantified by flow cytometry 1 h after adding this mix to the cells.To compare the efficacy of ligelizumab and omalizumab IgG to inhibit IgE binding to FcεRIα expressing cells, we cultured isolated primary human basophils at a density of 1 × 10® protein-treated human primary basophils and subsequently stimulated these cells with 100 ng/mL NIP7-BSA  in RPMI+/+-containing human 10 ng/ml recombinant human IL-3 and Flow CAST® kit antibody staining mix . The concentration of 0.68 nM JW8-IgE was subsequently used for pre-incubation with increasing concentrations (0.0064–20 nM) of ligelizumab and omalizumab in RPMI+/+ medium for 30 min at RT. Anti-IgE DARPin® protein-treated primary human basophils were then sensitized for 2 h at 37 °C with this mixture and stimulated with 100 ng/mL NIP7-BSA. Activation was determined by measuring CD63+ basophils using flow cytometry.To assess inhibition of IgE-dependent basophil activation, we first determined the concentration of JW8-IgE to be used to reach half-maximal activation. For this we titrated JW8-IgE (0.0064–20 nM) on 25,000 anti-IgE DARPin6 cells/ml) with either 0, 0.25, 0.5 or 1 µM of ligelizumab or omalizumab antibodies/fragments in RPMI+/+ supplemented with 10 ng/ml recombinant human IL-3. After 3 and 6 days of culture, the cells were stained with anti-IgE antibody for 15 min at RT and measured by flow cytometry.To investigate whether anti-IgE antibodies might actively remove FcεRIα-bound IgE from the cell surface, we cultured 50,000 isolated primary human basophils . One day before the experiment, the cells were split 1:2 in RPMI+/+ medium. Flow cytometry was performed using a BD FACSCanto device  and results were evaluated with FlowJo Version 10.1 .The human RPMI8866 leukemia B-cell line was kindly provided by Dr. Monique Vogel. The cells were cultured in RPMI3-4 on purified primary human basophils (purity > 90%), endogenous IgE was removed from 50,000 basophils per well by incubation with 5 µM disruptive anti-IgE DARPin® protein bi53_79 in RPMI+/+. After washing with PBS pH 7.4, the cells were reloaded with 100 nM JW8-IgE or C328 IgE-Fc3-4. Again the cells were washed with PBS pH 7.4. Then the cells were treated with 0.6–66.6 nM ligelizumab or omalizumab IgG in RPMI+/+ medium containing human 10 ng/ml recombinant human IL-3 for 30 min at 37 °C and 5 % CO2. Surface expression of human IgE was determined with an anti-human IgE FITC antibody and basophil activation was assessed using the Flow CAST® kit (Bühlmann Laboratories AG) by flow cytometry.To assess whether ligelizumab accelerates dissociation of FcεRI-bound IgE-Fc4 RPMI8866 cells per well were seeded in a 96-well plate  and incubated with biotinylated JW8-IgE for 1 h at 37 °C, 5% CO2 in RPMI+/+ medium. Subsequently, the cells were stained with streptavidin FITC , anti-CD23 and anti-CD19 antibodies for 20 min at 4 °C. For the IgE inhibition on CD23 expressing cells, 12.5 nM biotinylated JW8-IgE was pre-complexed with the anti-IgE antibodies  in RPMI+/+ medium for 30 min at RT. Subsequently, this mix was added to the RPMI8866 cells for 1 h at 37 °C, 5% CO2 in RPMI+/+ medium. Staining was performed as mentioned above.For IgE titration on CD23 expressing cells, 5 × 102 in RPMI+/+ medium. The cells were then washed three times with 150 μl of PBS pH 7.4 and centrifuged at 600 × g for 5 min at 4 °C, resuspended in RPMI+/+ medium containing 12.5 nM ligelizumab, omalizumab or control IgG and incubated for 30 min at 37 °C. Subsequently, the cells were washed two times with 200 μl of PBS pH 7.4 and stained with streptavidin, anti-CD19 and monoclonal mouse anti-human Ig κ light chain  at 4 °C. Blocking of CD23 receptor was performed by incubation of the RPMI8866 cells with 5 nM anti-CD23 DARPin® protein D89_86 (ref. 44) for 15 min at 37 °C, 5% CO2. Cells were then washed once with PBS before incubation with JW8-IgE and the anti-IgE antibodies as described above.To check for binding of anti-IgE antibodies to CD23-bound IgE RPMI8866 cells were incubated with 12.5 nM biotinylated JW8-IgE for 1 h at 37 °C, 5% CO+ DCs, we conjugated pH-sensitive rodamine (pH-Rodo) to NIP-specific JW8 IgE by the pHrodo Red Microscale Labeling Kit (Life Technologies). The fluorophore pH-Rodo increases emission at more acidic pH), which happens along the pathway of endocytosis/phagocytosis . First pH-Rodo IgE:antigen complexes were formulated with 25 nM pH-rodo IgE and 2.5 nM NIP(15)BSA  for 0.5 h at RT and were then supplemented with an 5 nM ligelizumab or omalizumab for additional 0.5 h at RT. Upon removing the endogenous IgE from basophils and BDCA1+ DCs by the disruptive anti-IgE DARPin® protein bi53_79 for 4 h at 37 °C, the cells were washed and pulsed with formulated IgE:antigen:anti-IgE antigen complexes for 2 h at 37 °C. After pulsing the cells, the cells were washed intensively and further incubated time-dependently  at 37 °C. The pH-Rodo JW8-IgE emissions were finally measured in the PE-channel by flow-cytometry.To assess the effect of ligelizumab and omalizumab on IgE:antigen complex internalization with BDCA16 cells/ml, and 1 × 105 cells per well were seeded in a 96-well round-bottom plate. Washing was performed once with 200 µl of PBS pH 7.4 at 500 × g for 5 min at 4 °C. The cells were then resuspended in 100 µl of RPMI+/+ medium containing 12.5 nM biotinylated JW8-IgE and incubated for 1 h at 37 °C, 5% CO2. Afterward, the cells were washed three times with 200 µl of PBS pH 7.4 at 500 × g for 5 min at 4 °C, followed by 30 min incubation at 37 °C, 5% CO2 in 100 µl of RPMI+/+ medium containing 12.5 nM ligelizumab, omalizumab or isotype IgG. Subsequently, the cells were stained with anti-CD23, anti-Ig κ light chain and streptavidin antibodies for 20 min at 4 °C. Binding of IgE and the anti-IgE antibodies was assessed using an Amnis® ImageStream®X MKII and the corresponding IDEAS® software .To assess the binding of anti-IgE antibodies to CD23-bound IgE by multispectral imaging flow cytometry, RPMI8866 cells were diluted to 1 × 10Peripheral blood mononuclear cells were isolated from fresh whole blood supplemented with 100 mM EDTA using Ficoll Paque PLUS  density gradient centrifugation. The blood was diluted 1:2 in PBMC wash buffer , overlaid onto Ficoll layer and centrifuged at 1800 rpm for 35 min at RT with brakes off. The peripheral blood leukocyte (PBL) layer was extracted and washed with PBMC wash buffer at 1200 rpm for 5 min, followed by two times washing with PBS pH 7.4 at 1000 rpm for 10 min.5 PBMCs were cultured in 96-well round-bottom plates at a density of 1 × 106 cells/ml in RPMI+/+ medium. The cells were stimulated with 30 ng/ml recombinant human IL 4 and 1 μg/ml anti-human CD40 antibody for 6 days at 37 °C, 5% CO2 in the presence of 0.5 μM anti-IgE antibodies. Unstimulated, untreated and monoclonal anti-human IgG1 antibody-treated cells served as controls. For the detection of IgE-producing B-cells, we used 96-well MultiScreen filter plates  and the human IgE ELISpotBASIC kit . Overnight incubation of the coating antibody (15 μg/ml) at 4 °C was followed by blocking for 2 h with RPMI+/+ medium the next day. The PBMCs were washed twice with PBS pH 7.4 before transfer to a MultiScreen plate and incubation in RPMI+/+ medium complemented with recombinant human IL-4 (30 ng/ml) and anti-CD40 (1 μg/ml) for 24 hours at 37 °C, 5% CO2. For IgE detection, Mabtech detection antibody mixture  and poly-HRP-conjugated streptavidin  were diluted in RPMI+/+ medium and incubated on ELISA plate shaker at RT. For HRP substrate, we used the AEC staining kit  and incubated the plate with 50 μl per well for 5 min. The colorimetric reaction was stopped by washing the plate with ddH2O. Washing steps were performed with PBS pH 7.4 or PBS pH 7.4/0.05% Tween. Spots were quantified using an ELISpot reader and the corresponding iSpot software v7.0 .A total of 2.5 × 106 cells/ml in RPMI+/+ medium at 37 °C, 5% CO2 in the presence of 0.5 µM anti-IgE antibodies and 0.5 µM of the anti-FcγRIIB DARPin® protein D11 (ref. 56), respectively. IgE production was induced by stimulation with 30 ng/ml recombinant human IL-4 and 1 μg/ml anti-human CD40 antibody for 12 days. On day 12, the supernatants were collected and soluble IgE was measured by ELISA. For the ELISA, anti-IgE antibody Le27 was immobilized on plastic surface of a 96-half-well plate  at a concentration of 30 nM by overnight incubation in PBS at 4 °C. The next day, the plate was blocked with PBS/0.15% casein. The culture supernatants were incubated for 1 h on the plate followed by washing twice with PBS pH 7.4 or PBS pH 7.4/0.05% Tween. IgE was detected using Mabtech detection antibody mixture  and poly-HRP-conjugated streptavidin  diluted in PBS/0.15% casein and incubated on ELISA plate shaker at RT. TMB  was used as a substrate for HRP and the reaction was stopped with 1 M sulfuric acid. Absorbance was measured at 450 nm wavelength using the standard ELISA reader SpectraMax M5 from Molecular Device LLC .To assess the effect of anti-IgE antibodies on the production of soluble IgE, we cultured PBMCs at 1 × 1020-BSA . Body-core temperature was measured before challenge (baseline) and every 10 min after antigen-challenge for 60 min and every 30 min until 120 min. Data are represented as measured temperature after challenge minus baseline temperature (Δ core body temperature) for each time point.Mice transgenic for human FcεRIα (huFcεRIα tg) on a mixed C57BL/6J–C57BL/6N background were obtained from Prof. J.-P. Kinet. All animal experimentation was approved by the local ethics committee (authorization BE66/18). Seven days before the experiment, huFcεRIα tg mice were subcutaneously implanted with an electronic temperature transponder (IPTT-300) from BMDS  to measure body-core temperature as instructed by the manufacturer. On day 0, mice received an intraperitoneal injection of PBS or 20 µg of anti-IgE antibody . Half an hour later, mice were passively sensitized with 20 μg of NIP-specific human JW8-IgE (in 200 µl) by intraperitoneal injection. On day 1, mice were challenged by intraperitoneal injection of 200 μg of NIPFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


EMSY and LRRC32 (leucine-rich repeat-containing 32) but the functional mechanisms affecting risk of AD remain unclear.Atopic dermatitis (AD) is a common, complex, and highly heritable inflammatory skin disease. Genome-wide association studies offer opportunities to identify molecular targets for drug development. A risk locus on chromosome 11q13.5 lies between 2 candidate genes, We sought to apply a combination of genomic and molecular analytic techniques to investigate which genes are responsible for genetic risk at this locus and to define mechanisms contributing to atopic skin disease.We used interrogation of available genomic and chromosome conformation data in keratinocytes, small interfering RNA (siRNA)–mediated knockdown in skin organotypic culture and functional assessment of barrier parameters, mass spectrometric global proteomic analysis and quantitative lipid analysis, electron microscopy of organotypic skin, and immunohistochemistry of human skin samples.EMSY; EMSY, LRRC32, and intergenic variants all appear to be within a single topologically associating domain. siRNA-knockdown of EMSY in organotypic culture leads to enhanced development of barrier function, reflecting increased expression of structural and functional proteins, including filaggrin and filaggrin-2, as well as long-chain ceramides. Conversely, overexpression of EMSY in keratinocytes leads to a reduction in markers of barrier formation. Skin biopsy samples from patients with AD show greater EMSY staining in the nucleus, which is consistent with an increased functional effect of this transcriptional control protein.Genomic data indicate active promoters in the genome-wide association study locus and upstream of EMSY in transcriptional regulation and skin barrier formation, supporting EMSY inhibition as a therapeutic approach.Our findings demonstrate an important role for   Atopic dermatitis , and EMSY.An association peak within an intergenic region on chromosome 11q13.5 was identified in the earliest AD GWAS.LRRC32 encodes the TGF-β activator LRRC32 (UniProtKB Q14392), previously termed glycoprotein A repititions predominant (GARP), a membrane protein that binds latent TGF-β1 on the surfaces of activated regulatory T cells.LRRC32 has been proposed as a causal gene for atopic skin inflammation,EMSY, also known as BRCA2-interacting transcriptional repressor and previously termed C11orf30, codes for the protein EMSY (UniProtKB Q7Z589), which is expressed in multiple human tissues, including cerebellum, breast, lung, ovary, uterus, and skin . EMSY has been characterized as a transcriptional regulator, either repressing transcription as part of a chromatin remodeling complex or activating transcription as part of a histone H3–specific methyltransferase complex.EMSY amplification is associated with DNA damage and increased risk of malignancy in breast and ovarian tissue.EMSY, LRRC32, or both, showed evidence of activity in human skin cells. We then investigated the mechanism of effect using functional and multi-omics analysis of organotypic skin, followed by immunostaining of AD biopsy samples.We set out to investigate, using genetic and genomic data, whether www.jacionline.org.All human tissues were obtained with written informed consent from donors under the governance of and with ethical approval from the NHS Research Scotland Biorepository in Tayside. Primary keratinocytes and donor-matched primary fibroblasts were extracted from skin samples discarded from plastic surgery procedures. AD skin samples were identified from the hospital pathology database as consecutive unselected cases. Demographic details are provided in the Normal human keratinocytes (NHKs) and normal dermal fibroblasts (NDFs) were isolated from healthy breast skin by means of sequential collagenase D and trypsin EDTA digestion, as previously described.2/1.1% NaCl; Sigma-Aldrich) were combined on ice, supplemented with 200,000 NDFs and aprotinin , and transferred to a 12-well plate. After 30 minutes of incubation at 37°C, the gels were covered in medium  and cultured overnight (day 1). The following day, medium was replaced with RM medium excluding EGF, 0.1 U/mL aprotinin, and 2 × 106 suspended NHKs (day 2). This was refreshed daily for the next 2 days (day 3 and 4) with RM medium containing 0.1 ng/mL EGF and 0.1 U/mL aprotinin. On day 5, gels were carefully removed from wells by using a plastic spatula and lifted onto custom-made steel grids lined with nylon gauze . RM medium supplemented with 0.1 ng/mL EGF and 0.1 U/mL aprotinin was added up to the base of the grid, enabling the fibrin gels to be nourished from below and the epidermis cultured at the air-liquid interface. Medium was refreshed every other day until day 12 when the cultures were analyzed. Where required, the epidermis was isolated from fibrin gel after hypertonic saline–induced split .Fibrin gel dermal equivalents were prepared by using a protocol adapted from published methods.6 suspended NHKs and transferred to the preprepared dermal substrate. A pool of 4 siRNA duplexes was used .NHKs were reverse transfected immediately before inclusion in the organotypic cultures by using RNAiMax transfection reagent , according to the manufacturer's instructions. Briefly, small interfering RNA (siRNA) complexes were formed in Opti-MEM medium (20 μmol/L siRNA and 5 μL of RNAiMAX) and, after 20 minutes of incubation, combined with 2 × 10C11orf30; catalog no. RC216916L4; OriGene) plasmid with Lipofectamine-3000 transfection reagent , according to the manufacturer's protocol, into 293T packaging cells for 16 hours. The next morning, the cells were washed twice with PBS to remove excess plasmid DNA, and the medium was replaced with virus-producing medium (20% FBS/DMEM). Forty-eight, 72, and 96 hours after transfection, the first, second, and third viral supernatants were harvested. Viral supernatants were spun down at 1200 rpm for 15 minutes and filtered with a 0.45-μm filter.A second-generation lentiviral system was used, as follows. Pseudoviral particles were prepared with psPAX2 packaging  and pMD2.G plasmid , which were cotransfected with the control pLenti-C-mGFP-P2A-puromycin–tagged cloning vector  plasmid or with the Lenti-ORF clone of mGFP-tagged-human chromosome 11 open reading frame 30  and 1 or 2 mL of viral supernatant and then plated onto 6-well plates and cultured overnight. The second transduction was performed in monolayer culture using RM medium without EGF, 10 μg/mL Polybrene, and 1 or 2 mL of viral supernatant. Cells were incubated for 90 minutes at 37°C, followed by centrifugation at 1200 rpm. To eliminate excess virus, cells were washed twice with PBS, and the medium was replaced with RM media . Fresh RM medium was replaced every second day, and samples were harvested on day 10 after transduction as a differentiated culture.Primary human keratinocytes from donor skin were transduced twice. The first transduction was in RM medium without EGF. After treatment, the keratinocytes with trypsin and 1 × 10t tests.Fifty microliters of 1 mmol/L Lucifer yellow dye (Sigma-Aldrich) was added to the epidermal surface of the organotypic culture and incubated at 37°C for 4 hours. Metal cloning rings were used to control uniform dosing on the epidermal surface. Lucifer yellow was removed, and the organotypic cultures were washed in PBS before formalin-fixed paraffin embedding under standard conditions. Four-micrometer sections were deparaffinized, counterstained with 4′-6-diamidino-2-phenylindole dihydrochloride , and imaged with a confocal Zeiss LSM710 microscope . Quantification of dye penetration in the upper dermis (average intensity in the upper 40 μm) was performed with Zeiss Zen software and compared by using paired Organotypic cultures were equilibrated at room temperature and atmospheric conditions for 30 minutes before transepidermal water loss (TEWL) was measured at 2 locations on the epidermal surface with an AquaFlux AF200 instrument  with a custom (5 mm in diameter) probe head. TEWL measurements were taken every second for a minimum of 60 seconds until a stable reading, as determined by using the software, was obtained.Organotypic cultures were equilibrated at room temperature and atmospheric conditions for 30 minutes before measurement of epidermal surface capacitance as a measure of water content with a Corneometer . Three measurements were recorded from each organotypic culture, and the mean was calculated.http://www.geneontology.org/page/go-enrichment-analysis . A volcano plot was generated in R/ggplot2 by using human proteins detected in all 4 of the replicates to calculate the fold change (nontargeting control/EMSY knockdown) for each donor with log10 transformation and a t test for significance.Network analysis was performed by using Ingenuity Pathway Analysis . Gene ontology (GO) enrichment analysis was performed in the Gene Ontology Consortium online tool by using PANTHER classification Frozen sections of skin organotypic samples were cut and air-dried onto slides before formalin fixation and rinsing with 60% isopropranol. Oil Red O (Sigma-Aldrich) working solution was freshly prepared, and sections were stained for 15 minutes, rinsed with 60% isopropanol, and then lightly counterstained with alum hematoxylin before a final rinse with distilled water.www.jacionline.org.Additional EMSY  and approximately 77 kb downstream of LRRC32 . The credible set identified by mapping this locus lies entirely within an intergenic region , supporting a possible functional interaction. Analysis of promoter-capture Hi-CLRRC32 and the intergenic SNP locus, but these data were not sufficiently detailed to determine whether EMSY also shows conformational interaction . However, EMSY is more highly expressed in skin than LRRC32 at the protein level (https://www.proteinatlas.org/), and it has not previously been studied in keratinocyte biology; therefore EMSY was selected for further detailed investigation.Gene expression dataEMSY expression was achieved by means of transfection of keratinocytes immediately before seeding onto the dermal equivalent. Knockdown was confirmed at the mRNA and protein levels, persisting to 10 days in organotypic culture (A-C). Equivalent effects were seen by using individual and pooled siRNAs (see www.jacionline.org). EMSY knockdown produced a marked phenotypic change (D), including thickening of the epidermal cell layer and stratum corneum (E). There was an increase in the number of layers within the stratum corneum and the frequency of corneodesmosomes (F). The stratum granulosum, the site of filaggrin expression,D), and increased filaggrin expression was confirmed by using quantitative PCR  and Western blotting .To investigate a functional effect of EMSY in skin, we used primary human keratinocytes seeded onto a dermal equivalent, which forms an organotypic model with stratified layers that effectively recapitulate the structure and gene expression patterns of human skin. culture , A-C. Eqc change , D, incl corneum , E. Thersmosomes , F. The www.jacionline.org). EMSY knockdown in the skin model resulted in a reduction in stratum corneum hydration (A), a reduction in TEWL (B), and a reduction in penetration of the Lucifer yellow dye (C and D), which is in keeping with enhanced and accelerated barrier development compared with control siRNA treatment.In our skin culture model stratum corneum hydration, TEWL, and Lucifer yellow dye penetration progressively decrease as the skin barrier is formed .In contrast to the phenotype observed with EMSY knockdown phenotype, we applied tandem mass spectrometric global proteomic analysis (MS/MS) to donor-matched control and EMSY knockdown organotypic experiments from 4 independent donors. Total epidermal protein extracts were fractionated by using high-pH reversed-phase chromatography before tandem mass spectrometry, which identified more than 9000 proteins per sample (see www.jacionline.org). qPCR and Western blotting confirmed knockdown of EMSY in each sample. A greater proportion of proteins showed increased rather than decreased expression on EMSY knockdown (A), indicating that EMSY's predominant role is as a transcriptional repressor in this tissue.To assess in more detail the nockdown , A, indiEMSY knockdown and showing reproducibility across different donors to minimize any effect of interindividual differences.Data were filtered for proteins showing changes in the same direction consistently across all 4 biological replicates and fold changes of 2.5 or greater (n = 154 proteins) or 0.5 or less (n = 130 proteins) in at least 3 of 4 of the biological replicates. Consistent direction of change was used as a criterion to focus analysis on changes of relevance to P ≤ .037). GO analysis of the downregulated proteins showed enrichment of cellular components termed cytosol (GO: 0005829), the mitochondrial membrane (GO: 0031966), and the Golgi membrane . The full lists of GO terms defined by using this analysis are shown in www.jacionline.org.GO analysis of the upregulated proteins showed enrichment for the biological processes termed establishment of the skin barrier (GO: 0061436), skin development (GO: 0043588), regulation of water loss through the skin (GO: 0033561), and cornification .www.jacionline.org.Pathway analysis  identified upregulation of pathways predicted to inhibit the development of dermatitis and ichthyosis , B.42, 4A), which is in keeping with the increase in levels of proteins involved with lipid metabolism . Mass spectrometric lipid analysis showed an increase in long-chain ceramides and esterified omega-hydroxy-ceramides species in organotypic samples with EMSY knockdown (B); these are the ceramides that have previously been reported to show a reduction in AD skin samples.Organotypic samples showed an increase in abundance of epidermal lipid staining with EMSY knockdown , A, whicnockdown , B; thesC), which is consistent with the observed increase in desmocollin 1 levels. An increase in the number of layers within the stratum corneum was also observed, which is consistent with the increase in corneodesmosin levels .Ultrastructural analysis demonstrated an increase in desmosome size , C, whicEMSY expression in human skin samples, we compared 18 normal control skin samples and biopsy specimens from 14 patients with spongiotic dermatitis, AD, or both. Immunohistochemistry showed considerable interindividual variability in EMSY staining, despite careful standardization of staining conditions, and there was no consistent difference in total intensity between cases and control subjects (see www.jacionline.org). However, all 14 AD cases showed greater nuclear than cytoplasmic staining, and 12 (86%) of 14 cases showed predominantly nuclear staining extending throughout the epidermis compared with 2 (11%) of 18 control subjects ; it is likely that these mechanisms also contribute to functional regulation in different cell and tissue types.Expression quantitative trait locus analysis is an approach that has been used to investigate genes responsible for risk mechanisms attributable to intergenic loci.Analysis of a single cell type, the keratinocyte, at different stages of terminal differentiation represents a reductionist view of the epidermis, but the skin organotypic model has proved to be a valuable model for investigation of molecular mechanisms controlling differentiation in this multilayered tissue.in vivo is predicted to improve skin barrier function and protect against AD. However, considerable further work is needed, including high-throughput screening to identify molecules capable of reducing the abundance of nuclear EMSY, followed by preclinical testing, before these findings can be assessed in clinical trials. The advent of biological therapy to target the immune component of AD has transformed care for patients with moderate-to-severe disease,•Genetic risk loci offer the opportunity for insight into the cause of complex disease, but the mechanisms require detailed molecular investigation.•EMSY and LRRC32.The AD-associated locus on chromosome 11q13.5 lies between 2 candidate genes: •EMSY expression in the control of skin barrier formation and function, which are of importance in patients with AD.Our genetic, proteomic, and immunohistologic analyses have together demonstrated a role for •Therefore EMSY represents a future therapeutic target in atopic disease.The work presented here has identified EMSY as a therapeutic target: knockdown


Pueraria lobata, coumestrol and puerarol, along with the cholinergic and amyloid-cascade pathways to mitigate Alzheimer’s disease (AD). Antioxidant activity was evaluated via 1,1-diphenyl-2-picryhydrazyl (DPPH) and peroxynitrite (ONOO−) scavenging ability further screened via ONOO−-mediated nitrotyrosine. Similarly, acetyl- and butyrylcholinesterase (AChE/BChE) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activities were assessed together with docking and kinetic studies. Considering DPPH and ONOO− scavenging activity, coumestrol  was found to be more potent than puerarol  followed by dose dependent inhibition of ONOO−-mediated nitrotyrosine. Coumestrol showed pronounced AChE and BChE activity with IC50 values of 42.33 and 24.64 µM, respectively, acting as a dual cholinesterase (ChE) inhibitor. Despite having weak ChE inhibitory activity, puerarol showed potent BACE1 inhibition (28.17 µM). Kinetic studies of coumestrol showed AChE and BChE inhibition in a competitive and mixed fashion, whereas puerarol showed mixed inhibition for BACE1. In addition, docking simulations demonstrated high affinity and tight binding capacity towards the active site of the enzymes. In summary, we undertook a comparative study of two similar isoflavonoids differing only by a single aliphatic side chain and demonstrated that antioxidant agents coumestrol and puerarol are promising, potentially complementary therapeutics for AD.The current study assesses the antioxidant effects of two similar isoflavonoids isolated from  Among elderly patients, the most prevalent disease of the modern age is Alzheimer’s disease (AD). It is comprised of amyloid plaques consisting of β-amyloid (Aβ) peptides and neurofibrillary tangles of hyperphosphorylated tau protein which results in neuronal cell loss with dementia like symptom . Of the Oxidation is responsible for the pathogenesis of various age-related degenerative diseases such as cancer, diabetes, macular degeneration, AD, and Parkinson’s disease, as reactive pro-oxidant species can damage proteins, lipids, carbohydrates, and nucleic acids over time . In addiPueraria lobata-derived antioxidant compounds.Despite the availability of cholinesterase (ChE) inhibitors like donepezil, galantamine, and rivastigmine for AD, adverse effects still outweigh the benefit of commercial drugs over natural products. The most common side effects related to cholinergic stimulation in the brain and peripheral tissues include, gastrointestinal, cardiorespiratory, extrapyramidal, genitourinary, and musculoskeletal symptoms, as well as sleep disturbance . DiffereCoumestrol is a phytoestrogen occurring in plants in the coumestan family of compounds; it shares a common structure with isoflavones and estradiol and exhibits estrogenic and antiestrogenic activity based on estrogen levels in the body . ResearcP. lobata-derived compounds illustrated obvious anti-AD  ethyl}-6-phenoxy quinazolin-3-ium (QUD), with a binding energy of −8.80 kcal/mol. As shown in The top binding configuration of puerarol against BACE1 (PDB ID: 2WJO) was similar to the catalytic ligand, 2-amino-3-{ by analyzing H-bonds and the interaction energy, suggesting that coumestrol upon interaction with estrogen receptor-α led to a strong substrate binding affinity . HoweverP. lobata as described by Seong et al.  (DTNB) and berberine chloride were purchased from Sigma-Aldrich Co. . All required reagent-grade chemicals used in this study were purchased from commercial sources. Coumestrol and puerarol were isolated from g et al. . The str−4 M). After gently mixing and standing at room temperature for 30 min, the optical density of the reactant was measured at 520 nm using a VERSAmax microplate spectrophotometer . The antioxidant activity of samples is expressed in terms of EC50 values , which was calculated from the log-dose inhibition curve. L-Ascorbic acid was used as a positive control.DPPH radical scavenging activity was evaluated using the method of Blois , with sl− scavenging activity was assessed using a modified Kooy’s method that involves the monitoring of highly fluorescent rhodamine 123, which is rapidly produced from non-fluorescent dihydrorhodamine (DHR) 123 in the presence of ONOO− [− (10 μM), dissolved in 0.3 N sodium hydroxide. The fluorescence intensity of oxidized DHR 123 was evaluated using a microplate spectrofluorometer  at excitation and emission wavelengths of 480 and 530 nm, respectively. Values of ONOO− scavenging activity were calculated as the final fluorescence intensity minus the background fluorescence via the detection of DHR 123 oxidation. L-penicillamine was used as a positive control.ONOOof ONOO− . In brie−-nitrotyrosine formation was evaluated using the method of Aulak et al. [− (200 µM). After a 10 min incubation period (room temperature), the sample solution was added to Bio-Rad 2X Laemmli Sample buffer with mercaptoethanol and then boiled for 7 min. The reactant was resolved in 10% polyacrylamide gel via electrophoresis and transferred to PVDF membranes. Pre-stained blue protein markers were used for molecular weight determination. Monoclonal anti-nitrotyrosine antibody (85 kDa) was used as a primary antibody and horseradish peroxidase-conjugated sheep anti-mouse secondary antibody was used as a secondary antibody. Antibody labeling was visualized using the Supersignal West Pico Chemiluminescent substrate . Densitometric analysis of the western blot results was determined using a CS analyzer v3.00 (ATTO Corp.).The inhibition of ONOOk et al. . Samples50 value  as calculated from the log-dose inhibition curve. Berberine was used as the positive control [The inhibitory activity of coumestrol and puerarol against ChEs was measured using the spectrophotometric methods reported by Ellman et al.  and Jung control  The testThe in vitro BACE1 enzyme assay was carried out according to the manufacturer’s recommended protocol with minor modifications. Briefly, a mixture of 10 mL of assay buffer , 10 mL of BACE1 (1.0 U/mL), 10 mL of substrate  and 10 mL of the test sample was dissolved in 10% DMSO and incubated for 60 min at 25 °C in the dark. Proteolysis of the two fluorophores (Rh-EVNLDAEFK-Quencher) by BACE1 was determined by monitoring formation of the fluorescent donor (Rh-EVNL) at wavelengths of 545 nm for excitation and 585 nm for emission. Fluorescence was measured with a microplate spectrofluorometer . Quercetin was used as a positive control .iK) were determined by secondary plots [To determine the mechanism of inhibition, AChE, BChE, and BACE1 inhibition was evaluated by monitoring the effects of different concentrations of substrate . Specifically, AChE, BChE, and BACE1 inhibition assays were performed as described above, but the substrate concentration was varied. Inhibition modes of coumestrol and puerarol against ChEs and BACE1 were determined by Lineweaver–Burk plots . Inhibitry plots .+ .To simulate the structure of the enzyme–inhibitor complex and to ensure accuracy, repeatability, and reliability of the docking results, we employed AutoDock 4.2 software. In our study, coumestrol was tested for ChEs inhibition, while puerarol was tested for BACE1 inhibition. For docking studies, the crystal structures of the AChE , BChE 4, and BACt-test were used to analyze statistics. Data are presented as the mean ± SEM of at least four independent experiments.ANOVA and Student’s


To investigate whether unilateral pedicle screw fixation is superior than bilateral pedicle screw fixation for lumbar degenerative diseases.Electronic databases including PubMed, Embase, and the Cochrane Library up to August 2018 were searched. All the high-quality randomized controlled trials (RCTs) and prospective clinical controlled studies comparing the unilateral pedicle screw fixation and bilateral pedicle screw fixation for lumbar degenerative diseases were enrolled in this study. Fusion rate was the main outcome. Stata 12.0 was used for the meta-analysis.P = 0.073), visual analog scale (VAS) at final follow-up, Oswestry Disability Index (ODI), Japanese Orthopedic Association scores (JOA), short-form health survey (SF-36), and length of hospital stay. Compared with bilateral pedicle screw fixation, unilateral pedicle screw fixation was associated with a reduction of the total blood loss and operation time (P < 0.05). Unilateral pedicle screw fixation was associated with an increase of the cage migration than bilateral pedicle screw fixation .Twelve RCTs including 808 patients  were included in our meta-analysis. There was a significant difference between unilateral pedicle screw fixation and bilateral pedicle screw fixation in terms of the fusion rate (risk ratio (RR) = 0.96, 95%CI , Unilateral pedicle screw fixation and bilateral pedicle screw fixation has similar fusion rate when treating for lumbar degenerative diseases. Our meta-analysis suggested that compared with bilateral pedicle screw fixation, unilateral pedicle screw fixation significantly reduced total blood loss and operation time for lumbar degenerative diseases. The use of unilateral pedicle screw for lumbar degenerative diseases increases the cage migration. Lumbar spinal fusion is recognized as an effective surgical procedure for degenerative lumbar diseases . Lumbar However, rigid fixation also has corresponding shortcomings. Rigid internal fixation may accelerate the degeneration of adjacent lumbar segments and cause device-related osteoporosis. Moreover, bilateral pedicle screw fixation was associated with greater blood loss, longer operative time, and involving greater costs .In 1991, Goel et al.  revealedHowever, a few controversies remain on whether this method could be applied in lumbar degenerative diseases for the lack of long-term results, such as the fusion rate, perioperative complications, and long-term functional outcomes . PreviouTherefore, to clarify these ambiguous findings, we performed a meta-analysis, comparing the two techniques for the treatment of degenerative lumbar diseases.Two reviewers performed an electronic literature search for RCTs or prospective clinical controlled studies comparing the unilateral pedicle screw fixation and bilateral pedicle screw fixation for lumbar degenerative diseases. The electronic databases include PubMed, Embase, and the Cochrane Library up to August 2018. No language or date restrictions were applied. The following terms were used as keywords:  AND bilateral pedicle screw fixation) AND lumbar degenerative diseases. In addition, further articles were obtained by reviewing the references of the selected articles. The detail retrieval process is shown in Fig. Randomized controlled trials were included if they met the PICOS criteria as follows: Population: patients with lumbar degenerative diseases; Intervention: unilateral pedicle screw fixation; Comparator: bilateral pedicle screw fixation; Outcomes: fusion rate, visual analog scale (VAS) at final follow-up, Oswestry Disability Index (ODI), Japanese Orthopedic Association scores (JOA), short-form health survey (SF-36), total blood loss, operation time, length of hospital stay, complications, and cage migration; Study design: RCTs or prospective clinical controlled studies. Fusion rate was assessed by the digital upright lateral radiographs according to the Bridwell-Lenke grading .Two reviewers independently retrieved the relevant data from articles using a standard data extraction form. The extracted data included publication date, authors, country, number of patients, mean age of patients, follow-up duration, type of operation, and surgical segments. For missing data, such as standard deviations, we tried to get it by contacting the original author first. If it did not work, we calculated the missing standard deviations from other available data such as standard errors or the formulas in Cochrane Handbook for Systematic Reviews of Interventions. Two reviewers extracted the data independently, and any disagreement was discussed until a consensus was reached.The methodological bias and quality of included studies were assessed by The Cochrane Collaboration’s tool for assessing the risk of bias according to the Cochrane Handbook for Systematic Reviews of Interventions. It is a two-part tool with seven specific domains: sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective outcome reporting, and other sources of bias.I2 and χ2 test. While I2 < 50% and P > 0.1, we used a fixed effects model to evaluate; otherwise, a random-effects was used. In addition, subgroup analysis  and sensitivity analysis were performed to explore the source of heterogeneity when heterogeneity existed.Stata 12.0  was used to perform the meta-analysis. We used weighted mean difference (WMD) and 95% confidence interval (CI) to assess continuous variable outcomes. For dichotomous outcomes, a risk ratio (RR) with a 95% CI was presented. Heterogeneity between studies was assessed by Initially, a total of 172 studies were searched via the databases and other sources . And 46 of the 172 studies were excluded by Endnote Software . After reading the full text of the 126 remaining studies in detail, 114 studies were excluded based on the titles and abstracts of these papers. Finally, 12 RCTs –20 with All the included trials were evaluated using the Cochrane Collaboration’s tool for assessing risk of bias in randomized trials and the Grading of Recommendations Assessment, Development and Evaluation Working Group grading scheme. The risk of bias of the RCTs is demonstrated in Figs. I2 = 0.0%, P = 0.994; Fig. P = 0.073; Fig. Ten studies enrolling 666 patients reported fusion rate postoperatively. No heterogeneity existed between the ten studies .JOA score was reported in four studies, and there was a large heterogeneity between the included studies  at final follow-up. There was no difference in SF-36 at final follow-up between the two groups . Compared with bilateral pedicle screw fixation, unilateral pedicle screw fixation was associated with a reduction of the operation time  with large heterogeneity .Figure Our meta-analysis comprehensively and systematically reviewed the currently available literature and found that (1) unilateral pedicle screw fixation compared with bilateral pedicle screw fixation significantly reduced total blood loss, operation time, and increase the cage migration and (2) unilateral pedicle screw fixation had no benefit on VAS, JOA, ODI, SF-36, and complications compared with bilateral pedicle screw fixation for lumbar degenerative diseases patients.Two relevant meta-analyses about unilateral pedicle screw fixation and bilateral pedicle screw fixation have been published. Although the main finding of our meta-analysis was consistent with most of the outcomes of previous meta-analyses, differences between ours and the previous ones should be noted. First, these previous meta-analyses included no more than six trials and 399 patients. In comparison, our current meta-analysis included 12 RCTs involving 808 patients. With the added statistical power of at least 409 cases, our current meta-analysis was the latest and the most comprehensive one, which generally concurs and further reinforces earlier results of previous meta-analyses. Second, we only included RCTs and thus the selection bias was avoided. Third, we performed subgroup analysis and sensitivity analysis to increase the robustness of our meta-analysis.We identify the fusion rate as the main outcome. The results showed that unilateral pedicle screw fixation has a similar fusion rate with bilateral pedicle screw fixation at the final follow-up. There is a heated discussion about the biomechanics stability of unilateral pedicle screw fixation. Han et al.  conducteWe then compared unilateral and bilateral groups in terms of the clinical and functional outcomes . The ODI was used to assess a patient’s disability during the activities of daily living. The results showed that unilateral pedicle screw fixation has similar outcomes with bilateral pedicle screw fixation. Lin et al.  found thOperation time, blood loss, and the length of hospital stay were took for evaluating surgical trauma and economic costs in this study. The results showed that unilateral pedicle screw fixation was associated with a reduction of the operation time and blood loss than bilateral pedicle screw fixation. No significant difference was found for the length of hospital stay. Since unilateral pedicle screw fixation only needs dissection on one side of the soft tissue and paravertebral muscles and insertion on the side of the pedicle screw, it can accordingly reduce the operation time and blood loss as compared with bilateral pedicle screw fixation . MoreoveThere was no significant difference between the occurrence of complication. There was no significant heterogeneity between the included studies. Eliades et al.  found thThe current study had several limitations. (1) Patients with different phases of follow-up were included. There was marked heterogeneity among the included studies in terms of total blood loss, length of hospital stay, and JOA. (2) The type of operation (including TLIF and MID-TLIF) and surgical segments varied between studies. (3) Blinding of the participant was unclear and this bias could not be avoided. (4) Some data for comparisons were not originally available but were calculated by estimation, leading to other bias.Unilateral pedicle screw fixation and bilateral pedicle screw fixation have similar fusion rates when treating for lumbar degenerative diseases. However, compared with the bilateral pedicle screw fixation, the unilateral pedicle screw fixation significantly reduced total blood loss and operation time for lumbar degenerative diseases. The use of unilateral pedicle screw for lumbar degenerative diseases increases the cage migration. There were no significant differences between the unilateral pedicle screw fixation and bilateral pedicle screw fixation in terms of the VAS at final follow-up, JOA, ODI, and SF-36. Unilateral pedicle screw fixation is recommended as the optimal surgical method for lumbar degenerative diseases.


On a micro-CT study, the material volume of the internal threads disappeared after destruction of its integrity due to repeated pedicle screw insertion and removal. Material integrity destruction of the internal threads decreased only the pull-out force and the insertion torque, but it did not affect the ISQ value. The ISQ value only decreased when the material volume of the internal threads disappeared, probably because the ISQ value reflects the resistance against a force in the perpendicular direction of the screw, unlike the conventional measures of fixation strength, such as pull-out force and insertion torque, which reflect axial load.Pull-out force and insertion torque have not been generally used as intraoperative measures for the evaluation of pedicle screw stability because of their invasiveness. On the other hand, resonance frequency analysis is a non-invasive and repeatable technique that has been clinically used in dentistry to evaluate implant stability e.g. by the Osstell apparatus. In this study, the characteristics of the implant stability quotient (ISQ) value obtained by the Osstell apparatus in the field of spinal surgery were investigated. Biomechanical test materials simulating human bone were used to provide a comparative platform for evaluating each fixation strength measure, including pull-out force, insertion torque, and the ISQ value. To perform pull-out force measurement and to repeat pedicle screw insertion and removal, loosening was artificially created, and its effect was investigated. The grade of loosening was quantified on a micro-CT image after pedicle screw removal. In the comparison of the 3 fixation strength measures, the correlations of the ISQ value with the pull-out force (R Implant stability is important in the field of orthopedics, because it secures implant fixation to the bone and is needed to avoid implant fixation failure –4. EspecPull-out force , 8–12 anSeveral studies have reported on the noninvasive measurement of orthopedic joint prosthesis stability during and after surgery –22. In pin vivo the temporal changes in implant stability that accompany the changes in bone structure around the implant and provide the diagnosis of implant loosening [In the field of dentistry, resonance frequency analysis (RFA) is performed widely for vibration analysis during surgery –27. The oosening , 30. Altoosening , and witThe aim of this study was to investigate, for the first time, the characteristics of the ISQ value in the field of spinal surgery, in comparison with the conventional methods of fixation strength measurements, such as insertion torque and pull-out force. In addition, we aimed to examine the relationship of these 3 fixation strength measures with the surrounding bone structure of the implants using micro-CT images by artificially creating implant loosening.3. These biomechanical test materials provided a homogeneous and consistent material that was similar to human cancellous bone [Five solid rigid polyurethane forms  were prepared as the test objects to represent human vertebrae. This material was constructed from rigid polyurethane foam with a density of 0.32 g/cmous bone . These mFive single-threaded, non-cannulated, titanium alloy (Ti-6Al-4V (ELI), ASTM F136), monoaxial pedicle screws  with length of 45 mm, outer threaded diameter of Ø 5.5 mm, inner thread diameter start point of Ø 3.8 mm – end point of Ø 4.6 mm, and screw pitch of 2.5 mm were used. To measure RFA with the Osstell ISQ® , 2 neodymium magnets  with a diameter of 4 mm, height of 2 mm, surface inductive flux of 331.7 mT, and magnetic attraction of 0.345 kgf were attached to the head of the pedicle screw using a cyanoacrylate-type of chemically reactive adhesive . The magnets’ magnetic poles were mounted face to face .A posthole was dug at the center of the 60 mm × 60 mm material. Before insertion, a pilot hole was drilled into the material using a 1.5-mm drill bit. A pedicle screw was inserted in its 40-mm length, leaving 5 mm not inserted. All screws were inserted in the same depth (40 mm) using a consistent depth gauge, and micro-CT examinations were performed to check the implanted screw depths .3). In the present study, it was used for converting the CT number to the calibrated value (arbitrary unit: a.u.) for quantification.The screw-inserted materials were scanned to check the depths of the pedicle screws  and the A total of 203 slices calibrated by the BMD phantom  were obtA digital torque gauge HTGA-5N  was used to measure the insertion torque  at RFA was conducted using a specific device  without contacting the screw after pedicle screw insertion was completed. The materials were not held by a fixture during measurement and were placed on a normal laboratory table instead. The ISQ value was obtained from the Osstell ISQ® and ranged from 0 to 100, depending on the resonance frequency (Hz) of the pedicle screws. The higher the ISQ value, the more stable the implant . The pedPull-out force measurements were performed according to the ASTM-F543-07 testing standards . The matThe same tests  were repeated at the same insertion path 10 times to intentionally create loosening .The measurements of the 3 fixation strengths  and the average screw insertion areas were collected from the 5 biomechanical test materials. These values were acquired in 10 steps for each biomechanical test material, because the screw insertion experiments were repeated 10 times in the manner described above .Statistical calculations were performed using SPSS statistics software version 24 . Box-and-whisker plots of the 3 fixation strength measures and the average screw insertion areas were drawn every time the insertions were repeated. One-way analysis of variance with post-hoc analyses for repeated measures were performed to evaluate the differences in the 3 fixation strength measures and the average screw insertion areas among the number of screw insertions.A simple regression model and a two-piece linear regression model with minimized sums of the squared residuals of the 2 types of regression were used to analyze the relationships among the 3 fixation strength measures and the average screw insertion areas. To assess the goodness of fit of these regression models, Akaike’s information criterion (AIC) was used , 41. Whe2 = 0.918, p < 1×10-26)  and the pull-out force  were not as strong as the relationship between peak torque and pull-out force (1×10-26)  in the sut force  in the sTo confirm the structural changes in the biomechanical test materials, micro-CT image analysis was performed . Even afSeveral studies have evaluated the stability of orthopedic implants using insertion torque and pull-out force , 43–49. With repeated screw insertion, the material integrity between the major and minor diameters of the internal thread is first compromised, but the material volume is preserved; however, at the end, material volume also disappears. Considering these mechanisms, peak torque and pull-out forces decrease along with the destruction of material integrity, whereas the ISQ value decreases along with the destruction of material volume that occurs after the destruction of material integrity. There was a strong relationship between peak torque and pull-out force, which suggested that the 2 were similar fixation strength measures. Meanwhile, the weak relationship of the ISQ value with both peak torque and pull-out force in the simple regression model was likely caused by the fact that the ISQ value was retained more than peak torque and pull-out force, even with repeated screw insertion and removal.In the present study, loosening was created artificially to perform pull-out force measurement and to repeat screw insertion and removal. All fixation strength measures decreased with loosening. Sanden et al reported in an animal study that in cases with pedicle screw loosening on X-ray images, the bone structure between the major and minor diameters was destroyed, and that the screw insertion areas were scaled to a cylindrical shape histologically . In the Pull-out force is the fixation strength measure that reflects axial load  and is dIn the dentistry field, the ISQ value has been reported to correlate negatively with the displacement of an implant after application of a lateral load , 30. Thiin vivo [In vivo, stress force against the implant is applied not only in the axial direction, but also in various directions . Therefoin vivo . In the in vivo , was shoThis study had some limitations. First, the Osstell ISQ® is a system for evaluating implant stability in the field of dentistry, and this method for measuring resonance frequency is not established in the field of orthopedics. Modification of the RFA system to make it suitable for orthopedic implants is needed. Since the Osstell ISQ® is one of the most popular devices used to evaluate implant stability in the dental field  and it iThe ISQ value reflects the fixation strength against the perpendicular direction, unlike conventional fixation strength measures.


Scientific Reports 10.1038/s41598-017-06706-x, published online 16 August 2017Correction to: C. glaucum’,This Article contains errors in the Results section, under the subheading ‘Analysis of interaction between CDPK and bHLH in “The three top ranking models of the CgCDPK-CgbHLH001 interaction complex were presented in Fig. 5c”should read:“The three top ranking models of the CgCDPK-CgbHLH001 interaction complex were presented in Fig. 6c”In addition,61-GKRLKS-66 was a putative action site for CgCDPK.”“Prediction of phosphorylation sites of CgbHLH001 revealed 39 potential amino acid residues , in which the motif of should read:91-GKRLKS-96 was a putative action site for CgCDPK.”“Prediction of phosphorylation sites of CgbHLH001 revealed 39 potential amino acid residues , in which the motif of


O-linked-β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on the hydroxyl group of serine or threonine residues. Unlike conventional protein glycosylation, O-GlcNAcylation is localized in the nucleocytoplasm and its cycling is a dynamic process that operates in a highly regulated manner in response to various cellular stimuli. These characteristics render O-GlcNAcylation similar to phosphorylation, which has long been considered a major regulatory mechanism in cellular processes. Various efficient chemical approaches and novel mass spectrometric (MS) techniques have uncovered numerous O-GlcNAcylated proteins that are involved in the regulation of many important cellular events. These discoveries imply that O-GlcNAcylation is another major regulator of cellular signaling. However, in contrast to phosphorylation, which is regulated by hundreds of kinases and phosphatases, dynamic O-GlcNAc cycling is catalyzed by only two enzymes: uridine diphospho-N-acetyl-glucosamine:polypeptide β-N-acetylglucosaminyl transferase (OGT) and β-D-N-acetylglucosaminidase (OGA). Many useful chemical tools have recently been used to greatly expand our understanding of the extensive crosstalk between O-GlcNAcylation and phosphorylation and hence of cellular signaling. This review article describes the various useful chemical tools that have been developed and discusses the considerable advances made in the O-GlcNAc field.The concepts of both protein glycosylation and cellular signaling have been influenced by  O-linked N-acetylglucosamine (O-GlcNAc) on the surfaces of living lymphocytes ) ,13. OGT lability .O-GlcNAc can also be phosphorylated, which implies a dynamic interplay between O-GlcNAcylation and phosphorylation . . et al. acceptor b. In recteolysis a. The OG13–15 in  with a met al. has introduced a high-throughput method termed as the azido-enzyme-linked immunosorbent assay (azido-ELISA) [Xanthomonas campestris OGT, (XcOGT) [XcOGT contains 5.5 TPRs followed by the GT domains that adopt the GT-B fold. The generic GT-B fold [N2-like reaction mechanism where the enzyme scaffold provides a general base that activates the incoming protein/peptide nucleophile (OH group of Ser/Thr) for the displacement of the leaving group (UDP portion of the donor) in a concerted manner.For a slightly different purpose, Bertozzi o-ELISA) . This me (XcOGT) ,160. XcOT-B fold ,161 consO-GlcNAc. In the vast majority of early studies of O-GlcNAc, O-GlcNAc was elevated by treatment of cells with inhibitors such as streptozotocin (STZ) [O-GlcNAc levels have been linked to the development of insulin resistance   and PUGNin (STZ) . These esee Ref. ,164,165,see Ref. ,166). Cesee Ref. ,168. A gsee Ref. ,170, butelldeath ,172. PUGelldeath . Due to tization ,175. In ncreases . The linO-GlcNAc cycling.In contrast to the availability of several useful specific OGA inhibitors, potent and selective OGT inhibitors are almost unknown. Clearly, intensive and continuous efforts focused on the development of simple and high-throughput OGT enzymatic assays and the design of the structure-inspired, OGT-specific inhibitors are essential to advance our understanding of the functional roles of OGT and O-GlcNAcylation participates in many important cellular processes via its extensive and dynamic interplay with phosphorylation [O-GlcNAc and have proven invaluable in elucidating our knowledge about cellular signaling. Nevertheless, important questions remain to be answered; one of the key questions is how OGT and OGA target so many substrates while retaining substrate specificity. Although it is now known that the substrate specificities of these enzymes can be determined, in part, by their transient interactions with many binding partners to form various substrate specific holoenzymes, an enhanced understanding of the molecular mechanisms by which they are regulated will remain a key focus for future research. Continuing efforts directed at enhancing the chemical arsenal with more direct and efficient chemical tools, such as the development of high-throughput yet robust OGT assays and isoform-specific OGA and OGT inhibitors, will lead to new discoveries about O-GlcNAcylation. In addition, the generation of site-specific O-GlcNAc antibodies and the elucidation of the enzymes’ crystal structures will be the subjects of future research to expand our understanding about the roles of O-GlcNAc. All of these tools will provide deeper insights into the cell signaling paradigm.rylation ,30,35,36


We evaluated the microbial communities in three Hawaiian forest soils along a natural fertility gradient and compared their distinct responses to long-term nitrogen (N) additions. The sites studied have the same elevation, climate, and dominant vegetation, but vary in age of development, and thus in soil nutrient availability and nutrient limitation to plant growth. Fertilized plots at each site have received 100 kg ha year N addition for at least 8 years. Soil parameters, water content, pH, and ammonium and nitrate availability differed by site, but not between control and N-addition treatments within a site at the time of sampling. Microbial biomass also varied by site, but was not affected by N addition. In contrast, microbial community composition  varied among sites and between control and N-addition plots within a site. These data suggest that microbial community composition responds to N addition even when plant net primary productivity is limited by nutrients other than N. This may have implications for the behavior of forests impacted by atmospheric N deposition that are considered to be “nitrogen saturated,” yet still retain N in the soil.