Ainsliadimer A is thus a natural product targeting the cysteine 46 of IKKα/β to block NF-κB signalling. Therefore, it has great potential for use in the development of anticancer and anti-inflammatory therapies.Aberrant activation of NF-κB is associated with the development of cancer and autoimmune and inflammatory diseases. IKKs are well recognized as key regulators in the NF-κB pathway and therefore represent attractive targets for intervention with small molecule inhibitors. Herein, we report that a complex natural product ainsliadimer A is a potent inhibitor of the NF-κB pathway. Ainsliadimer A selectively binds to the conserved cysteine 46 residue of IKKα/β and suppresses their activities through an allosteric effect, leading to the inhibition of both canonical and non-canonical NF-κB pathways. Remarkably, ainsliadimer A induces cell death of various cancer cells and represses  IKK is a key inducer of NF-κB, and has been targeted by several small molecule drugs. Here the authors show that a natural product from a Chinese medical herb inhibits NF-κB via covalent binding to a unique conserved region of IKK, and efficiently inhibits tumour growth and sepsis in mice. The evolutionarily conserved nuclear factor-κB (NF-κB) signalling pathway plays key roles in inflammatory and immune responses and in cell survival by regulating the transcription of numerous target genes123Canonical NF-κB activation depends on the degradation of IκB, which is rapidly phosphorylated by an active IκB kinase (IKK) complex. This complex is composed of IKKα and IKKβ catalytic subunits and a regulatory subunit, IKKγ/NEMO in vivo tumour growth and inflammatory responses. Therefore, strategies focused on reducing NF-κB activity by specific small molecule inhibitors could offer significant therapeutic value for the treatment of these diseases.Aberrant activation of the NF-κB signalling pathway is known to be involved in a variety of human diseases including cancer, autoimmune diseases and chronic inflammatory diseases21015191) tightly binds IKKα and IKKβ through the conserved cysteine residue 46(C46), leading to the inactivation of both the canonical and the non-canonical NF-κB signalling pathways triggered by multiple stimuli. To our knowledge, ainsliadimer A represents the first small molecule natural product targeting the functional C46 of IKKα/IKKβ. Ainsliadimer A leads to IKKα/IKKβ inactivation through a novel allosteric effect. Moreover, ainsliadimer A powerfully blocks LPS-mediated inflammatory responses and tumour growth in vivo. These discoveries not only indicate that ainsliadimer A indeed acts as a potent inhibitor of IKKα/β, but also reveal that ainsliadimer A could be used as a lead compound in the development of new therapeutic agents for the treatment of cancer and inflammatory disorders.Over the past decade, there has been a concerted effort to identify small molecule inhibitors of IKKβ because of its central role in the canonical NF-κB pathway. Some of the small molecule inhibitors that have been identified in these efforts have exerted promising inhibitory effects in various experimental models of tumour and inflammatory diseases121) is a structurally unique and complex sesquiterpene lactone dimer with an unprecedented carbon skeleton 241) has been previously shown to exhibit inhibitory activity against the production of nitric oxide (NO) in a mouse macrophage cell line RAW264.7 stimulated by lipopolysaccharide (LPS)1) on LPS-triggered inflammatory responses in vivo. Mice were pre-injected with or without Vehicle or ainsliadimer A (1), followed by administration of PBS or LPS. As shown in 1) but not in the Vehicle-pretreated mice. As such, it can be concluded that ainsliadimer A (1) has great potential as an anti-inflammatory agent in vivo. Furthermore, exogenously administered ainsliadimer A (1) (25 mg kg−1) was shown to cause a significant decrease in NF-κB activation in the spleen after intravenous injection (as compared with Vehicle-treated mice) following LPS challenge  prompted us to investigate how it modulates pro-inflammatory cytokine responses. As a well-defined agonist of TLR4 (refs 1) on LPS-induced NF-κB activation. Phosphorylation of IκB is a key step in the activation of the canonical NF-κB pathway. As expected, phosphorylated IκBα was detected in RAW264.7 cells after exposure to LPS for 10 or 20 min. Strikingly, pretreatment with 8 μM ainsliadimer A (1) could effectively block the phosphorylation of IκBα (1) exhibited much stronger inhibitory effects against LPS-induced NF-κB activation. Meanwhile, by assessing IFR3 phosphorylation, we found that ainsliadimer A (1) had no effect on LPS-induced type I IFN responses, indicating that ainsliadimer A (1) specifically targets LPS-trigged NF-κB signalling (1) markedly suppressed the phosphorylation of IκBα triggered by synthesized dsRNA poly(I:C), a TLR3 ligand1) affects a common signalling component shared in both TLR3- and TLR4-mediated NF-κB signalling. In addition to the TLRs, the engagement of death receptors with corresponding ligands including TNF-α is also known to trigger NF-κB activation. We found that ainsliadimer A (1) significantly inhibited TNF-α-induced phosphorylation of IκBα in various cell lines (1) abolished TNF-α-induced nuclear translocation of p65 (1) acts as a potent inhibitor of the NF-κB pathway induced by diverse stimuli.The potent anti-inflammatory activity of ainsliadimer A  completely abolished TRAF6-induced phosphorylation of IKKα/β, indicating that ainsliadimer A (1) disrupts the NF-κB pathway by intervening in the function of the IKK complex or that of upstream activators.TNF receptor-associated factors (TRAFs) are important intermediates in the NF-κB signalling pathway. For example, TRAF2 and TRAF5 regulate TNF-α receptor-triggered IKK activation, and TRAF6 is required for IL-1-induced IKK activation1) affected the non-canonical NF-κB pathway, which is activated via a subset of TNFR superfamily members, including the receptor activator for nuclear factor κB (RANK), a B-cell-activating factor belonging to the TNF family receptor (BAFFR) and a lymphotoxinβ-receptor (LTβR)1) in a dose-dependent manner. We further investigated the effect of ainsliadimer A (1) on the processing of p100 to p52 triggered by the ectopic expression of NIK. Consistently, addition of ainsliadimer A (1) prevented NIK-mediated production of p52 (1) also targets the non-canonical NF-κB pathway.We then tested whether ainsliadimer A (n of p52 . These r1) associated with the NF-κB pathway, we prepared chemical probes for affinity purification. Initial structure and activity relationship studies enabled by total synthesis revealed that the hydroxyl analogue 2 fully retained the biological activity observed with ainsliadimer A (1), which suggested that a biotin tag could be attached to this hydroxyl group  and a negative probe (NC) from 2 and 3, respectively, with biotin and a tri(ethyleneglycol) linker  of ainsliadimer A (yl group . In addigroup (3 . This in) linker . The bio) linker .1), we performed pull-down experiments using Probe and NC as positive and negative probes, respectively. The 293T-cell lysates were incubated with streptavidin agarose beads, which were pre-coupled with Probe or NC. The proteins precipitated by streptavidin agarose beads were resolved by sodium dodecyl sulfate–PAGE (SDS–PAGE) and stained with silver. As shown in (1) targets IKKα and IKKβ or a complex that contains IKKα and IKKβ.To identify cellular proteins that interact with ainsliadimer A  directly binds to IKKα and IKKβ, we generated recombinant IKKα and IKKβ proteins for further analysis. The 293T cells were transfected with Flag-IKKα or Flag-IKKβ plasmids, the recombinant Flag-IKKα or Flag-IKKβ was captured by anti-Flag agarose beads and then eluted off the beads using Flag peptide. The purified Flag-IKKα or Flag-IKKβ was incubated with DMSO or increasing concentrations of Probe, and then the mixtures were resolved by SDS–PAGE, followed by immunoblotting with streptavidin or anti-Flag antibodies. As shown in 1), but not by 3 (1) had no association with IKKγ/NEMO, another subunit of the IKK complex, and had no association with other components in the NF-κB pathway such as TAK1 or TAB1 (1) can efficiently and specifically interact with IKKα and IKKβ, and that the double bond is critical for its binding with IKKα and IKKβ.To determine whether ainsliadimer A (not by 3 . Notably or TAB1 . Taken t1) lost its inhibitory activity against NF-κB activation entirely when the enone moieties were saturated or in the presence of excess reducing agent dithiothreitol (DTT) (1) is most likely a reactive Michael acceptor, which forms a covalent bond with cysteine residues of its target proteins. Such a bond results in inactivation of the targeting enzymes, as previously demonstrated by natural products such as adenanthin1). We used BLAST analysis with the sequences of IKKα and IKKβ and found nine conserved cysteine residues (1), we individually mutated these nine cysteine residues of IKKβ into alanine residues (1), while the other eight mutants retained high-affinity binding for ainsliadimer A (1) in a manner similar to wild-type (WT) IKKβ (1). As expected, mutation of C46 into serine in either IKKα or IKKβ abolished its corresponding interaction with ainsliadimer A (As ainsliadimer A (ol (DTT) , the α,βresidues . To evalresidues  using thWT) IKKβ . These radimer A .1) in IKKβ, we incubated recombinant IKKβ protein with or without ainsliadimer A (1). After the reaction, the IKKβ protein was digested with trypsin and then analysed by LC-MS/MS. Database searching identified a peptide with calculated mass of 2,047.09 Da, which is 506.2 Da larger than the C46-containing peptide IAIKQCRQELSPR that has a calculated mass of 1,540.85 Da. The mass difference of 506.2 Da matches the molecular weight of a ainsliadimer A (1) molecule. Tandem mass spectrometry of this peptide revealed that a 506.2 Da mass shift occurred starting from the y8 to the y12 fragment ions (1). To further confirm this, two synthetic peptides derived from human IKKβ (#1 and #2) containing C46 were incubated with ainsliadimer A (1), and the resulting mixture was analysed by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry. As expected (1) or Probe after the reaction, indicating that ainsliadimer A (1) and Probe could modify C46 in vitro. When the C46 in peptide #1 and peptide #2 was replaced by alanine or serine , however, the mass of these peptides remained the same after the reaction with either ainsliadimer A (1) or Probe (1) or Probe (1) binds to IKKβ by covalently modifying its C46 residue.To determine the specific residue modified by ainsliadimer A  likely modifies IKKβ through Michael addition at C46. To verify the functional significance of this binding event, we performed in vitro kinase assays using WT-IKKβ or C46A-IKKβ recombinant proteins as the kinase, and purified Flag-IκBα protein as the substrate. As shown in 1), WT-IKKβ-induced phosphorylation of IκBα was significantly suppressed, whereas ainsliadimer A (1) did not affect the phosphorylation of IκBα triggered by C46A-IKKβ. Moreover, expression of WT-IKKβ or C46A-IKKβ in IKKα/IKKβ double-knockout MEF cells restored the phosphorylation and degradation of IκBα, and addition of ainsliadimer A (1) blocked the phosphorylation and degradation of IκBα induced by WT-IKKβ, but not that induced by C46A-IKKβ (1) inhibits IKKβ by direct modification of its C46 residue. Meanwhile, in vitro kinase assays using purified recombinant IKKβ and IκBα showed 2 μM of ainsliadimer A (1) could totally inhibit IκBα phosphorylation as BMS-345541 did (1) to affect the association of IKKβ with IκBα. As shown in 1) reduced the association of IKKβ with IκBα. Thus, ainsliadimer A (1) is proven to be able to block the function of IKKβ in the NF-κB pathway.Ainsliadimer A (46A-IKKβ . Collect5541 did . We next1) inactivates IKKα/β, we conducted molecular dynamics (MD) simulations using the first crystal structure of IKKβ (ref. 1) binding. According to the computational study, the binding mode shows that a stable hydrogen bond forms between the backbone amide group of C46 and the Michael acceptor carbonyl group of ainsliadimer A (1) during the simulation; this may be involved in stabilizing the transition state of a Michael addition reaction (1) forms favourable hydrophobic interactions with residues including Trp58, Ile62, Val79, Leu91, Pro92 and Leu94 (1) introduces significant conformational changes to the ATP binding-site in IKKβ by displacing the conserved structural αC-helix (1)-bound IKKβ, such an allosteric regulation mechanism was also reported in the crystal structures of PDK-1 kinase, where a covalent inhibitor binds with Cys148 in the same allosteric binding pocket and induces a positional change in the C-helix by moving away from the ATP-binding site  completely blocks solvent access to Trp58, which is located at the bottom of the allosteric pocket and interacts with ainsliadimer A (1) favourably throughout the simulation (1) to the allosteric pocket can be experimentally assessed using the tryptophan fluorescence quenching technique, which has been widely used to demonstrate allosteric effects when co-crystal structures are not available1). On the basis of the slope of the plot (1), which was 7.6709±0.2 and 11.076±0.2 M−1, respectively. This indicates that the binding of ainsliadimer A (1) to IKKβ partially protects W58 from the quencher by inducing conformational changes of the target protein. In other words, the process of ainsliadimer A (1) binding to IKKβ changes the disposition of W58 from being partially exposed to being in a more completely buried state. As expected, mutation of W58 into alanine in IKKβ abolished its corresponding interaction with ainsliadimer A (1) might lead to the inhibition of IKKβ. In addition, it is worth noting that the downward curvature in the Stern–Volmer plot existed in the presence of ainsliadimer A (1); this was conceivably caused by the existence of both drug-bound and drug-free receptor populations, whose tryptophan was exposed to differing extents. Together, our data suggest that W58 on the α C-helix is required for the interaction between ainsliadimer A (1) and IKKβ. As displacement of α C-helix by a small molecule inhibitor or regulatory protein is able to affect ATP binding1). The result indicated that ainsliadimer A (1) showed ATP-competitive inhibitory effect on IKKβ (1) significantly suppressed WT-IKKβ-induced phosphorylation of IκBα. Collectively, these data indicate that C46 of IKKα/β likely acts as a novel allosteric regulation site of these enzymes.Notably, ainsliadimer A , as the conventional IC50 value could not well characterize the potency and selectivity for irreversible covalent inhibitors that we observed. We detected the extent of IKKβ binding at several time points for a number of Probe concentrations1) at various concentrations showed time-dependent saturation (kobs) at each inhibitor concentration. Plotting these kobs values as a function of Probe concentration revealed a saturation curve (1), involving the noncovalent reversible binding of IKKβ to ainsliadimer A , which places the moderately reactive electrophile of ainsliadimer A (1) close to a specific nucleophile on the protein, followed by the second chemical step that results in specific covalent linkage (kinact) leading to the inhibited complex34kinact value for IKKβ binding with ainsliadimer A (1) was determined to be 7.743 min−1  was determined to be 30.25 nM (1) and IKKβ is effective (kinact/Ki=4.26 × 106 M−1 s−1), with good affinity (Ki=30.25 nM) and specific reactivity (kinact=7.743 min−1) (http://www.reactionbiology.com/) to further confirm this specific binding to IKKβ. As shown in 1) did not show significant enzyme inhibition effects as compared with the inhibitory effect against IKKβ.Next, we investigated the binding kinetics of ainsliadimer A  . We also1), we treated gastric adenocarcinoma BCG-823 cells with ainsliadimer A (1) alone, TNF-α alone or with a combination of ainsliadimer A (1) and TNF-α for a period of 24 h. As shown in 1) triggers apoptosis by measuring caspase-3, which is activated via proteolytic cleavage during apoptosis. In BCG-823 cells treated with ainsliadimer A (1), we detected cleaved caspase-3 that could be blocked by z-VAD, a pan-caspase inhibitor (1) suppressed the activation of NF-κB (1) on the NF-κB pathway facilitate apoptosis of cancer cells. Finally, we evaluated the in vivo effects of ainsliadimer A (1) in tumour growth. Nude mice bearing a BCG-823 human gastric adenocarcinoma xenograft were treated with Vehicle or ainsliadimer A (1)  or BMS-345541, a widely used IKKβ inhibitor . As shown in 1), as compared with the Vehicle-treated mice. Notably, ainsliadimer A (1) exerted a comparable anti-tumour effect to that of the therapeutic drug BMS-345541. Importantly, the mice did not display any discernible side effects or significant changes in body weight during administration of ainsliadimer A (1) resulted in significant death of human cancer cells and suppression of tumour growth with minimal undesired toxicities, thus implicating ainsliadimer A (1) as a novel IKK inhibitor with promising potential for anticancer and anti-inflammatory therapeutics.In addition to its crucial role in controlling immune responses, the NF-κB pathway has been shown to negatively regulate apoptosis and positively regulate the onset and progression of tumoursnhibitor . Furtherof NF-κB  and downof NF-κB . These radimer A . Collect1) specifically binds to IKKα/β and subsequently blocks both canonical and non-canonical NF-κB activation.NF-κB transcription factors play a critical function in the regulation of immune responses, inflammation, cell proliferation and survival. Aberrant activity of NF-κB is associated with tumour progression as well as auto-immune and inflammatory diseases. Therefore, crucial regulators in the NF-κB pathway such as IKKα/β are considered as promising therapeutic targets. We show here that a natural product ainsliadimer A (1) was originally isolated from a Chinese herb1) has been shown to suppress LPS-mediated NO production1) specifically blocked LPS-induced NF-κB activation, but not type I IFN responses. Moreover, ainsliadimer A (1) exerts potent inhibition of NF-κB activation triggered by different stimuli such as TLR3 ligand poly(I:C) and TNF. Using biotin-labelled 1 as a probe, we found that ainsliadimer A (1) directly targeted IKKα and IKKβ, but not other components of the NF-κB pathway such as IKKγ/NEMO, TAK1 and TAB1, suggesting that an exclusive binding mechanism is shared by IKKα and IKKβ.Ainsliadimer A (1) is a structurally unique and complex sesquiterpene lactone dimer with α, β-unsaturated enone moieties that may serve as Michael acceptors. Consistently, our mutagenesis and mass spectrometry results demonstrated that ainsliadimer A (1) formed a covalent bond with the conserved C46 of IKKα/β. Through both computational and experimental studies we further uncovered, remarkably, a novel putative allosteric binding pocket around C46 of IKKβ that could explain the high-binding specificity of ainsliadimer A (1) bound to IKKβ. Notably, the tryptophan fluorescence quenching results provided direct and strong evidence to support this novel allosteric effect. In addition, ainsliadimer A (1) was shown to be an irreversible inhibitor that exhibited specific and potent kinase inhibition activity against IKKβ with kinact /Ki of 4.26 × 106 M−1 s−1.Ainsliadimer A (1) represents the first small molecule that blocks NF-κB by directly targeting C46 of IKKα/β. The putative allosteric site around C46, most likely unique to IKKα/β, confers the high selectivity of ainsliadimer A (1) for IKKα/β, thereby tending to have low non-specific toxicity and a high therapeutic index. Further, in both in vitro and in vivo studies, ainsliadimer A (1) was shown to be a promising agent against the survival of cancer cells, tumour growth and endotoxin-mediated inflammatory responses. It is tempting to speculate that this unique inhibitory effect of ainsliadimer A (1) on IKKα/β should be further exploited for the elucidation of uncharacterized mechanisms of IKKα/β activation, as well as the development of new anticancer and anti-inflammatory therapies.Many small molecule inhibitors that interfere with IKKs have been previously described122, 3, the positive probe (Probe) and the negative probe (NC) were carried out according to 1) and 4 were synthesized as described in ref. The syntheses of 2, fetal bovine serum (FBS), BMS-345541, DTT, DMSO and LPS were purchased from Sigma. The Cell Titer-Glo Luminescent Cell Viability Assay kit was purchased from Promega. The Protein Assay kit was purchased from Bio-Rad. The HTRF KinEASE kit was purchased from Cisbio. IKKβ kinase protein was purchased from Carna Biosciences. RANKL was purchased from Sino Biological. Human IκBα full length protein was purchased from Abcam (ab59981).DMEM medium, phosphate buffered saline (PBS), trypsin and penicillin/streptomycin were purchased from GIBCO. ATP, MgClThe PCS2-3Flag-IKKα, PCS2-3Flag-IKKβ, PCS2-3Flag-TAK1, PCS2-3Flag-NEMO, PCS2-3Flag-TAB1, PCS2-3Flag-NIK, myc-p100, Flag-IκBα plasmids were kindly provided by Dr Feng Shao and his coworkers (NIBS).The following antibodies were used for western blotting: p-IκBα , IκBα , IKKα , IKKβ , p-IKKα/β , p100 , H1 , Biotin , Flag , myc , p-IRF3  and β-actin .2/95% air (v/v). All media were supplemented with 10% FBS (Invitrogen) and 100 units ml−1 penicillin/streptomycin (Hyclone).Human cervical cancer HeLa, human glioblastma T98G, human gastric adenocarcinoma BCG-823, human embryonic kidney 293 T and mouse macrophage Raw264.7 cells were obtained from ATCC. These were cultured in DMEM medium supplemented with 10% (v/v) heat-inactivated FBS in a humidified incubator at 37 °C and 5% COThe cell survival assay was performed using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) according to the manufacturer's instructions. Luminescence was recorded with a TecanGENios Pro plate reader.1) for 1 h before stimulation with the indicated concentrations of TNF-α, 20 μg ml−1 Poly(I:C) or 200 ng ml−1 LPS and then harvested at the indicated time points. Total cell extracts were tested by western blotting for the occurrence of IκBα phosphorylation. DMSO and BMS-345541 were chosen as negative and positive controls, respectively.Cells were pre-incubated with various concentrations of compound ainsliadimer A (1) for 1.5 h and then stimulated by 20 ng ml−1 TNF-α for 30 min. Cell treatment was terminated by washing with PBS, followed by fixation in freshly prepared 4% paraformaldehyde in PBS for 10 min. The fixed cells were washed three times with PBS and then permeabilized in 0.25% Triton X-100 in PBS for 10 min. After blocking with 2 mg ml−1 BSA for 1 h at room temperature, we added a P65 antibody at 1:1,000 dilution at 4 °C overnight, washed three times with PBS, then added 1:500 dilution of secondary AlexaFluor488 antibody for 1 h at room temperature in the dark. Finally DAPI was used to stain the nuclei at 37 °C for 30 min in dark. Microscopy was performed using a Nikon eclipse 80i.HeLa Cells were grown to 50–70% confluence in a glass chamber and pretreated with DMSO or 8 μM ainsliadimer A . The resuspended cell pellet or tissue was vortexed for 10 s, incubated on ice for 20 min, and centrifuged at 20,000 × g for 20 min. The supernatants were collected for western blot analysis or immunoprecipitation. Images have been cropped for presentation. Full-size images are presented in Cell pellets were collected and resuspended in lysis buffer  overnight at 4 °C. The beads were then washed three times in lysis buffer. The ainsliadimer A-bound agarose was then incubated with cell lysates overnight at 4 °C. The following day, the beads were washed six times with lysis buffer, then the immunoprecipitations were eluted off the beads using low pH elution buffer at 4 °C for 15 min. Acid elution was neutralized by adding 1/20 volume of 1 M Tris-HCl, pH 9.4, and the preparations were finally boiled in SDS loading buffer. The bead-bound proteins were separated by SDS–PAGE and visualized by silver staining. The protein-containing band in the gel was excised, followed by in-gel digestion and analysis by LC-MS/MS.Human IKKα or IKKβ were cloned into a PCS2 vector containing a 3 × Flag tag sequence at the N-terminal region. Site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit (Stratagene) using Flag tag-IKKα or Flag tag-IKKβ as a template. These proteins were expressed in 293 T cells and subsequently purified.The reaction mixtures of peptides or proteins were analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry with an Autoflex II MALDI-TOF/TOF mass spectrometer equipped with a nitrogen pulsed laser (Bruker). In brief the peptide mixtures were loaded on C18 Zip Tips separately, washed with 0.1%TFA, eluted with 2, 5-dihydroxybenzoic acid solution (Agilent), then spotted on a Bruker MTP 384 massive stainless steel MALDI target. The matrix spots were allowed to dry at room temperature. Peptide Mass Spectra were acquired in positive reflectron mode with pulsed ion extraction.The crystal structure of IKKβ (PDB entry: 3QA830) was used to generate initial binding complexes. The MD simulation was performed with the Gromacs 4.0.7 package35381) or DMSO was preincubated with 5 μM IKKβ for 90 min and titrated with 2.5 μl of 1 M acrylamide; we monitored the tryptophan florescence signal intensity by using 340 nm emission and 295 nm excitation. At the same time, the background signal was corrected by using a paired control that lacked protein. After using the Stern–Volmer equation to analyse F0/F, we compared the slopes from IKKβ with or without the treatment of ainsliadimer A (1). For the Stern–Volmer equation:Twenty-five micromolars of ainsliadimer A  to measure the ATP binding kinetics of IKKβ. After optimizing the conditions of IKKβ according to the kit protocol, the assays were carried out in the low volume, black 384-well plates , with a 10 μl assay volume containing varied concentration of ATP, non-limiting STK Substrate 2-biotin and 1 ng μl1) moves close to specific nucleophile residues of IKKβ to form the initial encounter complex IKKβ: ainsliadimer A (1), and Ki is the apparent dissociation constant for the initial IKKβ: ainsliadimer A (1) complex; in the second chemical step, the formation of specific covalent linkage (kinact) between ainsliadimer A (1) and IKKβ resulted in the inhibited complex and kinact the rate constant for IKKβ- ainsliadimer A (1) formation at saturating ainsliadimer A (1). To get the Ki and Kinact value, we incubated IKKβ with a large excess of ainsliadimer A (1) for different time periods and then analysed the streptavidin-HRP signal intensity quantified by densitometry. During this process, we performed scanning at the optimal exposure time to ensure that band intensity was proportional to the concentration of protein present. Under the pseudo-first order experimental conditions, the reaction proceeds as in these equations:The specific interaction between IKKβ and Probe followed two steps: in the initial binding step, ainsliadimer A  concentrations by fitting to kinact and Ki by fitting kobs values to equation (3)We initially obtained the reaction rate constants of GAPDH.Cells were lysed and total RNA was extracted using the TRIzol Reagent (Invitrogen). Synthesis of cDNA was done with 1 μg of RNA from oligo (dT)-20 primers using the Superscript II first strand synthesis system (Invitrogen). Real-time PCR was performed using specific primers and the SYBR Green ROX Mix (Thermo Scientific). The sequence of the primers is given in the 1) and LPS+BMS-345541 groups. The mice were administered intravenously with ainsliadimer A (1) (25 mg kg−1) (LPS+ ainsliadimer A group), BMS-345541(25 mg kg−1) (LPS+BMS-345541group), Vehicle (LPS+Vehicle group) or PBS(PBS group) for 90 min, and then the mice  were given intraperitoneal injection of LPS (1.6 mg kg−1). Mice in those groups were killed and their serum was separated from clotted blood at 3.5 h following administration of LPS. Serum was stored at –80 °C, and concentrations of cytokine IL-1β and IL-6 were measured by sandwich ELISA using commercially available reagents according to the manufacturer’s instructions (Beijing Gersion Bio-Technology). The concentration of cytokine TNF-α was measured using a Mouse Inflammatory Cytometric Bead Array  according to the manufacturer’s protocol. For each group, plasma samples were obtained from at least six mice and analysed in duplicate.Cytokines were assessed by measuring serum concentrations. C57 Male mice at 7–8 weeks of age were randomly assigned into five groups: the PBS, LPS, LPS+Vehicle, LPS+ ainsliadimer A  or BMS-345541, a widely used IKKβ inhibitor . Control mice were treated with the same amount of vehicle under the same protocol. Tumour volume was calculated by using the formula mm3=4/3πr3, where r=(length+width)/4, measuring the two largest perpendicular axes of the tumour.Female nude (nu/nu) mice (6–7 weeks old) were purchased from Vital River Company and acclimated for 1 week before the start of the experiment. All animal experiments were performed in accordance with protocols by the Institutional Animal Care and Use Committee at National Institute of Biological Sciences (NIBS). Exponentially growing BCG-823 cells were suspended in PBS and mixed in a 1:1 ratio with Matrigel 0.1 ml suspension containing 2.5–5 × 10t-test . Graph Pad Prism was used for analysis.All data are expressed as the mean ±s.e.m. Results are representative examples of three individual experiments. Inferential statistical analyses were performed with a Student’s X.L. and S.H. designed the study; T.D. and X.W. performed and analysed the biological experiments; C.L. and L.D. performed chemical synthesis; X.Z. performed and analysed the xenograft studies; R.C. performed the docking experiments under the guidance of N.H.; L.L. and S.C. performed MS experiments; X.L., S.H., T.D. and C.L. wrote the manuscript. X.L. and S.H. guided all of the aspects of this study.How to cite this article: Dong, T. et al. Ainsliadimer A selectively inhibits IKKα/β by covalently binding a conserved cysteine. Nat. Commun. 6:6522 doi: 10.1038/ncomms7522 (2015).Supplementary Figures 1-28, Supplementary Tables 1-2, Supplementary Notes 1-2, Supplementary Methods and Supplementary References.


KIN10 and some cold-acclimation related genes (HOS1 and ICE1s) from the cold-resistant wild banana ‘Huanxi’ (Musa itinerans) were cloned and their expression patterns under different temperature treatments were analyzed. Thirteen full-length cDNA transcripts including 6 KIN10s, 1 HOS1 and 6 ICE1s were successfully cloned. Quantitative real-time PCR (qRT-PCR) results showed that all these genes had the highest expression levels at the critical temperature of banana (13 °C). Under chilling temperature (4 °C), the expression level of KIN10 reduced significantly but the expression of HOS1 was still higher than that at the optimal temperature . Both KIN10 and HOS1 showed the lowest expression levels at 0 °C, the expression level of ICE1, however, was higher than control. As sucrose plays role in plant cold-acclimation and in regulation of KIN10 and HOS1 bioactivities, the sucrose contents of wild banana under different temperatures were detected. Results showed that the sucrose content increased as temperature lowered. Our result suggested that KIN10 may participate in cold stress response via regulating sucrose biosynthesis, which is helpful in regulating cold acclimation pathway in wild banana.Banana cultivars may experience chilling or freezing injury in some of their cultivated regions, where wild banana can still grow very well. The clarification of the cold-resistant mechanism of wild banana is vital for cold-resistant banana breeding. In this study, the central stress integrator gene The online version of this article (doi:10.1186/s40064-015-1617-z) contains supplementary material, which is available to authorized users. Musa spp.) is one of the most important nutrient-rich crops, staple foods and ornamental plants cultivated in tropical and subtropical regions where temperature is relatively high. Nonetheless, considerable interests still exit in exploring banana cold-resistant genes and developing cold tolerant banana cultivars due to the chilling or freezing injuries they might experience at some of their cultivated regions , ICE1 (inducer of CBF expression1), HOS1  and so on  genome data will be surely helpful for identification and characterization of these genes  genes were identified by searching the Malaysian wild banana genome data and were cloned by RT-PCR and/or RACE technologies from cold-resistant wild banana ‘Huanxi’ (Musa itinerans). Their expression patterns under different temperature, together with the sucrose content in leaves, were also analyzed. Our study could be helpful in understanding the cold acclimation responses of wild banana under different temperature and in exploring the function of KIN10 in cold response.Wild banana is widely distributed in all prefecture-level cities in Fujian Province, China , 1 HOS1 and 6 ICE1 (ICE1-1–ICE1-6) genes were successfully cloned from cold-resistant wild banana ‘Huanxi’. To the best of our known, this is the first report about cloning these genes from Musa spp. The generated sequences were submitted to GenBank, and the corresponding accession numbers were granted to KC127685, KC127686, KC127687, KC127688, KC127689, KC127690, JX678611, KC157569, KC157570, KC157571, KC157572, KC157573 and KC157574, respectively.In total, 6 KIN10-1, KIN10-2 and KIN10-3 had similar ORF sequences that were 95.51 % identical to the KIN10 of Malaysian wild banana (Musa acuminata) (GSMUA_Achr10 G09220_001). But the ORF sequences of KIN10-4, KIN10-5 and KIN10-6 shared lower identity (only 76.58 %). These sequence variations may be due to differences between genes or species. Bioinformatics prediction result revealed that all the 6 KIN10s were basic, hydrophilic, and unstable proteins possessing transmembrane domains with predicted location in the nucleus or in membranes. Moreover, 21–26 phosphorylation sites were found in KIN10s  with the Malaysian wild banana HOS1 (GSMUA_Ach1G14640_001). The major difference between the two species was the presence of a 140 bp insertion in the upstream region of the ‘Huanxi’. On the basis of bioinformatics prediction analysis, HOS1 was shown to be a nuclear-localized, hydrophilic unstable protein without signal peptide. And 57 phosphorylation sites and a specific ELYS-like conserved domain were found in HOS1 (Table Phoenix dactylifera HOS1 showed the closest relationship with wild banana ‘Huanxi’ HOS1.The ilarity 9.95 % witICE1-1–ICE1-4 genes shared higher identity (97.52 %) with Malaysian wild banana ICE1 (GSMUA_Achr10 G18380_001) compared with ICE1-5 and ICE1-6 (92.08 %). A 75 bp sequence, which was almost exactly the same size as that of ICE1 introns in Malaysian wild banana, was missing from the middle region of ICE1-1–ICE1-4 in wild banana ‘Huanxi’. Other missing sequences in wild banana ‘Huanxi’ were a 16 bp sequence absent from the upstream region of ICE1-3 and ICE1-4 and a 19 bp sequence deleted from the termination codon region of ICE1-1 and ICE1-3. ICE1-5 and ICE1-6 were 9 bp longer in wild banana ‘Huanxi’. Interestingly, compared with the Malaysian wild banana ICE1, ICE1-5 of wild banana ‘Huanxi’ contained one more intron and one fewer exon and ICE1-6 possessed two additional introns, which might be results of alternative splicing in evolution , critical temperature (13 °C), chilling temperature (4 °C) and freezing temperature (0 °C) were detected. Banana is basically a tropical crop growing at temperatures ranging from 13 to 38 °C. Below 13 °C or above 38 °C, banana growth ceases and 13 °C is thought to be the critical low temperature for banana growth  were obtained from the Wild Banana Germplasm Nursery of the Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, China. For temperature treatments, 25-day-old plantlets growing at 28 °C were subjected to 0, 4 and 13 °C for 36 h with plantlets growing at 28 °C as control.Plants of cold-resistant wild banana ‘Huanxi’ (http://www.ncbi.nlm.nih.gov) and in the Malaysian wild banana (Musa acuminata) genome-wide data (http://banana-genome.cirad.fr) were downloaded and used as reference sequences for determination of the conserved regions and for primer design. Information of all the used primers was listed Additional file http://www.ncbi.nlm.nih.gov).Homologous gene sequences in GenBank database (http://www.ebi.ac.uk/interpro/scan.html)   for 36 h and used for the qPCR analysis were analyzed for sucrose content. The sucrose contents in leaves were measured by using the method of Xue .


Tankyrases, multifunctional poly(ADP-ribose) polymerase (PARP) superfamily members with features of both signaling and cytoskeletal proteins, antagonize Wnt/β-catenin signaling. We found that tankyrases interact with CD2-associated protein (CD2AP), a protein essential for kidney ultrafiltration as CD2AP-knockout (CD2AP−/−) mice die of kidney failure at the age of 6–7 weeks. We further observed that tankyrase-mediated total poly-(ADP-ribosyl)ation (PARylation), a post-translational modification implicated in kidney injury, was increased in mouse kidneys and cultured podocytes in the absence of CD2AP. The data revealed increased activity of β-catenin, and upregulation of lymphoid enhancer factor 1 (LEF1) (mediator of Wnt/β-catenin pathway) and fibronectin (downstream target of Wnt/β-catenin) in CD2AP−/− podocytes. Total PARylation and active β-catenin were reduced in CD2AP−/− podocytes by tankyrase inhibitor XAV939 treatment. However, instead of ameliorating podocyte injury, XAV939 further upregulated LEF1, failed to downregulate fibronectin and induced plasminogen activator inhibitor-1 (PAI-1) that associates with podocyte injury. In zebrafish, administration of XAV939 to CD2AP-depleted larvae aggravated kidney injury and increased mortality. Collectively, the data reveal sustained activation of the Wnt/β-catenin pathway in CD2AP−/− podocytes, contributing to podocyte injury. However, we observed that inhibition of the PARylation activity of tankyrases in the absence of CD2AP was deleterious to kidney function. This indicates that balance of the PARylation activity of tankyrases, maintained by CD2AP, is essential for normal kidney function. Furthermore, the data reveal that careful contemplation is required when targeting Wnt/β-catenin pathway to treat proteinuric kidney diseases associated with impaired CD2AP.Inappropriate activation of the Wnt/ Disruption of the filtration barrier leads to proteinuria, which may progress to renal failure. Podocytes are terminally differentiated cells with a large cell body and interdigitating foot processes, which rest on the glomerular basement membrane. Podocyte injury has been shown to have an important role in the development of proteinuria as the loss of function of several podocyte proteins has been shown to lead to podocyte foot process effacement and impaired glomerular ultrafiltration.2 CD2AP was originally characterized as an interaction partner of T-cell adhesion molecule CD2,3 and as a protein upregulated during mesenchyme to epithelium differentiation in the developing kidney.4 In the glomerulus, CD2AP interacts with nephrin,5 the key component of the slit diaphragm interconnecting the neighboring podocyte foot processes.6 CD2AP regulates cytoskeletal remodeling,7 apoptosis8 and vesicular trafficking.9Adapter protein CD2-associated protein (CD2AP) is essential for the maintenance of glomerular filtration.11 They catalyze poly(ADP-ribosyl)ation (PARylation), a post-translational modification targeting tankyrases themselves or their interaction partners, subject the target proteins to ubiquitination and proteasomal degradation,10 and thereby modulate diverse cellular processes.12 During recent years, several new interaction partners and functions have been characterized for tankyrases;15 however, the possible involvement of tankyrases in regulating podocyte function has remained unknown. PARylation activity of tankyrases is involved in regulating the activity of the canonical Wnt/β-catenin signaling pathway.16 When the pathway is inactive, β-catenin is phosphorylated and targeted to degradation by the β-catenin destruction complex. Tankyrases PARylate AXIN in the destruction complex, stimulate its degradation, and thus enable dephosphorylation of β-catenin and its consequent translocation to the nucleus where it together with T-cell factor/lymphoid enhancer factor (TCF/LEF) family of co-activators drives Wnt-specific transcriptional programs.17 In the adult kidney, Wnt/β-catenin pathway has been considered to be silent. Inappropriate activation of the pathway has been indicated in podocyte dysfunction and injury,18 and in many kidney diseases such as diabetic nephropathy and adriamycin-induced nephropathy.22Tankyrases 1 and 2 are members of the poly(ADP-ribose) (PAR) polymerase family.β-catenin accompanied by elevation of total PARylation associates with podocyte injury in the absence of CD2AP. However, inhibition of tankyrases in the absence of CD2AP enhances the expression of podocyte injury markers and aggravates kidney damage.Here, we show that tankyrases are novel interaction partners of CD2AP, and that CD2AP is a negative regulator of tankyrases. We demonstrate that activation of 2 terminus of CD2AP containing three SH3 domains. The screening identified a clone that covered amino acids 1–190 of tankyrase 2. Pull-down assays with the NH2 terminus or individual SH3 domains of CD2AP fused with GST  treatment of WT and CD2AP−/− cell lysates confirmed that the slower migrating form of tankyrase 1 is phosphorylated in podocytes  by 3–4 weeks of age. Kidney failure results in the death of the animals by the age of 6–7 weeks.8 We examined whether we observe an increase in total PARylation, shown to associate with kidney injury in diabetes,29 in the kidneys of CD2AP-deficient mice at 3 weeks of age. Indeed, we observed that CD2AP deficiency leads to increased total PARylation in the kidneys compared with WT kidneys  revealed that there is more interaction between LEF1 and active β-catenin in CD2AP−/− podocytes compared with WT podocytes , fibroblast-specific protein 1 (FSP-1), matrix metalloproteinase 9 (MMP-9) and fibronectin, have been shown to be upregulated in kidney injury.20 However, we observed no significant difference in the expression levels of Snail1, PAI-1 and FSP-1 between CD2AP−/− and WT podocytes .XAV939 treatment had no effect on odocytes , and fibodocytes . Furtherodocytes . As alsorylation . XAV939 rylation . Also inrylation . Additiorylation , whereasrylation . Full-lerylation . XAV939 rylation . The datin vivo, we knocked down cd2ap with morpholino antisense oligonucleotides (MOs) in zebrafish, previously shown to result in pronephric kidney dysfunction,32 coupled with tankyrase inhibition. At 5 days post fertilization (dpf), 60–68% of cd2ap-MO- and cd2ap-MO and tankyrase inhibitor-treated larvae exhibited pericardial edema indicative of kidney dysfunction  that is required for fucose synthesis,35 which bind tankyrases and inhibit their activity, but are not PARylated.Increased PARylation in podocytes has been shown to have a role in glomerular injury associated with type 2 diabetes and general PARP inhibitors ameliorate this pathological situation.β-catenin pathway in the absence of CD2AP. In cells deficient of CD2AP, active β-catenin is stabilized and translocated to the nucleus. This is coupled with upregulation of its downstream targets, LEF1 and fibronectin, whereas several other typical Wnt/β-catenin target genes associated with kidney injury were not affected. Notably, upregulation of LEF1 and fibronectin occurred without exogenic Wnt stimulation. Our data propose that the major enhancer of the pathway is enhanced tankyrase PARylation activity . Podocyte cultures were supplemented with 10 U/ml IFN-γ (Sigma-Aldrich). HEK293T cells were transiently transfected with tankyrase 1 , flag-tagged tankyrase 2 and flag-tagged ankyrin domains of tankyrase 2  using Lipofectamine 2000 . Proliferating WT and CD2AP−/− podocytes were cultured to 70% confluency and treated with 3 μM XAV939 (Sigma-Aldrich), 3 μM ABT-888  or DMSO for 18 h when indicated.Conditionally immortalized podocytes derived from CD2AP−/− mice and WT littermates51 mouse anti-PARP1 and rabbit anti-Poly(ADP-ribose) , mouse anti-active-β-catenin , rabbit anti-LEF1 and rabbit anti-phospho p38 MAPK  and mouse anti-WT1 , guinea pig anti-p62/SQSTM1  and mouse anti-Caspase-1 .Antibodies used were rabbit anti-tankyrase 1/2 and mouse anti-Bcl-2 , rabbit anti-podocin and mouse anti-tubulin (Sigma-Aldrich), goat anti-tankyrase 2, rabbit anti-BAX and rabbit anti-fibronectin , rabbit anti-CD2AP 1774 or 1764,52 Tissues and cells were lysed as described.53 Pooled zebrafish larvae were sonicated in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS supplemented with 1 mM PMSF and protease inhibitor cocktail . Nuclear extracts were prepared as described.54Glomerular and tubular fractions were isolated from rat kidney cortices using graded sieving.55 or the NH2-terminus (amino acids 1–330) of mouse CD2AP was performed as described.5 Pull-down assay for PARylated proteins was performed with a WWE Affinity Resin kit  according to the manufacturer's instructions, and immunoprecipitation as described.5 Immunoblotting was performed as described.51Pull-down assay with glutathione-S-transferase (GST)-tagged SH3 domains49 Sections were analyzed with a Nikon Eclipse 800 microscope .Mouse kidneys were fixed with 10% formalin, dehydrated and embedded in paraffin. Immunoperoxidase staining was performed with a VectaStain Elite kit  as described.μm) were fixed with acetone and cultured podocytes were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100. The stainings were carried out as described.53 AlexaFluor 594 donkey anti-rabbit and AlexaFluor 488 donkey anti-goat or donkey anti-mouse IgGs were used as secondary antibodies. Samples were analyzed with Leica TCS SP2 or SP8 confocal microscopes .Mouse kidney frozen sections .The total PARylation was measured with HT PARP in situ PLA was carried out according to the manufacturer's instructions . Fluorescence images were captured using Zeiss Axioplan2 microscope . Quantification was performed using the Duolink Image Tool (Olink Bioscience).Duolink et al.56 Primer sequences for Snail1, MMP-9, FSP-1, PAI-1, LEF1, TCF-1, TCF-3 and TCF-4 are shown in β-actin using the comparative Ct method. Reverse transcriptions were performed with three individual RNA preparations and quantitative RT-PCRs were performed in triplicate.Quantitative PCR was performed as described in Wang cd2ap (5′-CATACTCCACCACCACCTCAACCAT-3′)32 and a standard control MO were purchased from GeneTools, LLC . Zebrafish embryos were injected with 4 nl of 100 μM MOs using a Narishige MN-153 microinjector . Embryos were allowed to develop until 3 days post fertilization (dpf) and then transdermally exposed to 5 μM XAV939 or an equivalent volume of DMSO (0.05%) in E3 medium for 2 days. The phenotype of larvae on 5 dpf was examined under a Leica dissecting microscope.MO blocking translation of μm sections were stained with hematoxylin and eosin.Zebrafish larvae at 5 dpf were fixed with 4% PFA in PBS overnight at 4 °C and embedded in JB-4 . In all, 4 Statistical analysis was performed with one-way ANOVA. Results are presented as mean±S.E.M.


We tested (1) whether the PQRST method, involving Preview (P), Question (Q), Read (R), State (S), and Test (T) phases, is effective in enhancing long-term memory in patients with mild memory problems due to prefrontal cortex lesions, and (2) whether patients also benefit from a more self-initiated version of the PQRST. Seven patients with prefrontal lesions encoded new texts under three different conditions: the Standard condition, requiring to read texts repeatedly, the PQRST-Other condition, in which the experimenter formulated questions about the text (Q phase), and the PQRST-Self condition, in which patients formulated the relevant questions on their own. Compared to the Standard condition, both the PQRST-Other and the PQRST-Self condition resulted in higher immediate and delayed recall rates, as well as a higher ability to answer questions about the texts. Importantly, the two PQRST conditions did not differ in efficacy. These results confirm that the PQRST method is effective in improving learning of new material in brain-injured populations with mild memory problems. Moreover, they indicate that the PQRST proves effective even under conditions with higher demands on patients’ autonomy and self-initiation, which encourages its application to real-life situations. Long-term memory disorders are among the most challenging cognitive impairments following acquired brain damage, and may have a profound impact on patients’ daily living, ranging from minimal forgetfulness to a pervasive inability to learn new information and cope with life demands. For this reason, there is great interest in developing methods aimed at restoring or compensating memory impairment.Memory is a multicomponential process. Consequently, memory deficits are multifaceted, and call for different treatment options. Lesions to the medial temporal lobe may result in an inability to explicitly encode and retrieve new information, while implicit memory is spared . Damage While treatment options for severe amnesia include techniques based on spared implicit memory processes and memory aids , patientPreview , Question (formulate main questions about the text), Read , State (summarize the main information), and Test (test your knowledge) (PQRST is the acronym of owledge) . This meowledge) .The first aim of this study is to test the efficacy of the PQRST method in a group of patients with mild memory problems due prefrontal cortex lesions. Given that prefrontal cortex supports strategic encoding and retrieval processes , and thaTo these aims, a group of patients with prefrontal lesions memorized texts in three different encoding conditions: a Standard condition, requiring to read the text repeatedly (see below), and two different PQRST conditions; in one, the questions were created by the experimenter (PQRST-Other condition), whereas in the other questions were created by the patients themselves (PQRST-Self condition). To foreshadow the results, we found that, compared to the Standard condition, both the PQRST-Other condition and the PQRST-Self condition resulted in better memory for the texts. Importantly, the PQRST-Self condition proved as effective as the PQRST-Other condition.Table 1 for demographic and clinical data). Patients had a mean age of 45 years (range 32–60), and a mean education of 11.57 years (range 8–13). Time since injury was, on average, 7.4 years (range 1–20). Patients were recruited at the Centre for Studies and Research in Cognitive Neuroscience of the University of Bologna, in the context of a routine neuropsychological assessment, which highlighted, in all cases, long-term memory deficits. All patients complained about memory deficits in real life, and participated voluntarily to the study. Included patients were not receiving psychoactive drugs, and had no other diagnosis likely to affect cognition or interfere with the participation in the study  as determined by history. Participants gave written informed consent to participate in the study according to the Declaration of Helsinki  with lesions to the prefrontal cortex due to anterior communicating artery (AcoA) aneurysm or TBI (see Helsinki  and the M = 26.7). Patients performed normally also in several tests assessing attentional and executive functions, such as selective attention  = 3. Note that the ES ranges from 0 = impaired performance, and 1 = borderline performance, to 2–4 indicating normal performance; M = 87.4). Performance in immediate recall of word lists , covered a single topic, and was divided into 28–30 idea units for scoring purposes. For each passage, four questions were developed, covering the main aspects of the story . The 24 prose passages were randomly divided into three sets of eight passages, matched for number of words  and units . The assignment of the three sets to the different experimental conditions  was counterbalanced across participants.Twenty-four prose passages were selected and adapted from various online media  as well as the reading comprehension section of a high school book. Each passage was between 145 and 190 words in length , and the order of administration of each prose passage within each set was determined randomly for each participant. Depending on the experimental condition, participants received different encoding instructions.In the Preview (P) phase, the experimenter read the passage aloud, to make the participants get a general idea of the material. The Question (Q) phase was different in the PQRST-Other and PQRST-Self conditions: in the PQRST-Other condition, the experimenter read the four (other-generated) questions about the text . In the PQRST-Self condition, the patient formulated four questions regarding the text (self-generated questions). The experimenter stressed the need to formulate four questions that covered the whole story. In both PQRST conditions, the four questions were written on a card that was placed on the desk, in front of the participant, and remained there throughout the Read phase (R), in which participants read the material carefully to look for the answers to the questions. In the following State (S) phase, patients stated the answers, and, if necessary, read the text again. The whole “study session” lasted 10 min on average. Immediately afterward, the Test (T) phase began, in which the experimenter tested memory for the text by (1) asking the same questions that had been embedded in the study phase , (2) asking for free recall of the passage, and (3) asking for delayed free recall of the passage, after 10 min of non-interfering activities . In the PQRST-Self condition, after delayed recall of the self-generated questions, patients were also asked the other-generated questions, i.e., those commonly used in the PQRST-Other condition. This was done to verify whether the improvement in answering questions about a text was limited to those questions that were part of the procedure, or generalized to untrained questions.The standard condition was designed to (1) be representative of patients’ usual encoding strategies and (2) last as long as the PQRST-based conditions. A preliminary, informal interview with each patient and a relative revealed that patients’ most common strategy to learn new material was to re-read texts over and over again, and in some cases highlight the relevant parts. In a second session, the experimenter asked patients to memorize a text, which confirmed that this was indeed the most frequent learning strategy they adopted. The Standard enconding condition was designed to mimic patients’ spontaneous strategies. First, similar to the two PQRST conditions, the experimenter read the passage aloud (Preview phase). Patients were then left free to read the text over and over again for 10 min, during which they could take notes and underline the most important parts (Study phase). Importantly, also in the Standard condition patients had the four questions about the passage introduced early on, which remained in front of them for the entire duration of the Study phase see also . PatientA scorer blind to the aim of the study and to the experimental hypotheses evaluated, for each prose passage, the frequency of passage units recalled correctly and the frequency of questions answered correctly. The scorer was instructed to consider an answer, or a passage unit, correct when it conveyed the relevant information completely and unambiguously, no matter whether verbatim or not.p > 0.20 in all cases) and were analyzed with parametric tests.Six dependent variables were considered: the frequency of passage units recalled immediately after the study phase and after the delay, and the frequency of correct answers given to the four questions, both immediately and after the delay. For the PQRST-Self condition, we also evaluated the frequency of correct answers given to the other-generated questions. In all cases, the variables were distributed normally  = 11.72, p = 0.01], such that participants recalled more units at the immediate compared to the delayed test, and a significant effect of Condition . Post hoc Newmann–keuls tests showed that patients recalled more units in the PQRST-Other condition and in the PQRST-Self condition compared to the Standard condition (p < 0.05 in both cases), with no difference between the two PQRST conditions . The Condition × Test interaction was not significant (p = 0.82).An analysis of variance (ANOVA) on free recall rates with Test  and Condition  as within-subject factors showed a significant effect of Test , such that correct answers were more frequent immediately after study than after a delay, and a significant effect of Condition . Post hoc tests showed that patients answered more questions in the PQRST-Other condition and in the PQRST-Self condition compared to the Standard condition (p < 0.0003 in both cases), with no difference between the two PQRST conditions (p = 0.79) . The Condition × Test interaction was not significant (p = 0.57).Similar results were found on the ability to answer questions about the texts. The ANOVA on the frequency of correct answers with Test and Condition as factors showed a significant effect of Test , which was qualified by a Condition X Test interaction . Post hoc tests showed that in the immediate testing session correct answers were more frequent in the PQRST-Other condition compared to the Standard condition (p < 0.0001), but there was no difference between the PQRST-Self and the Standard conditions (p = 0.15) . However, in the delayed testing session, correct answers were more frequent in both the PQRST-Other and the PQRST-self condition compared to the Standard condition (p < 0.0004 in both cases) . The PQRST-Other condition proved more effective than the PQRST-Self condition both immediately and after a delay (p < 0.006 in both cases).We also ran the same ANOVA using the frequency of correct answers to the other-generated questions also for the PQRST-Self condition. There was a significant effect of Condition [p < 0.05), we run non-parametric correlation analyses.In order to shed light on the type of patients that would benefit the most from the PQRST method, and that would benefit differentially from the classic, PQRST-Other method vs. the PQRST-Self method, we ran correlation analyses between patients’ results at standard neuropsycological tests and (1) the difference between scores attained in the PQRST-Other condition and the Standard condition, and (2) the difference between scores attained in the PQRST-Other condition and the PQRST-Self condition (in both cases collapsing across immediate and delayed conditions). Given that, in some cases, the variables were non-normally distributed  and WMS scores , and the improvement in free recall correlated negatively with semantic fluency . Thus, patients with weak memory and executive functioning benefited the most from a well-organized plan to encode new material. We next investigated which aspect of patients’ neuropsychological profile predicted performance differences between the PQRST-Other and PQRST-Self conditions. We found that differences in the ability to answer questions in the PQRST-Other vs. PQRST-Self condition correlated negatively with WMS scores , and differences in free recall correlated negatively with semantic  and phonemic fluency . Thus, in individuals with more preserved memory and executive functioning the PQRST-Self condition tended to be as effective as the PQRST-Other condition.We first investigated which aspect of patients’ neuropsychological profile predicted the degree of improvement in the PQRST-Other vs. Standard condition. We found that the improvement in answering questions about the texts in the PQRST-Other vs. Standard condition correlated negatively with standardized prose recall scores . This suggests that the PQRST method actually improved patients’ ability to encode and store new material.It has often been hypothesized that patients with prefrontal cortex lesions exhibit problems in learning new information due to an impairment in engaging effective encoding strategies spontaneously . As discThe systematic series of encoding operations probed by the PQRST method provided patients with a unique opportunity to process incoming information optimally for learning. Multiple mechanisms may underlie the efficacy of PQRST. First of all, PQRST favors deep  encoding of incoming information , requiriIn addition to the (classic) PQRST-Other condition, patients in the present study encoded texts through a modified PQRST-Self condition, in which they formulated the questions on their own. The rationale behind this choice was that, under these encoding instructions, patients would select the passages of the text that were relevant to them, motivating them to inspect the text carefully. Information that is more personally meaningful generally undergoes greater elaboration and organization at encoding, resulting in higher recall rates compared to information that does not have the same relevance e.g., . MoreoveThe fact that the two PQRST procedures were equally effective in patients is of great importance for rehabilitation. As anticipated, the ultimate goal of rehabilitation is the ability to transfer the trained skills to other contexts than the laboratory, such as real life. The present finding that a PQRST procedure based on self-formulated questions is effective in ameliorating memory performance suggests that patients with prefrontal lesions could be trained to apply this method in real life, for example to keep track of news or to re-learn some aspects of their autobiography.We have confirmed that the PQRST is effective to promote new learning in patients with mild memory impairment, and shown that patients may benefit even from alternative versions of the procedure requiring higher levels of self-initiation. Future studies should verify whether patients can generalize the use of PQRST to untrained, and real-life situations. In our laboratory, we are currently investigating whether repeated encoding via the PQRST-Self procedure improves learning of untrained materials.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cell signaling pathways are the mechanisms by which cells transduce external stimuli, which control the transcription of genes, to regulate diverse biological effects. In cancer, distinct signaling pathways, such as the Wnt/β-catenin pathway, have been implicated in the deregulation of critical molecular processes that affect cell proliferation and differentiation. For example, changes in β-catenin localization have been identified in Human Papillomavirus (HPV)-related cancers as the lesion progresses. Specifically, β-catenin relocates from the membrane/cytoplasm to the nucleus, suggesting that this transcription regulator participates in cervical carcinogenesis. The E6 and E7 oncoproteins are responsible for the transforming activity of HPV, and some studies have implicated these viral oncoproteins in the regulation of the Wnt/β-catenin pathway. Nevertheless, new interactions of HPV oncoproteins with cellular proteins are emerging, and the study of the biological effects of such interactions will help to understand HPV-related carcinogenesis. This review addresses the accumulated evidence of the involvement of the HPV E6 and E7 oncoproteins in the activation of the Wnt/β-catenin pathway. Signaling pathways are the mechanisms by which cells decide their fate and communicate with other cells and their environment. The binding of ligands to cell receptors can activate protein cascades and consequently affect gene transcription levels. Via these complex processes, cells transform external stimuli into biochemical signals that control biological effects, such as proliferation, differentiation, and death.Many signaling pathways have been identified as being deregulated in cancer. Consequently, numerous elements targeting these pathways have been proposed as therapeutic targets. Consistent alterations of some important pathways controlling cell proliferation and apoptosis, such as PI3K/Akt, ERK/MAPK, Notch, and Wnt/β-catenin, have been identified in different types of cancer. In particular, the activation of the Wnt signaling pathway has been implicated in osteosarcoma , hepatocCervical cancer is the fourth most common cancer in women worldwide and is one of the leading causes of cancer death in women in developing countries . PersistThis review addresses the participation of the Wnt/β-catenin signaling pathway in HPV-related cancers and the possible mechanisms by which HPV E6 and E7 oncoproteins induce the activation of this pathway.Drosophila melanogaster proved to be a homologue of Int-1 2+ pathwa Wnt1, 2 , and 10b Wnt1, 2 ,26. The  Wnt1, 2 , 5a [28, Wnt1, 2 , and 11  Wnt1, 2  ligands. Wnt1, 2 .The non-canonical Wnt/PCP, also known as the Wnt/JNK pathway, is important in various processes including wound healing , the cor2+ pathway, secondary messengers, such as IP3 and DAG, liberate calcium ions from the endoplasmic reticulum [In the Wnt/Caeticulum  and subseticulum  and PKC eticulum .The processes that are triggered by the activation of this non-canonical pathway include the following: the regulation of convergent extension movements , the reoThe Wnt canonical signaling pathway is the best understood Wnt signaling cascade. In the absence of Wnt ligands (OFF-STATE), β-catenin is mainly located at cellular junctions. Nevertheless, a small amount remains in the cytoplasm and binds to a complex responsible for the degradation of β-catenin via the proteasome. This degradation complex consists of the scaffold protein Axin which recruits essential elements during this process such as GSK3β , CK1 4545, APC , YAP/TAZAs a consequence of the Wnt ligand binding to the Fz receptor and LPR5/6 co-receptor  ON-STATB. These Persistent infection with Human Papillomaviruses (HPVs) has been implicated in the carcinogenesis of the uterine cervix . OropharThe carcinogenic potential of HPV is mainly due to the expression of E6 and E7 viral proteins, which are directly involved in cellular transformation ,68. The More than 170 HPV types have been identified . HPVs caPersistent HR-HPV infection is a crucial event in cellular transformation, but additional events are required to complete the malignant phenotype. Other mechanisms implicated in HPV-related cancers include the activation of multiple cellular pathways such as the Hedgehog , Erk/MAPThe HPV genome consists of a double-stranded circular DNA of approximately 8000 bp that contains genes that are expressed early (E) or late (L) during the viral life cycle and whose transcription and replication are mediated by the long control region (LCR) .L1 and L2 are the HPV structural proteins ,78. SpecE1 is the viral DNA helicase, and E2 a transcriptional activator and repressor that also complexes with E1 as a critical component of the HPV replisome . E2 protThe E4 coding sequence is contained within the E2 open reading frame (ORF). Although E4 is located in the early region, it is expressed as a late gene and is regulated by a promoter that is responsive to differentiation transcription factors. Moreover, the properties of E4 have not been fully characterized, but several studies implicate E4 in virion release via its association with keratin filaments .E6 and E7 are considered the most important viral oncoproteins: they play a clear role in cellular transformation . Among sDuring the normal viral life cycle, the HPV genome exists in host cells as an episome. However, the viral genome may be incorporated into the host genome in rare cases. Viral genome integration is closely tied to the development of cancer because most HPV-induced cervical cancer cases contain an integrated form of the HPV genome. Viral episome rupture during integration frequently occurs in a zone that includes E1 and E2. The consequent loss of E2 causes the uncontrolled expression of the E6 and E7 oncoproteins, which increases the likelihood of HPV-induced carcinogenesis ,86.E6 and E7 are small proteins that localize to the nucleus and cytoplasm and the interaction of both E6 and E7 immortalizes primary cells in a highly efficient manner .Moreover, the expression of E6 and E7 in organotypic raft cultures results in cellular changes that are similar to those observed in high-grade squamous intraepithelial lesions . AccordiDuring HPV infection, E6 and E7 induce the proliferation of undifferentiated and differentiated suprabasal cells and also inhibit apoptosis. These actions promote the accumulation of DNA damage and mutations that can result in cell transformation and the development of cancer . Table 1The E6 protein consists of approximately 150 amino acids and contains an LXLL motif in the amino terminal region that is required to interact with the ubiquitin ligase E6AP. Moreover, several proteins also bind to E6 via its LXLL motif, such as E6BP, IRF3, Tuberin, and Paxillin. Another critical E6 motif found in the carboxyl terminus is the S/TXV PBM (PDZ-binding motif), which mediates the interaction with specific domains on cellular proteins known as PDZ domains, specialized in protein-protein interactions . E6 inteThe E7 protein consists of 98 amino acids separated in three conserved regions: CR1, CR2, and CR3. CR2 includes a conserved LXCXE motif that mediates high-affinity binding to pRB . The CR3Growing evidence suggests that HPV oncoproteins can modulate cell signaling pathways to contribute to the carcinogenesis. Upon initial infection, this modulation may be necessary to complete the viral cell cycle and form infective viral particles. Nevertheless, the consistent high-level expression of viral oncoproteins may eventually alter the normal functions of the cell, triggering an uncontrolled transformation process. Via their different cellular interactions, E6 and E7 may be deregulating the different cell signaling pathways implicated in HPV-related cancers. Some of these pathways are involved in cell proliferation and apoptosis, such as PI3K/Akt, Ras/Raf, Notch, and Wnt/β-catenin .Several mutations in different components of the Wnt/β-catenin pathway have been described in various types of cancer . In contFurthermore, patients with oral and lung cancers that express high levels of the β-catenin-binding partner FOXM1 exhibit worse overall and relapse-free survival than patients with tumors that express low levels of FOXM1; interestingly, this effect is significantly enhanced by the presence of HPV DNA sequences . TherefoEpigenetic changes that suppress the activity of the negative regulator of the Wnt pathway have also been identified. Specifically, methylation markers in the APC and SFRP3 promoters have been found in ovarian cancer samples, but only in cases in which HR-HPV genomic sequences were detected . MoreoveMicroarray expression studies of cervical cancer-derived tumors and cell lines have identified the over-expression of genes involved in Wnt pathway maintenance and regulation, such as JUN, MYC, FZD2, RAC1, GSK3β, Dvl-1, and CTNNB1 ,132. Spei.e., WNT5A, FZD1, FAS, MYC, FZD6, CCND1, and PDGFRA, to facilitate cell proliferation and invasion.Additionally, a systematic study in cervical cancer samples showed an alteration in the expression of miRNAs involved in Wnt/β-catenin pathway modulation . In thisClear evidence indicates that the Wnt pathway is hyperactivated in HPV-related cancers. Currently, HPV oncoproteins are known to bind and alter the function of several cellular targets associated with Wnt pathway regulation, including hTERT, p53, p300/CBP, Dvl, and PP2A see , and infThe activation of the canonical Wnt pathway represents a second requirement for the malignant transformation of the HPV-infected epithelium ,135. SpeIn HPV-positive oropharyngeal cells, β-catenin expression is strongly localized in the cytoplasm and nucleus, whereas β-catenin is mainly detected in the membranes of HPV-negative cells .In these HPV16-positive oropharyngeal cancer cell lines, E6 and E7 repression was shown to significantly decrease the β-catenin cytoplasmic and nuclear protein levels as well as the β-catenin mRNA levels. Moreover, both E6 and E7 expression were confirmed to up-regulate β-catenin expression and to enhance TCF-mediated transcription. This effect was attributed to a decrease in the ubiquitin ligase type 3 Siah-1 protein (seven in absentia homologue-1 protein), which acts as β-TrCP to induce β-catenin degradation. Because p53 mediates Siah-1 transcriptional activation , the dowin vitro study showed that the HR and LR-HPV E6 proteins can distinctly augment the TCF response, with the highest activity observed for the HR-HPV E6 proteins [An proteins . In contSubsequently, E6AP was confirmed to act as a novel Wnt signaling regulator that cooperates with E6 . SpecifiAnother element participating in the Wnt signaling pathway is FOXM1, which is also regulated by the HPV E6 oncogene. FOXM1 can induce β-catenin nuclear translocation by directly binding to β-catenin . In cellIn vivo studies of transgenic mice support the role of the HPV E6 oncogene in the Wnt signaling pathway. In the K14E6 transgenic mice, the nuclear accumulation of β-catenin depends on the E6 PDZ-binding motif [in vitro studies showed that both E6 forms enhanced TCF transcriptional activity due to the interaction of E6 with Dvl2, which is responsible for the disassembly of the β-catenin degradation complex [ng motif . In this complex . These rOther assays of double-transgenic mice expressing E7 and a constitutively active β-catenin indicated that the co-expression of both proteins promotes invasive cervical cancer, supporting that the activation of the Wnt/β-catenin pathway in premalignant lesions may be partly due to HPV .Although the role of the E7 oncoprotein in the regulation of Wnt signaling has not been studied as well as that of E6, some findings suggest that this protein is involved in this pathway. Specifically, PP2A participates as a negative regulator of Wnt signaling. This phosphatase induces GSK3β activation via its dephosphorylation at the Ser 9 residue, which results in β-catenin degradation . In a moNotably, the above-described findings strongly support a role for HPV oncoproteins in the Wnt canonical pathway. Nevertheless, HPV has not been conclusively linked to the Wnt non-canonical pathway regulation, although some E6 targets, such as WNT7A and Dvl, are known to participate in the activation of Wnt non-canonical pathways.Evidence supporting the role of HPV in the modulation of the Wnt signaling pathway is shown in Persistent infection with high-risk HPV types is clearly a main factor in cervical cancer development, and such infections are also implicated in the development of other types of cancer. HPV infection and the activation of diverse cellular processes such as signaling pathways are required to induce a malignant phenotype. Moreover, the Wnt/β-catenin pathway is deregulated in various neoplasias, and has been implicated in HPV-related cancers.Several studies support the role of HPV oncoproteins in the activation of the canonical Wnt/β-catenin pathway, which may be involved in the onset, progression and maintenance of transformed cells.Deciphering the precise mechanisms by which HPV oncogenes participate in Wnt/β-catenin modulation will help to elucidate HPV-related carcinogenesis. This information could eventually aid in identifying biomarkers of prognosis and contribute to the design of more effective targeted therapeutics.


The wingless-type MMTV integration site (Wnt) signaling is a group of signal transduction pathways. In canonical Wnt pathway, Wnt ligands bind to low-density lipoprotein receptor-related protein 5 or 6 (LRP5 or LRP6), resulting in phosphorylation and activation of the receptor. We hypothesize that canonical Wnt pathway plays a role in the retinal lesion of age-related macular degeneration (AMD), a leading cause of irreversible central visual loss in elderly.Ccl2/−−/Cx3cr1/−−/rd8 and Ccl2/−−/Cx3cr1gfp/gfp mouse models with AMD-like retinal degeneration to further explore the involvement of Wnt signaling activation in the retinal lesions in those models and to preclinically evaluate the role of Wnt signaling suppression as a potential therapeutic option for AMD.We examined LRP6 phosphorylation and Wnt signaling cascade in human retinal sections and plasma kallistatin, an endogenous inhibitor of the Wnt pathway in AMD patients and non-AMD subjects. We also used the Ccl2/−−/Cx3cr1/−−/rd8 mice than that in wild type. Intravitreal administration of an anti-LRP6 antibody slowed the progression of retinal lesions in Ccl2/−−/Cx3cr1/−−/rd8 and Ccl2/−−/Cx3cr1gfp/gfp mice. Electroretinography of treated eyes exhibited larger amplitudes compared to controls in both mouse models. A2E, a retinoid byproduct associated with AMD was lower in the treated eyes of Ccl2/−−/Cx3cr1/−−/rd8 mice. Anti-LRP6 also suppressed the expression of Tnf-α and Icam-1 in Ccl2/−−/Cx3cr1/−−/rd8 retinas.We found higher levels of LRP6  protein phosphorylation and transcripts of the Wnt pathway-targeted genes, as well as higher beta-catenin protein in AMD macula compared to controls. Kallistatin was decreased in the plasma of AMD patients. Retinal non-phosphorylated-β-catenin and phosphorylated-LRP6 were higher in Ccl2 and Cx3cr1 deficiency, and intravitreal administration of anti-LRP6 antibody could be beneficial by deactivating the canonical Wnt pathway.Wnt signaling may be disturbed in AMD patients, which could contribute to the retinal inflammation and increased A2E levels found in AMD. Aberrant activation of canonical Wnt signaling might also contribute to the focal retinal degenerative lesions of mouse models with The online version of this article (doi:10.1186/s12967-015-0683-x) contains supplementary material, which is available to authorized users. Age-related macular degeneration (AMD) is a common cause of irreversible central blindness in the elderly . PatholoThe wingless-type MMTV integration site (Wnt) signaling is a group of signal transduction pathways including the canonical pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway . In the Ccl2/−−/Cx3cr1/−− mice on rd8 background (Ccl2/−−/Cx3cr1/−−/rd8) develop a broad spectrum of dry AMD-like pathology with early onset and high penetrance -1-(2-hydroxyethyl)-4--pyridinium) is the major component of lipofuscin fluorophores generated from the visual cycle flux of all-trans-retinol. The molecule is particularly correlated with retinal aging and AMD pathogenesis . A2E wasWestern blot analysis detected the phosphorylated level of LRP6. Mouse retina was dissected and homogenized for protein extraction. Equal amounts (50 μg) of total protein from each sample were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked with 5 % nonfat milk and separately blotted with primary antibodies . After thorough washes, the membrane was incubated with the peroxidase-conjugated horse anti-rabbit or horse anti-mouse antibody (Vector Lab). The signal was developed with the enhanced chemiluminescence (ECL) system . Membranes were stripped and re-blotted with anti-β-actin  as the loading control. Densitometry of the signal bands on digital images was quantified using FluorChem Q software .−ΔΔCt analysis method were used to determine relative amounts of product using Gapdh as an endogenous control. Each sample was analyzed in triplicate.cDNA synthesis was described as above. The primers/probes were purchased from Applied Biosystems as inventoried TaqMan gene expression reagents. Relative quantitative real time PCR was performed according to ABI’s instructions. To determine the Ct values, the threshold level of fluorescence was set manually in the early phase of PCR amplification. ABI SDS 1.3.1 software and the 2p <0.05. The collective ERG amplitudes were calculated by the area under curve (AUC) and compared by T-test between the groups. Comparison of plasma kallistatin levels between AMD patients were performed by independent-samples T test.Multiple means were compared by paired T-test. Differences were considered significant when CYCLIN D, C-MYC, AXIN 2 and VEGF, which are the target genes of β-catenin, were also detected in the AMD macular lesions compared to age-matched non-AMD controls  were found  were performed in ice Fig. a, whereaice Fig. b. The clion Fig. c, a repeice Fig. a.Fig. 4SCcl2/−−/Cx3cr1/−−/rd8 are more abundant in ventral retina  ERG also exhibited larger amplitudes in both a- and b-waves in the treated eyes compared to control mIgG treated eyes  family –34. PrevThe molecular analysis of mouse retinal tissues indicated that the development of lesions correlated with canonical Wnt pathway activation in the retina at the post-translational but not the transcriptional level, as we did not detect altered mRNA expression of Wnt components in an mRNA array but did find altered protein phosphorylation of canonical Wnt/β-catenin proteins. This finding provided the clue for proper targeting of the pathway to manage the retinal lesions. The Wnt pathway is known to mediate various physiological and pathological processes including angiogenesis, fibrosis, and inflammation , 36. WntMultiple Wnt-relevant pathways were activated during advanced AMD, when angiogenesis and inflammation play important roles , 45. HowCcl2/−−/Cx3cr1/−−/rd8 mice express a spectrum of immunological features of AMD, including increased deposition of complement factors, altered secretion of chemokines and cytokines, and the accumulation of activated microglia and macrophages in the retinal lesions [Ccl2 and Cx3cr1 rather than the rd8 mutation alone [Ccl2 and Cx3cr1, the fact that two animal models with these two deletions show similar results supports the involvement and the importance of Wnt signaling pathway in AMD pathogenesis.The Wnt signaling pathway has several intersections with other pathways including inflammatory processes such as TNF-α and NF-kB signaling , 50, 51. lesions , 52–54. on alone , 55. AltTnf-α levels have been measured in the Ccl2/−−/Cx3cr1/−−/rd8 mouse retina treated with omega-3, AREDS2 diet, TSG-6, PEDF, or Naloxane [This study demonstrates that the Wnt pathway activation is associated with focal retinal degeneration in animal models and human AMD. Blockage of Wnt signaling using a specific antibody for LRP6 improved the retinal structure and function, likely through reducing oxidative stress and inflammation. Significant lower ocular Naloxane , 46, 47.Naloxane . This fiCcl2 and Cx3cr1 deficiency, and intravitreal anti-LRP6 antibody therapy might be beneficial via deactivation of the canonical Wnt pathway.In summary, the balance of Wnt signaling activation may disturb homeostasis and allostasis in the normal aging retina, which could be linked to AMD pathogenesis due to allostatic overload. The activation of canonical Wnt signaling might contribute to the focal retinal degeneration of mouse models with This study reported strong evidence of Wnt signaling involvement in AMD. A beneficial effect was found on retina lesions by intravitreal administration of anti-LRP6 antibody to deactivate the canonical Wnt pathway in two related mouse models with focal retinal degeneration mimicking certain features of human AMD.