Epoxy adhesives are widely used in various industries because of their high heat and chemical resistance, high cohesion, and minimal shrinkage. Recently, epoxy adhesives have been applied in the automotive industry as structural adhesives for lightweight vehicles. However, the brittleness of the epoxy is an obstacle for this application, since the automotive industry requires epoxy-based structural adhesives to have a high level of high-speed impact resistance. Hence, we used phenol-terminated polyurethane (PTPU) as a toughening agent for epoxy adhesives and compared the results with those that were obtained with carboxyl-terminated butadiene acrylonitrile copolymer (CTBN). The high-energy impact resistance of the epoxy adhesives was measured by the impact wedge-peel (IWP) test, and the shear strength was measured by the single lap joint test. As a result, the 20 wt % PTPU-modified epoxy adhesive showed remarkably higher total absorbed energy (25.8 J) during the IWP test and shear strength (32.3 MPa) as compared with the control epoxy adhesive . In particular, the total absorbed energy of the PTPU-modified epoxy adhesive was much larger than that of the CTBN-modified epoxy adhesive (5.8 J). When more than 10 wt % PTPU was added, the modified epoxy adhesives showed stable crack growth and effectively transferred external stress to the substrate. These results were explained by changes in the glass transition temperature, crosslinking density, and morphology due to the toughening agents. Structural adhesives are used for structural bonding that requires high adhesion strength, elasticity, and environmental resistance. Particularly, structural adhesives are emerging as a fastening method for replacing bolts and nuts in the field of transportation vehicles in order to reduce carbon emissions and energy consumption. To be used as a structural adhesive for automobiles, high shear strength as well as high peel, bending, impact, and fatigue strengths are required . TherefoIt is common to introduce core-shell rubber (CSR) particles or soft toughening agents to improve the toughness of epoxy resins. Butadiene acrylonitrile (BN) liquid rubbers are the most prevalent compounds used as toughening agents ,7,8,9. Bg-CTBN) by ring-opening reaction between epoxide group of soybean oil and carboxyl group of CTBN to improve the fracture toughness and mechanical properties of the epoxy [g-CTBN modified epoxy resin was improved by 67.7% when compared to neat epoxy through the reduction of Tg and crosslink density.Kunz-Douglass et al. used CTBNs with different acrylonitrile contents  to know the relationship between the CTBN particle size and epoxy toughness . AccordiTg) and reactive groups are also used as toughening agents. For example, thermoplastic polyurethane (TPU), which exhibit various properties depending on the polyols, are also used as toughening agents for epoxy resins [gT like CTBN; the Tg of TPU is known to have a value of −80 to−40 °C, depending on the type of polyols [Tg, and toughness of the TPU/epoxy blends depend largely on their final morphologies, which, in turn, are determined by the phase separation [In addition, thermoplastics with a low glass-transition temperature  and aromatic amine-terminated polyurethane with different macroglycols and evaluated their toughening effect after blending with epoxy . AccordiOn the other hand, a typical method for measuring the impact resistance of an epoxy resin is the Izod or Charpy impact test, which applies a relatively low-speed impact ,35,36,37Tg, crosslinking density and viscosity of the epoxy adhesives according to the addition of PTPU and CTBN were measured, and fracture surface and surface morphology analyses were performed in this study.Therefore, in this study, phenol terminated polyurethane (PTPU), a thermoplastic polyurethane, and carboxyl-terminated butadiene acrylonitrile copolymer (CTBN), a reactive liquid rubber, were used as toughening agents for structural adhesives prepared with diglycidyl ether of bisphenol-A (DGEBA) epoxy as a main component, and their effects on the physical properties of the structural adhesives were examined according to their content. The resistance to cleavage fracture, an index of impact resistance, was determined by the IWP test using CR340, which is used for automobile pillars , and theN,N-dimethyl-N-phenylurea accelerator (Omicure U-405) were purchased from AlzChem Group  and DyStar Group , respectively. For the fillers, calcium carbonate (Omyacarb 10) was used and supplied by OMYA Company . Additionally, glass beads with a diameter of 0.2 mm to control the thickness of the adhesive were supplied by UNITECH . The bisphenol-A type of epoxy resin , the carboxyl-terminated butadiene copolymer with 26% acrylonitrile (Hypro 1300X13 CTBN), and the dimer acid-modified epoxy resin used for diluent (YD-171) were purchased from Kukdo Chemical Company . Phenol-terminated polyurethane (DY-965) was purchased from Huntsman Corporation (USA). A 40 wt % core-shell rubber (CSR) dispersed in DGEBA epoxy resin (MX-154) was purchased from Kaneka Corporation . Dicyandiamide hardener (Dyhard 100) and 3 were added and mixed in the paste mixer at 2000 rpm for three minutes. Finally, 1 wt % DAME by weight was added, mixed in the paste mixer, and defoamed at 2000 rpm under 1.0 kPa vacuum condition for three minutes.The ISO 11343 Standard does not specify the dimensions to be used for the IWP specimens. Hence, we adopted the widely used Ford specimen design . Two specimens were prepared and the surface of each specimen was wiped with acetone to remove dust. The mixture was applied to an area of 30 mm × 20 mm on the specimen surface, and a small amount of glass beads was spread onto the well-coated mixture. Each specimen was nested, as shown in We used a specific lap shear specimen to prepare for the single lap joint (lap shear) test, as described in the ISO 4587 Standard  . In the DMA specimens were prepared by casting the epoxy adhesives in silicone mould. The rectangular specimens  were employed for DMA measurement. The epoxy adhesive mixtures were poured into the silicone mould and degassed for an hour in an 80 °C vacuum oven and cured 28 min. in a 180 °C conventional oven. After curing, the specimens were cooled down to room temperature and separated from the silicone mould.The viscosity of the epoxy mixture was measured by a Brookfield DV1 viscometer . The viscosity measurements were conducted at room temperature while using a HB-07 spindle at 2.0 rpm.The IWP tests of the epoxy adhesive test specimens were performed using an impact drop tower  at room temperature. Two bent steel plates were bonded using the epoxy adhesives , and the force was measured when the adhesive layer was cleaved by the wedge at a velocity (v) of 2.0 m/s, as per ISO 11343. The cross-section morphologies of the epoxy adhesives after the IWP tests were observed by FE-SEM .The single lap shear tests of the epoxy adhesive test specimens were conducted using a dual-column universal testing machine . The crossed specimens were bonded using the epoxy adhesives , and the shear stress measurements were performed at room temperature at 5 mm/min. crosshead displacement, as per the ISO 4587 standard.ρ) and glass transition temperature (Tg) of the epoxy adhesives were measured by a dynamic mechanical analyser . The rectangular specimens  were prepared and tested in tension mode from −100 to 250 °C with 5 °C/min. ramp rate at a frequency of 1 Hz.The crosslink density (The molecular weights of PTPU and CTBN were measured by gel-permeation chromatography (GPC) . As a control, polystyrene with a PDI of approximately 1 was prepared. Subsequently, the sample was dissolved in THF for HPLC at 1 wt %. After that, the solution was placed in a cylinder and filtered by a syringe filter with 0.2 μm pore size. Finally, each sample was injected into GPC and then measured at 40 °C.Tg) and crosslink density (ρ) of the cured epoxy adhesives were measured by a dynamic mechanical analyser (DMA) in order to determine the effect of the toughening agents. Tgs and crosslink density of the epoxy adhesives as a function of the PTPU content. The glass-transition temperature was determined by the peak value of the tan δ curves, and the crosslink density was calculated based on the rubber elasticity theory [Tg and storage modulus of the epoxy adhesives decreased as the PTPU content increased. Tg decreased by approximately 8 °C for every 5 wt % increase in PTPU. The crosslink density remarkably decreased after the initial addition of PTPU, but the extent of decrease after the further addition of PTPU was diminished.The glass transition temperature . The crosslink density was also changed in a manner that differed from that of PTPU; it was constantly decreased with the CTBN content. When CTBN exceeded 15 wt %, the crosslink density became lower than that of the PTPU-modified sample. This result seemed to be due to the differences in the size, miscibility, and functional groups of CTBN and PTPU.CTBN led to results that were similar to those of PTPU, as shown in nM: 6800) than PTPU . CTBN may be more uniformly distributed in the epoxy network, causing a reduced crosslink density as compared with that caused by PTPU, owing to its small size and high miscibility. This observation is supported by the CTBN domain size, which is relatively smaller than that of PTPU. On the other hand, the reduced crosslink density of the PTPU-toughened epoxy adhesives could be explained, as follows. Based on the crosslink density and morphology of the PTPU-toughened epoxy adhesives, the sharp decrease in the crosslink density of the PTPU-toughened epoxy (at 5 wt % of PTPU) was believed to result from the hindrance of the curing reaction by the PTPU domains. However, as the PTPU content increased, more PTPU-epoxy adducts were formed and included in the network structure, alleviating the decrease in the crosslink density. In conclusion, it is believed that the crosslink density is determined by the degree of phase separation and the ease of toughening agent-epoxy adduct formation.The GPC results show that CTBN has a much lower molecular weight . Therefore, epoxy adhesives have three times more CTBN molecules than PTPU molecules, even at the same wt %.Based on the fracture surface analysis, most of the samples showed cohesive failure of the epoxy adhesives, except for the samples in It is well known that the separate domain formed by the toughening agent during the curing reaction plays an important role in improving the toughness of the epoxy. The number and size of the domain depend on the solubility parameter and chemical interaction between the toughening agent and epoxy ,49,50.Tg and ρ values, the drastic decrease in the shear strength of the CTBN-toughened epoxy adhesives is attributed to a decrease in the interfacial adhesion and the crosslink density.The shear strength of the epoxy adhesives was measured and is shown in Polyurethane (PTPU)- and butadiene-acrylonitrile rubber (CTBN)- based toughening agents were used to improve the high-speed impact resistance of epoxy-based adhesives, and the following conclusions were obtained.Tg reduction was highly dependent on the Tg of the toughening agent itself, and the crosslink density depends on the phase separation and the ease of formation of toughening agent-epoxy adducts.The glass-transition temperatures and crosslink densities of the epoxy adhesives changed differently, depending on the toughening agent (PTPU or CTBN). The degree of From the results of the IWP test, stable crack growth is observed if the epoxy adhesive effectively transfers external stress to the substrate, which means that the adhesive strength with the substrate and the toughness of the adhesive are very important. PTPU has very good elongation and recovery properties, showing excellent effects on improving toughness, and the urethane group is a site for H-bonding for the substrate.The IWP test does not show meaningful results in the case of CTBN, which is known to have a toughness improving effect. This finding suggests that the mechanism for improving toughness by CTBN is not effective for high-energy impacts due to the decrease in the crosslink density and interfacial adhesion.


Our data showed that GFAT1 can regulate β-catenin expression via modulation of the O-GlcNAcylation process. TOP/FOP-Flash and real-time qPCR analysis showed that GFAT1 knockdown inhibited β-catenin activity and the transcription of its downstream target genes CCND1 and MYC. Ectopic expression of a stabilized form of β-catenin restored the suppressive roles of GFAT1 knockdown on PDAC cell proliferation and invasion. Collectively, our findings indicate that higher GFAT1/HBP/O-GlcNAcylation exhibits tumor-promoting roles by maintaining β-catenin activity in PDAC.Reprogrammed glucose and glutamine metabolism are essential for tumor initiation and development. As a branch of glucose and metabolism, the hexosamine biosynthesis pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and contributes to the O-GlcNAcylation process. However, the spectrum of HBP-dependent tumors and the mechanisms by which the HBP promotes tumor aggressiveness remain areas of active investigation. In this study, we analyzed the activity of the HBP and its prognostic value across 33 types of human cancers. Increased HBP activity was observed in pancreatic ductal adenocarcinoma (PDAC), and higher HBP activity predicted a poor prognosis in PDAC patients. Genetic silencing or pharmacological inhibition of the first and rate-limiting enzyme of the HBP, glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), inhibited PDAC cell proliferation, invasive capacity, and triggered cell apoptosis. Notably, these effects can be restored by addition of UDP-GlcNAc. Moreover, similar antitumor effects were noticed by pharmacological inhibition of GFAT1 with 6-diazo-5-oxo-l-norleucine (DON) or Azaserine. PDAC is maintained by oncogenic Wnt/ Reprogrammed energy metabolism is emerged as a hallmark of cancer cells . DiffereThe first and rate-limiting step of the HBP is catalyzed by glutamine:fructose-6-phosphate amidotransferase (GFAT) that converts fructose-6-phosphate to glucosamine-6-phosphate , 11. Theβ-catenin activity was maintained by GAFT1-mediated O-GlcNAcylation. The activity of Wnt/β-catenin signaling and expression of its downstream targets were suppressed by GFAT1 knockdown.In this study, we performed a pan-cancer analysis of the HBP and uncovered that HBP activity is upregulated in PDAC and increased HBP activity predicts a poor prognosis in PDAC patients. Blockade of HBP activity by genetic or pharmacological inhibition of GFAT1 significantly suppressed PDAC cell proliferation and invasive potential. Mechanistically, we revealed that v/v, Gibco, USA) and 1% streptomycin-penicillin . All cells were incubated in a humidified atmosphere with 5% CO2 at 37°C. LY294002, rapamycin, and ICG-001 were obtained from Selleck . The GFAT1 inhibitor 6-diazo-5-oxo-L-norleucine (DON) and Azaserine were purchased from Sigma-Aldrich . OSMI-1 was purchased from Selleck . OSMI-1 was treated at 30 μM for 24 h before functional experiment analysis.The pancreatic cancer cell lines AsPC1, BxPC3, Capan1, MiaPaca2, PANC1, and SW1990 used in this study were all obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science . All cells were cultured in Dulbecco's modified Eagle's medium  or Roswell Park Memorial Institute- (RPMI-) 1640 medium supplemented with 10% fetal bovine serum (http://www.tcga.org/) database was used. The online available analyzer GEPIA2 (http://gepia2.cancer-pku.cn/) was conducted for correlation analysis. The mean value of the log2(TPM + 1) is used to calculate the signature score as reported previously [For pan-cancer analysis of the activity of the hexosamine biosynthetic pathway (HBP) and its prognostic value, data derived from The Cancer Genome Atlas . The negative control was nonhomologous to any human genome sequences. In brief, BxPC-3 and SW1990 cells were seeded in 6-well plates at a density of 3‐5 × 105 cells per well. When reached 50-70% confluence, cells were transfected with siRNAs or overexpression vector using RNAimax or Lipofectamine  according to the manufacturer's instructions. After incubation for 48 h, cells were collected to analyze the knockdown efficiency or subjected for indicated cell experiments.Two specific siRNAs against Glutamine-Fructose-6-Phosphate Transaminase 1 (GFAT1) and the v/v) and transferred to nitrocellulose membranes . Then, the nitrocellulose membranes were blocked with 5% nonfat milk, followed by probing corresponding primary antibodies: anti-mTOR , anti-p-mTOR , anti-Akt , anti-p-Akt , anti-β-catenin , anti-O-GlcNAc , and anti-β-actin antibody . After incubation with primary antibodies overnight at 4°C, the membranes were incubated the horseradish peroxidase-conjugated secondary antibodies in the next day. Finally, the antigen-antibody complexes were detected by enhanced chemiluminescence . Protein quantification was performed by ImageJ software (NIH Image), and the β-actin was used as an internal control.Cell total protein was extracted using RIPA lysis buffer containing protease inhibitor cocktail . The protein concentration was detected using a BCA Protein Assay Kit (Pierce Biotechnology). Thirty micrograms of protein were separated using 6-10% SDS-PAGE . Protein concentration from cell supernatants was estimated by a BCA Protein Assay Kit (Pierce Biotechnology). Whole-cell extracts were subjected to immunoprecipitation with protein A/G-linked The Cell Counting Kit  was used to determine cell viability. In brief, cells were seeded into 96-well plates in triplicate at a density of 3,000 cells per well. Cell viability was measured every 24 h using the CCK-8 according to the manufacturer's instructions. The absorbance at 450 nm was measured using a multifunctional microplate reader (Bio-Rad). For cell apoptosis, indicated cells were starved for 48 h and then cells were collected and subjected for Apo-ONE Homogeneous Caspase-3/7 Assay . The presented data were normalized to protein content.μm) according to the manufacturer's instructions . Before experiment, 100 μl Matrigel  was preincubated in the upper chamber and allowed to solidification. Then, the upper chamber was incubated with 1 × 104 cells in 100 μl culture medium without serum. The lower chamber was supplemented with 700 μl culture medium containing 10% FBS (v/v). After incubation for 24 h at 37°C, the noninvaded cells were removed, and the invaded cells on the lower surface were fixed with 4% paraformaldehyde (w/v) and stained with 0.25% crystal violet (w/v). The number of invaded cells was counted in six randomly selected microscopic fields.Transwell invasion assays were performed in 24-well transwell plates (8 β-actin). All reactions were run in triplicate. The primer sequences were shown as follows: GFAT1 forward, 5′-GGAATAGCTCATACCCGTTGG-3′; GFAT1 reverse, 5′-TCGAAGTCATAGCCTTTGCTTT-3′; CCND1 forward, 5′-CAATGACCCCGCACGATTTC-3′; CCND1 reverse, 5′-CATGGAGGGCGGATTGGAA-3′; MYC forward, 5′-GTCAAGAGGCGAACACACAAC-3′; MYC reverse, 5′-TTGGACGGACAGGATGTATGC-3′; ACTB forward, 5′-CATGTACGTTGCTATCCAGGC-3′; and ACTB reverse, 5′-CTCCTTAATGTCACGCACGAT-3′.The methods for real-time qPCR analysis is reported elsewhere . In brieBriefly, BxPC-3 and SW1990 cells were transfected with si-ctrl and si-GFAT1 in combination with the TOP-Flash and FOP-Flash vectors using Lipofectamine 2000 reagent  for 24 h. After 24 h incubation, luciferase activity was assessed with the Dual-Luciferase Reporter Assay Kit . The intensity values of blank wells were substrated from experimental data, and the presented data were normalized to control wells (with control siRNA).t-test or one-way ANOVA test. Statistical analyses were performed by using SPSS 13.0 statistical package software  or GraphPad Prism . P values of less than 0.05 were considered statistically significant.All the experiments in this study were performed in triplicate and repeated at least 3 times. All data are presented as the means ± SDs. Statistical significance was determined by the two-tailed Student's B4GALNT2, DPAGT1, SLC35A3, GFAT1, MGAT1, AMDHD2, PGM3, NAGK, RENBP, GNPNAT1, GNPDA1, UAP1, UAP1L1, and GFAT2) from the Molecular Signatures Database  was used for determining HBP activity. Combined the data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx), we calculated a HBP score for each sample and found that HBP activity was significantly and specifically increased in diffuse large B-cell lymphoma (DLBC), pancreatic adenocarcinoma (PAAD), and thymoma (THYM) compared with their normal counterparts  or Azaserine (50 μM) also significantly inhibited cell proliferation and invasive potential and triggered cell apoptosis  inhibition by OSMI-1 (β-catenin downstream targets (β-catenin (β-catenin Ser33Y), largely rescued the inhibitory effect of GFAT1 knockdown on cell proliferation and invasion (Figures β-catenin signaling activity through enhancing β-catenin O-GlcNAcylation to promote PDAC cell proliferation and invasion (Using the Hallmark gene sets from MSigDB, we found that HBP gene signature was positively associated with the activity of PI3K/AKT/mTOR and Wnt/ignaling . This re in PDAC . Next, w in PDAC . Surpriscylation . To furtnockdown . UDPGlcNy OSMI-1 . As a re targets . Moreove Figures  of BxPC3invasion .PDAC is a leading cause of cancer-related deaths worldwide with a 5-year survival less than 8% . Like moIt has been well documented that HBP/O-GlcNAcylation process is critical to tumorigenesis . Accumulβ-catenin [β-catenin [β-catenin activity, nuclear accumulation, and subsequent transcription of its target genes [β-catenin to promote cell proliferation [β-catenin suggesting that elevated O-GlcNAcylation contributing to PDAC malignant phenotypes likely acts in part through maintenance of the oncogenic Wnt/β-catenin signaling pathway.It has been reported that the PI3K/Akt pathway stimulates glucose metabolism and provides more UDP-GlcNAc , 28. Con-catenin –33. For -catenin . Many reet genes , 34–36. feration . Here, wβ-catenin O-GlcNAcylation in PDAC cells. Thus, PDAC cells are addicted to increased HBP activity and suppression of the HBP by targeting GFAT1 may serve as a novel therapeutic intervention in PDAC.In conclusion, the present study demonstrated that HBP activity is increased in many human cancers and can act as a prognostic factor. Blockade of GFAT1 function exhibits remarkable antitumor effects via disruption of the HBP flux and


A series of innovative thermosetting polymer nanocomposites comprising of polysiloxane-imide-containing benzoxazine (PSiBZ) as the matrix and double-decker silsesquioxane (DDSQ) epoxy or polyhedral oligomeric silsesquioxane (POSS) epoxy were prepared for improving thermosetting performance. Thermomechanical and dynamic mechanical characterizations indicated that both DDSQ and POSS could effectively lower the coefficient of thermal expansion by up to approximately 34% and considerably increase the storage modulus (up to 183%). Therefore, DDSQ and POSS are promising materials for low-stress encapsulation for electronic packaging applications. The chemical structures of the SiBZ monomer, DDSQ epoxy, and POSS epoxy are displayed in Phenyltrimethoxysilane (97%), dichloromethylsilane (97%), triethylamine (99.5%), isopropyl alcohol (99.5%), and allyl glycidyl ether (99%) were purchased from Sigma-Aldrich . Sodium hydroxide was purchased from UniRegion Bio-Tech . Tetrahydrofuran (95%) was purchased from Fisher Chemical . Toluene (99.5%) was obtained from J.T. Baker . Tris(dibutylsulfide)rhodium trichloride was obtained from Gelest . N,N-dioctadecylmethylamine was supplied by Fluka . Activated charcoal powder was obtained from Showa . Siloxane-imide-containing benzoxazine monomer (SiBZ) was provided by Industrial Technology Research Institute . Octa-functionalized POSS epoxy (70 wt%) in cyclopentanone was purchased from Hybrid Plastics . DDSQ epoxy was prepared with reference to previous literature ,40. The The weight ratios of the prepared DDSQ epoxy/PSiBZ and POSS epoxy/PSiBZ nanocomposites are displayed in 1H-NMR) analysis was performed using an NMR spectrometer  with CDCl3 as the solvent and tetramethylsilane (TMS) as a reference. Differential scanning calorimetry  was conducted under a nitrogen atmosphere. Approximately 10 mg of the testing sample was sealed in an aluminum pan. The samples were heated from 40 to 250 °C at a heating rate of 10 °C/min. The onset temperature, peak temperature, and enthalpy of the exothermic peak of the cured compositions were recorded. Thermal degradation properties were measured using a thermogravimetric analyzer  from 30 to 800 °C at a heating rate of 10 °C/min. Dynamic mechanical analysis  was performed at a heating rate of 5 °C/min from −80 to 280 °C with a fixed frequency of 1 Hz in the single cantilever mode. The dimensions of the test species were 17.5 mm (L) × 10.0 mm (W) × 0.5 mm (T). Thermomechanical analysis  was conducted at a heating rate of 10 °C/min from 30 to 250 °C, and a force of 0.05 N was applied.Proton nuclear magnetic resonance  was 419 °C for the pure PSiBZ and slightly decreased to 413 °C and 410 °C for 20 wt% and 40 wt% DDSQ epoxy/PSiBZ, respectively. The decrease in Td5% should originate from the reacted two epoxy groups in the DDSQ Epoxy/PSiBZ nanocomposites. In the case of the POSS epoxy/PSiBZ nanocomposites, Td5% for 20 wt% and 40 wt% POSS epoxy/PSiBZ was 392 °C and 396 °C, respectively, reasonably due to the presence of eight reacted epoxy groups of POSS with PSiBZ, leading to a lower thermal stability than the DDSQ epoxy/PSiBZ nanocomposites. The char yield of the DDSQ epoxy/PSiBZ and POSS epoxy/PSiBZ nanocomposites also increased with the increase of the DDSQ or POSS. As a result, the DDSQ epoxy/PSiBZ nanocomposites exhibited better thermal stability than the POSS epoxy/PSiBZ nanocomposites.The thermal stability of the pure PSiBZ, the DDSQ epoxy/PSiBZ nanocomposites and the POSS epoxy/PSiBZ nanocomposites was analyzed by TGA under air atmosphere as displayed in The dynamic mechanical properties of the DDSQ epoxy/PSiBZ and POSS epoxy/PSiBZ nanocomposites from −100 to 280 °C were characterized through DMA. The storage modulus and tanδ of the DDSQ epoxy/PSiBZ and POSS epoxy/PSiBZ nanocomposites are presented in Tg from the 157 °C of pure PSiBZ to 155 and 140 °C, respectively. The eight bulky phenyl groups of the DDSQ epoxy disturbed the chain stack to increase larger free volume in the cross-linked structure and thus reduce the Tg [Tg of 226 °C. Notably, Tg becomes not obvious when the POSS epoxy content was 40 wt%. The increased Tg can be attributed to the increased cross-linking caused by the presence of eight epoxy groups of POSS. Furthermore, at 40 wt% POSS epoxy, the apparent nano effect caused by strong interaction between nanoscale POSS epoxy and PSiBZ was found; consequently, Tg became not obvious. Similar nano effect phenomena have been previously observed [The peak temperature from the tanδ curves of the DDSQ/PSiBZ nanocomposites revealed that the addition of 20 and 40 wt% of DDSQ lowered e the Tg . By contobserved ,49.Tg (directly related to the crosslinking density) of the DDSQ/PSiBZ and POSS/PSiBZ nanocomposites networks. In the case of DDSQ/PSiBZ nanocomposites, the storage modulus increased with increasing the DDSQ content due to the rigidity of DDSQ; however, the increased DDSQ simultaneously resulted in a lower Tg, which would lead to the decrease of modules because of the decreased crosslinking density. The very close modulus found from the specimen of the 20 or 40 wt% of DDSQ epoxies could thus be considered as the competition of these two factors. On the other hand, in the POSS/PSiBZ system, both of the rigidity and Tg increase with the increase of the POSS epoxy. The eight epoxy groups in POSS could provide more crosslinking points in the POSS/PSiBZ network. As a consequence, the superior reinforcement effect could be obtained for the POSS/PSiBZ nanocomposites.There are two major factors that led to the reinforcement of the DDSQ/PSiBZ and POSS/PSiBZ nanocomposites: (i) the rigidity of DDSQ and POSS and (ii) the Tg). Tg at a temperature range of 40–75 °C) considerably decreased with an increase in DDSQ content. From 0 to 40 wt% of DDSQ, the decreases in CTE 1 were approximately 34%. Notably, unlike the DDSQ epoxy system, the POSS epoxy/PSiBZ nanocomposites show the estimated changes in CTE 1 were approximately 14% (Tg, the molecules lose energy under the glassy state, only short-range molecular movement occurred, and the larger core dimension and the phenyl substituents at the vertex of the DDSQ frame efficiently restricted the chain mobility of PSiBZ. Thus, noticeable variation was observed in CTE 1. The results indicate that both adding 40 wt% DDSQ epoxy and POSS epoxy demonstrated the significant CTE 1 reduced the effect of PSiBZ; furthermore, a DDSQ epoxy exhibited more of a CTE 1 reduce effect than the POSS epoxy.The CTEs of the DDSQ epoxy/PSiBZ and POSS epoxy/PSiBZ nanocomposites were investigated through TMA and compared with that of pure PSiBZ. tely 14% b. The prIn this study, DDSQ epoxy and POSS epoxy were added to PSiBZ to achieve superior thermomechanical and dynamic mechanical properties. The addition of 40 wt% DDSQ epoxy resulted in a 34% reduction in CTE 1 and 158% increase in the storage modulus. The introduction of the 40 wt% POSS epoxy increased the storage modulus by 183%. This study not only serves as proof of the design concept but also experimentally demonstrated the potential of the DDSQ and POSS epoxy as functional additives for PSiBZ for lowering the CTE while enhancing the thermomechanical properties.


Sufu), a negative regulator of Hh signaling, (SufuOC) and a forced endogenous activation of Smo (SmoM2OC) under the control of osteocalcin (OC) promoter-driven Cre recombinase. Interestingly, cellular cementum apposition was remarkably reduced in both mutants. Consistently, matrix formation and mineralization ability were down-regulated in OCCM-30, a cementoblast cell line, following treatment with a pharmaceutical Smo agonist. In addition, reductions in Osx expression and β-catenin activity, which are critical for cellular cementum formation, were also detected in vitro. Furthermore, the compound mutant mice designed for the stabilization of β-catenin with both Hh-Smo signaling activation in cementoblasts revealed a complete restoration of defective cellular cementum. In addition, Wnt antagonists such as Sostdc1 and Dkk1 were also induced by Smo activation and played a role in the reduction of Osx expression and β-catenin activity. Collectively, our data demonstrated that Hh signaling negatively regulates cementum apposition in a Wnt/β-catenin/Osx-dependent manner.Hedgehog (Hh) signaling plays a broad role in the development of many organs including bone and teeth. It is noted that sustained Hh activity in osteoblasts negatively regulates postnatal development in mice. However, it remains unknown whether Hh signaling contributes to cementum formation. In this study, to define the roles of Hh signaling in cementum formation, we analyzed two kinds of transgenic mouse models for Hh signaling activation designed by the inactivation of Suppressor of Fused ( Developmentally, cementum is formed as a heterogeneous connective tissue that differs with respect to location, structure, function, rate of formation, chemical composition, and degree of mineralization2. Acellular extrinsic fiber cementum is mainly found on the cervical and middle portions of the root. Cellular intrinsic fiber cementum mainly located apically is a unique avascular hard tissue that does not undergo continuous remodeling but continuously grow in thickness throughout life1. It has been known that cementum has the dynamic and highly responsive features critical for maintaining occlusal relationships, the integrity of the root surface, and its function in tooth support1.Cementum covers the tooth root and allows for the attachment of periodontal ligaments between the roots and alveolar bone and plays adaptive roles in supporting the tooth in its proper occlusal position6. Excessive cementum formation occurs in transgenic mice where β-catenin is constitutively stabilized7. β-catenin induces cementoblast differentiation and boosts matrix secretion through a reciprocal interaction with Osx during cellular cementum formation6. It is suggested that a local balance between Osx and PPi is related to the determination of the cementum type in which local PPi suppresses matrix accumulation and further mineralization. We have been reported an antagonistic interaction of PPi with Osx and that their relationship is under the synergistic influence of FGF signaling during cementum formation8.Recently, there has been increasing evidence to support key interactions between Osterix (Osx), β-catenin, and pyrophosphate (PPi) in cementum development and homeostasis. Osx has been reported as an important factor orchestrating overall tooth root formation. Osx regulates odontoblast differentiation, maturation, and root elongation including cementum formation10. The Hh ligand binds to an Hh receptor, patched (Ptc), stopping the suppression of the transmembrane protein, smoothened (Smo), which stabilizes the glioma-associated oncogene (Gli) transcription factor. Interestingly, Hh signaling targets Ptc1 and Gli1 in vertebrates, and the presence of Ptc1 and Gli1 transcripts indicates the functional activation of Hh signaling12. It is well-known that Suppressor of Fused (Sufu) negatively regulates Hh signaling by direct binding to the Gli protein13. When the cells lack Sufu, the Hh signaling pathway is maximally activated in a ligand-independent manner14. However, the regulation system for Hh signaling through Sufu seems having more complicated connection with other signaling. Sufu may act as a dual regulator of Hh and Wnt/β-catenin signals and integrate the multiple pathways during development15. It has been previously reported that Sufu plays a role on the repressive regulation of Hedgehog activity in preosteogenic mesenchyme to ensure osteogenesis in calvarial bone formation16. We have reported that sustained Hh activity in osteoblasts inhibits postnatal bone development by suppressing the gene expression of bone formation regulatory factors in mice17.The roles of Hedgehog (Hh) signaling pathway has been known to regulate the growth and morphogenesis in the development of several organsThe aim of this study was to investigate the developmental regulation of Hh-Smo signaling in cementum formation during tooth development. We investigated the role of Hh-Smo signaling in cementum formation using multiple approaches and demonstrated that the activation of Hh-Smo signaling in cementoblasts leads to defective cementum formation through the inactivation of Osx and β-catenin.Sufu (SufuOC) and constitutively active for Smo (SmoM2OC) in OC-positive dental mesenchyme. We confirmed the Hh gain-of-function in the dental tissue of SufuOC mice through Sufu and Ptc1, a downstream target of Hh signaling, expression by immunohistochemical (IHC) staining   than in the control mice (8.2 μm/day)  controlled under the same OC-Cre recombinase. Different with our expectation, SmoOC mutant mice exhibited normal development of cellular cementum whereas SmoM2OC mutant mice exhibit clear reduction in cellular cementum apposition  Fig.  and H-E 28) Fig. . There i28) Fig. . More drP56 Fig. . FurtherP84 Fig. . To addray) Fig. . To clarGli1, Gli2, and Ptc1, were increased by the treatment of 1 μM SAG while the transcription of Sufu was mildly reduced , collagen 1a2 (Col1a2), OC, and Opn, were reduced by the treatment of SAG in a concentration-dependent manner  staining of the mandibular first molar from control, SmoM2OC, and SufuOC mutant mice revealed that most of the TRAP-positive (TRAP +) osteoclasts were detected at the marginal area of alveolar bone in all three types of mice, while TRAP + osteoclasts around the apical cellular cementum region were barely detected  mice, which have molars with a thicker cellular cementum layer on the root surface including the cervical region7. To test whether constitutively active β-catenin in cementoblasts could restore cellular cementum defects from elevated Hh-Smo signaling in vivo, we generated and analyzed Catnb:SufuOC and Catnb:SmoM2OC mice, which corresponded to forced β-catenin signaling with Hh-Smo signaling activation in cementoblasts. Interestingly, the apical cellular cementum of Catnb:SufuOC and Catnb:SmoM2OC mice at P28 were dramatically restored by constitutively active β-catenin when compared to the corresponding single mutants 18 and analyzed Osx promoter activity. As shown in Fig. Osx promoter activity by β-catenin S33Y and a reduction in Osx promoter activity by SAG treatment. However, the SAG treatment did not inhibit Osx promoter activity in the cells with higher β-catenin activity by β-catenin S33Y transfection. In addition, we treated SAG to OCCM-30 cells after transfection with mouse Osx and β-catenin S33Y. Forced β-catenin activity and Osx expression in OCCM-30 cells restored the mRNA transcript level of Bsp, a marker of cementum, to the level of control when compared with SAG-treated cells  and an inactivation of an antagonistic molecule, Sufu (SufuOC). Thus, we provide genetic evidence that Hh-Smo signaling activation in cementoblasts results in a common defective formation of cellular cementum following down-regulation of matrix formation and mineralization ability. We also show that Hh signaling inhibits cementum apposition by repressing Wnt/β-catenin signaling and Osx expression and that possible downstream mediators of this process are Sostdc1 and Dkk1 26 mice with Osteocalcin (OC)-Cre transgenic mice27. Catnblox(ex3)/+(Catnblox/+) has been previously described28. To generate OC-Cre:Catnblox/+:Sufufl/fl(Catnb:SufuOC) mice, OC-Cre;Sufufl/+ mice were crossed with Catnblox/+:Sufufl/fl mice. To generate OC-Cre:Catnblox/+:SmoM2(Catnb:SmoM2OC) mice, OC-Cre mice were crossed with Catnblox/+:SmoM2 mice. We also analyzed conditionally inactivated allele mice of Smoothened, SmoOC under the same Cre regulation29. The offspring were genotyped by polymerase chain reaction (PCR) analysis using previously described primers. Sufufl/fl or SmoM2 mice were used as controls. At least three independent littermates were used for each experimental group .All procedures were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals. All experimental procedures were approved by the Animal Welfare Committee of Chonbuk National University. All the mice were housed in a temperature-controlled environment with 12 h light/dark cycles. We generated a conditionally regulated mouse by crossing an activated allele mice of Smoothened, p < 0.001).For micro-CT (μCT) analysis, the mandibles were dissected at P28, bisected at the symphysis, and fixed in 4% PFA . The mandibles were scanned using a desktop scanner  and analyzed using the CTAn software (Skyscan). For double-fluorochrome labelling, calcein  was injected twice at P28 and P56. The mice were sacrificed at P58. Fifty-micrometer cross-sections were cut perpendicularly, passing through the midsection of the mandibular first molar, and viewed under a model LSM510 confocal laser scanning microscope . Cellular cementum thickness (μm) was measured as the shortest vertical distances beginning from the fluorochrome-labeled proximal line to another distal line using the analySIS Pro imaging system (Soft Imaging System). Eight start points of the proximal line for measurement were randomly selected within the apical part of the root. For thickness evaluation, the average cementum mineral apposition rate (μm/day) was acquired as the distances divided by the number of days between injections. The experiments were performed three times with representative slides from each group, and the differences of the values were evaluated statistically , β-catenin , Osx , Dmp1 , Axin2 (Abcam), Sufu , Ptc1 (Abcam), and Sostdc1 (Abcam). The HistostainPlus Rabbit Primary (DAB) kit  was used following the manufacturers’ instructions. The average cellular cementum area was calculated usingthree measurements of five representative individual slides in each group at the indicated age using the analySIS Pro imaging system .30. SAG (N-Methyl-Nʹ-(3-pyridinylbenzyl)-Nʹ-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane; Calbiochem, San Diego, CA, USA), a cell-permeable Smo agonist, was used to treat cells under the concentration of 1μM for up to 72 hours, in which cell toxicity was not detected. To induce cell differentiation and mineralization, 95% confluent cells were cultured in osteogenic medium (OM), which consisted of the medium supplemented with 2% fetal bovine serum, 50 μg/ml ascorbic acid (Sigma Aldrich), and 10 mM β-glycerophosphate (Sigma Aldrich), for up to 4 days. Recombinant mouse Sclerostin domain-containing protein 1  and Dickkopf related protein 1  were used to treat cells at the indicated concentrations for 24 or 48 hours in osteogenic medium.OCCM-30, a mouse cementoblast cell line, was provided by Dr. Martha J. Somerman  and cultured as described previouslyp < 0.05 was considered statistically significant. Detailed descriptions for other experimental materials and methods are provided in Supplementary Methods.Data are presented as mean±standard error of the mean (SEM) of three or more separate experiments. Normal data with equal variance were analyzed using Student’s t-test and Supplementary information.


Mechanistically, LZZ-02 degrades the expression of β-catenin by stabilizing axin 2, thereby diminishing downstream proteins levels, including c-Myc and cyclin D1. LZZ-02 also inhibits the growth of colonic carcinoma cell harboring constitutively active β-catenin. More importantly, LZZ-02 effectively shrinks tumor xenograft derived from colonic cell lines. Our study successfully identified a novel tankyrase 1/2 inhibitor and shed light on a novel strategy for developing inhibitors targeting the WNT/β-catenin signaling axis.Aberrant activation of the WNT/β-catenin signaling pathway is implicated in various types of cancers. Inhibitors targeting the Wnt signaling pathway are intensively studied in the current cancer research field, the outcomes of which remain to be determined. In this study, we have attempted to discover novel potent WNT/β-catenin pathway inhibitors through tankyrase 1/2 structure-based virtual screening. After screening more than 13.4 million compounds through molecular docking, we experimentally verified one compound, LZZ-02, as the most potent inhibitor out of 11 structurally representative top hits. LiCl-induced HEK293 cells containing TOPFlash reporter showed that LZZ-02 inhibited the transcriptional activity of β-catenin with an IC The highly conserved WNT/β-catenin signaling pathway plays pivotal roles in the context of development, differentiation and cellular self-renewal ,2,3. Notc-Myc -7,8-dihydro-5H-thiopyrano  pyrimidin-4-ol], were obtained from the Protein Data Bank . Energy minimization was performed by the Powell gradient algorithm with the Tripos force field [DOCK 6.5 directly. For docking with Autodock, compounds were papered with prepare_ligand4.py script in Autodock tools.The XAV939, ABT-888 and LDW6433D structures of these compounds were constructed using the Sketch Molecule module in the ce field  and the ce field . More thce field ,61. ThesDOCK 6.5 [Autodock 4.2 software, respectively. The job was performed on a Dawning A620-F cluster of 16 processors-each 2.6G AMD Opteron at the Gansu Computational Center.The molecular docking program DOCK 6.5  was utilLipofectamine 2000, and dual-specific luciferase assay kit (Promega), ECL Western Blotting Substrate (Millipore), Cell Counting Kit-8 (Dojindo), chemical compounds were purchased from Topscience , other compounds come from the J&K company . SW480, DLD-1, and HEK293 were obtained from ATCC . Mouse antibodies against β-actin (Sigma), GAPDH (Abcam). Rabbit antibodies against c-Myc (Abcam), axin 2 (CST), β-catenin (CST), Cyclin D1 (CST). Super TOPFlash (ST-Luc) .HEK293, DLD-1 and SW480 cells were cultured in DMEM (Dulbecco’s modified Eagle’s Medium)  or RPMI 1640 Medium , supplemented with 10% FBS  . 100 units/mL penicillin, and 100 mg/mL streptomycin and incubated at 37 °C in a 5% COHEK293 cells were transfected by the standard calcium phosphate precipitation method, and DLD1 cells were transfected with lipofectamine 2000 according to manufacturer’s instruction. An empty amount of vector plasmid was served as control in each transfection. To normalize for transfection efficiency, 0.1 μg Renilla luciferase reporter plasmid was added to each transfection. Luciferase assays were performed using a dual-specific luciferase assay kit.3 per well. Cell proliferation was evaluated using the Cell Counting Kit-8 according to the manufacturer’s instructions. Briefly, 10 µL of the CCK-8 solution were added to culture medium and incubated for 2 h, and the absorbance at a 450 nm wave length was determined.Cells were seeded in 96-well plates at 1 × 10Cells were seeded in 6-well plates at 200 or 1000 per well and maintained in a medium containing 0.5% FBS. Sixteen hours later, LZZ-02 were added at the indicated concentrations. The medium was replenished every two days until colony formation was observed. The cells were then washed twice with PBS, fixed with cold methanol, and stained with 0.5% crystal violet.The cells were washed twice with ice-cold PBS and then lysed with ice-cold RIPA lysis buffer. Lysates were kept on ice for 30 min and then centrifuged at 13,000 rpm for 5 min at 4 °C. The supernatant was subjected to 10% SDS/PAGE and followed by immunoblot analysis with the indicated antibodies.6) suspended in a 100 μL mixture of equal volumes of PBS and Matrigel were implanted subcutaneously into the flank of 6-week-old female BALB/c nude mice. When the tumors had reached a volume of about 80 mm3, the 12 mice were then randomly divided into two groups. Animals received the compound by oral gavage, whereas the control group received vehicle solution. LZZ-02 were orally gavaged at 30 mg/kg once daily for 18 consecutive days. Tumor volumes and the body weight of animals were measured every three days. Tumor volume (tumor volume (cm3) = D × d2/2, where D is the longest and d is the shortest diameter, respectively) were monitored once every three days after 18 days up to the end of the experiment.All mice were housed in a pathogen-free environment in Jinan University. All experimental protocols were approved by the Institutional Committee for Animal Care and Use at Jinan University. All animal work was performed in strict accordance with the approved protocol. DLD1 cells (2.5 × 10p < 0.05 was considered statistically significant.Statistics were performed using GraphPad Prism 7.04, and the student t-test was used to compare differences between the two experimental groups. The data are presented as means ± SD and In conclusion, tankyrase 1/2 structure-based virtual screening was performed successfully to identify inhibitors of the WNT/β-catenin pathway from the ZINC Database. In total, 11 compounds were selected to test their in vitro inhibitory activities against the WNT/β-catenin pathway. Among them, LZZ-02 showed the most significant inhibitory potency against LiCl-, Wnt3a- or β-catenin-induced TOPFlash activity in HEK293 cells. Meanwhile, LZZ-02 showed remarkable inhibition of TOPFlash activity in DLD-1 cells, which express constitutively activated β-catenin. Furthermore, LZZ-02 stabilized axin 2 protein level and restrain β-catenin, c-Myc and Cyclin D1 expression in DLD1 and SW480 cells. LZZ-02 also inhibits the colonic cancer cells’ viability and colony formation. In the whole process of treatment, the mice were active, LZZ-02 showed antitumor function in vivo and the bodyweight of mice remains constant after treated. LZZ-02 is therefore worthy of further characterization in clinic. Equally important, LZZ-02 could serve as a lead candidate for developing highly potent tankyrase inhibitors, but the precise mechanism requires more investigation.


These demonstrate CBP blockage can be developed for the treatment of CML independent of BCR-ABL mutation status including T315I.The treatment of chronic myeloid leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKIs), such as imatinib, has yielded clinical success. However, the direct targeting of BCR-ABL does not eradicate CML cells expressing mutant BCR-ABL, especially the T315I mutation in BCR-ABL. Moreover, increasing mutations were identified in BCR-ABL domain, resulting in TKIs resistance recently. It is necessary to find BCR-ABL-independent target for treating CML patients with various mutations, including T315I mutation in BCR-ABL. The dichotomous behavior of CREB binding protein (CBP) and E1A protein (p300), recruited by β-catenin associated with self-renewal and differentiation, have been identified in hematopoietic stem cells, respectively. In this study, CBP was aberrantly expressed in CML cells on the basis of Oncomine dataset. The β-catenin bound with much more CBP than p300 in CML cells. Down-regulation of CBP inhibited cell proliferation capacity and increased the binding of β-catenin to p300, thus promoting cell differentiation and p53-dependent cell senescence in CML cells with either wild type or T315I mutant BCR-ABL  Chronic myeloid leukemia (CML) is a clonal malignant disorder of pluripotent hematopoietic stem cells (HSCs), characterized by the presence of Philadelphia chromosome (Ph) translocation t9;22)q34;q11), leading to the formation of the novel BCR-ABL fusion gene Despite the high degree of homology and similar patterns of expression, CBP and p300 play unique and distinct roles in gene regulation , 9. Distin vitro and in vivo models.In the current study, more p300 is bound to beta-catenin when CBP is knock-down. Down-regulation of CBP increased the binding of β-catenin to p300, thus promoting cell differentiation and p53-dependent cell senescence and inhibiting cell proliferation capacity in CML cells with either wild type or T315I mutant BCR-ABL Nitrobluetetrazolium (NBT) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich . Antibodies against β-catenin (#8480), p21(#2947S), phosphorylated Rb (#9307S), and CyclinD1 (#2922S) were purchased from Cell Signaling Technology . CBP (sc-369), p300 (sc-584), JUN (sc-45) were purchased from Santa Cruz Biotechnology Inc. . p16 (ab117443) and recombinant mouse Wnt3a protein was purchased from Abcam . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (822051) was purchased from Kangchen Co. . Cell protein Extraction Kit and Nucleoprotein Extraction Kit was purchased from Beyotime . The senescence detection kit was purchased from Beyotime .The human CML cell line K562 was obtained from ATCC and the murine 32D myeloid cells stably expressing either wild-type BCR-ABL (32D-WT) or T315I mutant BCR-ABL (32D-T315I) were obtained from Professor Pan JX . BCR-ABL expression in these cells was checked by Western-blot for verification every 3 to 4 months routinely. Cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum , 1 unit/ml penicillin and 1 mg/ml streptomycin at 37°C and 5% CO2, 32D-WT, and 32D-T315I cells were additionally supplemented with 0.5 ng/ml WNT3a to imitate the feature of activated Wnt signaling in CML cells.For depletion of CBP and p300, the pSIH-H1-puro-construct  containing CBP or p300 short hairpin RNAs (shRNA) was generated by cloning the following CBP specific or p300 specific RNAi target sequences into pSIHF-H1:CBP shRNA: 5-GATCCCGTTTACCATGAGATCCTTATCTTCCTGTCAGAATAAGGATCTCATGGTAAACGTTTTTG-3’.P300 shRNA:5’- GATCCTAACCAATGGTGGTGATATTACTTCCTGTCAGATAATATCACCACCATTGGTTATTTTTG-3’.Briefly, CMLs silencing CBP or p300 were generated by infection with CBP shRNA lentiviruses or p300 shRNA lentiviruses, and selected by treatment with 0.5 μg/ml puromycin for 10 days, beginning from 48 h after infection.K562, 32D-WT, and 32D-T315I cells receiving different treatment were cultured in 96-well plates for 72 h. The NBT reduction assay was performed as Collins’description .For the SA-ß–galactosidase assay, the SA-ß-galactosidase activity was determined using a senescence detection kit (Beyotime) according to the manufacturer’s instructions. Senescent cells were identified as blue-stained cells under an Olympus IX71S8F inverted microscope.Cell specimens were washed twice with PBS and total cellular protein was extracted with RIPA. Nuclear proteins were extracted by nucleoprotein extraction buffer and were used when detecting expression of ß-catenin. Equal amounts of protein (50–100 ug) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to PVDF membranes, blocked and incubated overnight with primary antibodies at 4°C. After the secondary monoclonal antibodywas applied to visualize proteins using Kodak X-AR film, the results were analyzed by Gel-Pro Analyzer.3) was calculated as L×W2×0.5 (L indicates the length-diameter and W the width-diameter of the tumor) . To test the incidence of tumors and proliferation ability, 32D-WT or 32D-T315I cells with CBP or p300 knockdown were subcutaneously injected into left subaxillary of mice, respectively. Tumor volume (mme tumor) .via boiling and centrifugation. The immnoprecipitated proteins were then analyzed by standard immune blotting , and the supernatant of cell lysate corresponding to1mg of total protein was precleared by Protein G Plus/Protein A-agarose beads  to minimize nonspecific binding, followed by the addition of either mouse IgG  or β-catenin overnight. The bound proteins were washed thrice with lysis buffer and dissociated with the beads blotting .p<0.05 was considered statistically significant.Data analysis was performed using the SPSS 16.0 software package. All experiments were repeated at least three times, and data are presented as Means ± SEM. The mean values were compared using unpaired student’s t-test (two groups). A value of www.researchdata.org.cn), with the approval RDD number as RDDB2020000942.The authenticity of this article has been validated by uploading the key raw data onto the Research Data Deposit public platform (p=0.002) and β-catenin (p=0.001) expression was significantly up-regulated in 76 cases of CML samples compared with 74 cases of normal samples (p=0.900), suggesting that β-catenin/CBP is preferentially expressed in malignant proliferative CMLs. To further evaluate the significance of CBP and p300 on cell growth or differentiation in CMLs, we silenced endogenous CBP and p300 using lentiviral short -hairpin RNA (shRNA) in K562, 32D-WT, and 32D-T315Icells, respectively  showed that CBP  was performed and indicated that the down-regulated expression of CBP increased the binding of β-catenin to p300 in both BCR-ABL wild-type and T315I mutant CMLs , has been successful in 5%–10% AML patients but not in CML patients . Under tWnt signaling pathways regulate a variety of processes including cell growth, oncogenesis, and development. Upon Wnt signaling, β-catenin accumulates in the nucleus and binds to transcription factors of the TCF/LEF family to activate transcription and constitutive activation of downstream target genes, such as surviving, c-myc and cyclinD1 by the TCF/LEF-β-catenin complex, which is essential for balancing cell survival and differentiation , 23, 24.Our results indicate that β-catenin recruits more p300 after CBP expression is silenced in CMLs expressing wild-type and T315I mutant BCR-ABL. The repression of CBP decreased the expression of cyclinD1 , which ivia a switch to β-catenin binding to p300 by targeting CBP, and to ultimately eliminate CMLs, including expressing T315I mutant BCR-ABL CMLs.Cellular senescence disables proliferation in damaged cells and is relevant for cancer and aging. This involvement of tumor-suppressor pathways led to the identification of senescence as potent tumor-suppressor mechanism. Several studies have revealed that, adult stem cells could escape cellular senescence by avoiding both differentiation and the activation of checkpoint responses that arrest the cell cycle in cell homeostasis. The key proteins that induce senescence are members of the p53/p21 and p16/p-Rb tumor suppressor networks that function to arrest proliferation and contribute to the irreversibility of the senescent state. In our study, we used SA-β-Gal staining to identify senescent cells because it is a proven and reliable marker of senescence in many settings. To determine whether p53/p21 or p16/p-Rb were required for β-catenin/p300 mediated senescence, we examined these markers and mediators of senescence. The results indicated that the increased β-catenin/p300 mainly activated p53/p21 signaling mediated cell senescence. This offers a possibility to promote cell differentiation and senescence and limit proliferation These results suggest the binding of β-catenin to CBP or p300 decides CML cell fate, proliferation or differentiation, which is independent of BCR-ABL mutation status. The inhibition of CBP could be a promising strategy for the treatment of CMLs including BCR-ABLT315I mutant CMLs Figure 5The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Sun Yat-Sen University.in vitro experiments and analyzed data. XKW, LC, XS, XPW, and QH performed the animal experiments. ZC analyzed data and provided technical assistance. KY wrote the paper. LF and Z-SC provided technical assistance and revised the manuscript. All authors contributed to the article and approved the submitted version.LF designed the research. KY, FW, and HZ performed the This work was financially supported by the National Science & Technology Major Project Key New Drug Creation and Manufacturing Program, China (2018ZX09711002), the Natural Scientific Foundation of China , the Guangdong Provincial Special Fund for Marine Economic Development Project (GDNRC [2020]042), the Science and Technology Foundation of Guangdong Province , and the Scientific and Technological Leading Talent Project of Guangdong Province . Author QH was employed by Guangzhou Handy Biotechnological Co., Ltd.The remaining 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.


Glycine max (L.) Merr.] requires a large amount of P during growth and development. Improvement of P efficiency and identification of P efficiency genes are important strategies for increasing soybean yield.Phosphorus (P) is an essential element in maintaining high biomass and yield in crops. Soybean [Glyma.10 g018800, which contained SPX and RING domains and was named as GmSPX-RING1. Furthermore, the function research of GmSPX-RING1 was carried out in soybean hairy root transformation. Compared with their respective controls, P concentration in GmSPX-RING1 overexpressing transgenic hairy roots was significantly reduced by 32.75% under +P condition; In contrast, P concentration in RNA interference of GmSPX-RING1 transgenic hairy roots was increased by 38.90 and 14.51% under +P and -P conditions, respectively.Genome-wide association analysis (GWAS) with NJAU 355 K SoySNP array was performed to identify single nucleotide polymorphisms (SNPs) significantly associated with three shoot P efficiency-related traits of a natural population of 211 cultivated soybeans and relative values of these traits under normal P (+P) condition and P deficiency (−P) condition. A total of 155 SNPs were identified significantly associated with P efficiency-related traits. SNPs that were significantly associated with shoot dry weight formed a SNP cluster on chromosome 11, while SNPs that were significantly associated with shoot P concentration formed a SNP cluster on chromosome 10. Thirteen haplotypes were identified based on 12 SNPs, and Hap9 was considered as the optimal haplotype. Four SNPs  located on chromosome 10 were identified to be significantly associated with shoot P concentration under +P condition in two hydroponic experiments. Among these four SNPs, two of them (AX-93636685 and AX-93932874) were also significantly associated with the relative values of shoot P concentration under two P conditions. One SNP AX-93932874 was detected within 5′-untranslated region of GmSPX-RING1 affects soybean phosphorus efficiency by negatively regulating soybean phosphorus concentration in soybean hairy roots. The SNPs and candidate genes identified should be potential for improvement of P efficiency in future soybean breeding programs.This study shows that the candidate gene  Glycine max (L.) Merr.]. From 1961 to 2013, the total amount of agricultural phosphate fertilizer used in the world increased from 4.6 to 17.5 million tons per year. And the use of phosphate fertilizer per unit cropland area increased by about 3 times in the same period [2PO4− and HPO42−) can be directly absorbed and utilized by plants [Phosphorus (P) is one of the essential mineral elements in plant growth and reproduction  and it iArabidopsis  [Oryza sativa) [Glycine max (L.) Merr.]  , respect) Merr.] . The SPX) Merr.] .Nitrogen-Limited Adaptation (NLA), has been identified in Arabidopsis. Arabidopsis nla mutant was identified to be unable to accumulate anthocyanin and showed accelerated senescence compared with WT in the absence of nitrogen  [The values of three P efficiency-related traits of 211 soybean accessions under +P and -P conditions and the relative values of two traits were used for genome-wide association analysis (GWAS). The manhattan and quantile-quantile (QQ) plots for the GWAS results were shown in Fig. Sat_251) . Twelve att487)] . Among tn = 112) formed the largest group, Hap9 (n = 31) was the second largest group, Hap5 (n = 20) was the third largest group, Hap2 (n = 18) was the fourth largest group and the other nine haplotype classes constituted minor groups. The soybeans with Hap5 or Hap9 had significantly higher SP under +P condition, compared with that of Hap1 or Hap2; and the SP of soybean with Hap9 was significantly higher than that of Hap5  was detected in 5′-untranslated region of Glyma.10 g018800. Therefore, Glyma.10 g018200 and Glyma.10 g018800 were selected for further functional analysis.According to the calculation of the linkage disequilibrium distance , genes wGlyma.10 g018200 and Glyma.10 g018800) were affected by low P stress, the relative expression levels were examined in soybean accessions Kefeng No. 1 and Nannong 1138–2  in Arabidopsis , common bean (Phaseolus vulgaris), alfalfa (Medicago truncatula), grape (Vitis vinifera) and rice (Oryza sativa) to find homologous proteins with E values less than 10− 100. Phylogenetic tree was drawn after multiple sequence alignments of homologous proteins in six species  of https://www.ncbi.nlm.nih.gov/), it was found that At1g02860 named AtNLA in Arabidopsis and LOC_Os07g47590 named OsNLA1 in rice had been studied. AtNLA and OsNLA1 were involved in maintenance of phosphate homeostasis  accessions with diverse geographic origins were collected , and the other three replicates were grown in one-half Hoagland’s solution supplemented with 0.005 mM KH2PO4 . Under -P condition, KH2PO4 was replaced by equal concentration of KCl. One-half Hoagland’s solution was composed of macroelements  and microelements . The pH of the nutrient solution was adjusted to 5.8–6.0. They were grown in a green house with a 12 h/12 h photoperiod and a temperature of 30–32 °C/22 °C (day/night). The 20 L hydroponic tanks  were used and for each tank a supporting plate containing 59 holes was used to plant soybean seedlings. A randomized blocks design was employed. Nutrient solution was changed every three days. The shoots (cotyledon node to the top growth point) were sampled at 15d after transfer. Two independent hydroponic experiments were conducted: The first experiment (E1) was from April to June, 2015, and the second experiment (E2) was from May to July, 2016.The hydroponic culture experiments were conducted in a greenhouse at Nanjing Agricultural University . Seeds were sterilized in Cl3 was added to the samples (about 0.1 g) and the samples were digested with a microwave digestion system (Milestone Ethos) for 25 min. After cooling, the digested sample was transferred to a volumetric flask to a volume of 25 mL. And 10 mL from the 25 mL sample was transferred to the tube and stored at − 20 °C for further measurement. The shoot P concentration (SP) of the samples was measured using Perkin Elmer Optima 8000 ICP-OES system. Shoot P concentration (SP) under +P condition was abbreviated as SPP; shoot P concentration (SP) under -P condition was abbreviated as SPM; the ratio of SP under -P condition to +P condition was abbreviated as SPR. The total P content in shoot was named as shoot P accumulation (SPA) = shoot dry weight (SDW) × shoot P concentration (SP); shoot P accumulation (SPA) under +P condition was abbreviated as SPAP; shoot P accumulation (SPA) under -P condition was abbreviated as SPAM; the ratio of SPA under -P condition to +P condition was abbreviated as SPAR.The samples (shoots of 211 soybean accessions) were oven-dried at 105 °C for 1 h and then at 65 °C to constant weight to weigh shoot dry weight (SDW) . Shoot dh2) was defined by Shi et al. . The LD decay rate of these 211 cultivated soybeans was 130 Kb , common bean (Phaseolus vulgaris), alfalfa (Medicago truncatula), grape (Vitis vinifera) and rice (Oryza sativa), at Phytozome v12.1 website . Eleven gene structures in six species were predicted by GSDS (http://gsds.gao-lab.org/index.php). The phylogenetic tree was drawn using MEGA 5.02 by the neighbor-joining method with bootstrap probabilities based on 1000 replicates. Nucleic acid sequence and amino acid sequence alignment was done with DNAman software. Prediction of gene conserved domains was performed online using NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The molecular weight and pI of candidate gene GmSPX-RING1 was predicted by ExPASY (https://web.expasy.org/compute_pi/). Sequence analysis was performed using online program PLACE (http://www.dna.affrc.go.jp/PLACE/).Using the full-length protein sequence of the candidate gene GmSPX-RING1 in different tissues of soybean, the roots, stems, leaves, flowers, pods and seeds of Nannong 1138–2 were sampled, and each tissue included three biological replicates. The sampling time of roots, stems and leaves was V4 stage of soybean vegetative growth, the sampling time of flower was R2 stage of soybean reproductive growth, and the sampling time of pods and seeds was 45 days after flowering.To analyze the expression pattern of GmSPX-RING1 was amplified from cDNA of Nannong 1138–2, and cloned into pSuper1300 vector with GFP. The recombinant plasmid GmSPX-RING1-pSuper1300 and empty control were expressed in tobacco (Nicotiana benthamiana) leaves by agrobacterium-mediated infiltration (strain EHA105) as described previously [The coding sequence (without terminate codon) of eviously . The GFPGmSPX-RING1 was cloned from cDNA of Nannong 1138–2, a P low efficiency soybean accession, and the verified PCR product was cloned into pMDC83 with the CaMV 35S promoter using double restriction enzyme digestion and recombination to obtain GmSPX-RING1 overexpression vector (GmSPX-RING1-OE). A specific 405-bp fragment of GmSPX-RING1 was amplified from GmSPX-RING1-OE, which cloned into pB7GWIWG2 (II) vector to obtain RNA interference of GmSPX-RING1 vector (GmSPX-RING1-RNAi) using Gateway technology with the Clonase II Kit . The coding sequences of GmSPX-RING1 in three soybean accessions, Jack, Kefeng No. 1 and Nannong 1138–2, were amplified and found to have only one SNP (C/A) with no difference in amino acid sequence i.e., is a synonymous mutation  were independently transformed into Agrobacterium rhizogenes strain K599 for soybean hairy root transformation. Jack is a soybean accession with high transformation efficiency. Soybean hairy root transformation was conducted using Jack as described previously [GmSPX-RING1-OE, GmSPX-RING1-RNAi and their corresponding empty plasmids. The photoperiod and temperature were consistent with the hydroponic conditions of the natural population, but soybean hairy root required high humidity about 2–3 weeks. When the soybean hairy roots were about 2–10 cm in length, the primary roots were cut off. Then, soybean lines without primary roots after injected with GmSPX-RING1-OE, GmSPX-RING1-RNAi or its corresponding empty plasmid were transferred to +P or -P nutrient solution to culture for 15 days, each of which had 10–14 lines. Both pMDC83 and pB7GWIWG2 (II) vectors contained independently expressed green fluorescent protein (GFP). The GFP was observed by Luyor 3415 RG excitation source to screen soybean positive hairy root. After positive screening, there were fewer hairy roots. In order to ensure sufficient samples for further experiments, two soybean hairy root lines were combined into a biological replicate. Screened transgenic positive hairy roots were used for the measurement of GmSPX-RING1 expression and P concentration.eviously . Five-da-ΔΔCt, ΔΔCt =  genotype- calibrator [The total RNA was isolated using RNAsimple Total RNA Kit . The first strand of cDNA was synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser  . qRT-PCR was conducted with the ABI 7500 system . The 20 μL reaction system consists of 10 μL 2 × RealUniversal PreMix , 50 ng first-strand cDNAs and 0.6 μL each of 10 μM gene-specific primers. Data was analyzed using ABI 7500 system Sequence Detection System (SDS) software v.1.4. The relative expression was calculated as 2librator . TubulinAdditional file 1: Figure S1. Box plot of three P-efficiency related traits in natural population in two independent hydroponic experiments. SDWP: shoot dry weight under +P condition, SDWM: shoot dry weight under -P condition, SDWR: the ratio of shoot dry weight under -P condition to +P condition; SPP: shoot P concentration under +P condition, SPM: shoot P concentration under -P condition, SPR: the ratio of shoot P concentration under -P condition to +P condition; SPAP: shoot P accumulation under +P condition, SPAM: shoot P accumulation under -P condition, SPAR: the ratio of shoot P accumulation under -P condition to +P condition. E1/E2: first/second independent hydroponic culture.Additional file 2: Table S1. Correlation coefficients of three P-efficiency traits in natural population under two P conditions. SDW: shoot dry weight; SP: shoot P concentration; SPA: shoot P accumulation. The correlation coefficients of natural populations under +P (0.5 mM KH2PO4) and -P (0.005 mM KH2PO4) conditions were listed in the upper right and lower left of the table, respectively. ns: not significant; *, **and *** significant at 0.05, 0.01 and 0.001 probability levels, respectively.Additional file 3: Figure S2. Quantile-quantile (QQ) plots of three P efficiency related traits. SDWP: shoot dry weight under +P condition, SDWM: shoot dry weight under -P condition; SPP: shoot P concentration under +P condition, SPM: shoot P concentration under -P condition; SPAP: shoot P accumulation under +P condition, SPAM: shoot P accumulation under -P condition. E1/E2: first/second independent hydroponic culture. QQ plot of the same P efficiency traits in E1, E2 and mean of E1 and E2 were all in the same figure, dark green line represented four P-efficiency traits in E1, darkblue line represented four P-efficiency traits in E2, and green line represented four P-efficiency traits in mean of E1 and E2.Additional file 4: Figure S3. Manhattan and QQ plots of relative values of two P efficiency related traits. SPR: the ratio of shoot P concentration under -P condition to +P condition; SPAR: the ratio of shoot P accumulation under -P condition to +P condition. The red line indicated the significance threshold (−log 10 (P) =5.32).Additional file 5: Table S2. Details of SNPs significantly associated with two P efficiency-related traits in only one hydroponic experiments. SDWP: shoot dry weight under +P condition, SDWM: shoot dry weight under -P condition, SDWR: the ratio of shoot dry weight under -P condition to +P condition; SPP: shoot P concentration under +P condition, SPM: shoot P concentration under -P condition, SPR: the ratio of shoot P concentration under -P condition to +P condition; SPAP: shoot P accumulation under +P condition, SPAM: shoot P accumulation under -P condition, SPAR: the ratio of shoot P accumulation under -P condition to +P condition. Chr.: chromosome; P value: significant at P ≤ 4.82 × 10− 6; R2: percentage of phenotypic variation explained by the SNP; Exp.: experiment; E1/E2: first/second independent hydroponic culture. Mean: average of the two experiments.Additional file 6: Figure S4. Amino acid sequence alignment of homologous genes of Glyma.10 g018800 and conserved domain analysis of Glyma.10 g018800. (a) Amino acid sequence alignment of Glyma.10 g018800 in Arabidopsis, rice and soybean. (b) Conserved domain analysis of Glyma.10 g018800. Red line (2–138) stands for SPX domain, and green line (210–261) stands for RING domain.Additional file 7: Figure S5. Expression pattern of GmSPX-RING1 in different tissues of soybean. Data were the mean values of biological replicates mean ± standard deviation (SD) (n = 3). Statistical significance was detected by a two-tailed t-test. * and ** significant at 0.05 and 0.01 probability levels, respectively.Additional file 8: Figure S6. Positive screening of soybean transgenic hairy roots by green fluorescence. GmSPX-RING1-OE: soybean transgenic hairy roots with GmSPX-RING1 overexpression vector, Control 1: soybean transgenic hairy roots with overexpression empty vector; GmSPX-RING1-RNAi: soybean transgenic hairy roots with RNA interference of GmSPX-RING1 vector, Control 2: soybean transgenic hairy roots with RNA interference empty vector. Negative Control: normal soybean roots without genetic transformation.Additional file 9: Figure S7. Fresh weight of GmSPX-RING1 transgenic hairy roots. (a) Fresh weight of GmSPX-RING1-OE transgenic hairy roots and Control 1 under +P and -P conditions. (b) Fresh weight of GmSPX-RING1-RNAi transgenic hairy roots and Control 2 under +P and -P conditions. GmSPX-RING1-OE: soybean transgenic hairy roots with GmSPX-RING1 overexpression vector, Control 1: soybean transgenic hairy roots with overexpression empty vector; GmSPX-RING1-RNAi: soybean transgenic hairy roots with RNA interference of GmSPX-RING1 vector, Control 2: soybean transgenic hairy roots with RNA interference empty vector. Data were the mean values of biological replicates mean ± standard deviation (SD) (n = 3). Statistical significance was detected by a two-tailed t-test. * and ** significant at 0.05 and 0.01 probability levels, respectively.Additional file 10: Table S3. Geographic origins of 211 soybean accessions.Additional file 11: Table S4. Primers used in this study.Additional file 12: Figure S8. The coding sequence and amino acid sequence alignment of GmSPX-RING1 in three soybean accessions. (a) Nucleic acid sequence of GmSPX-RING1 in Jack, Kefeng No. 1 and Nannong 1138–2. (b) Amino acid sequence of GmSPX-RING1 in Jack, Kefeng No. 1 and Nannong 1138–2.


A new type of protein nanostructure as a chemodynamic therapy nanoagent was fabricated via proper assembling and crosslinking techniques.The as-fabricated nanostructures could cross the blood–brain barrier, possessing excellent long circulating times and accumulation properties at the tumor site.The RBC@Hb@GOx NPs can be regarded as ‘pure particles of drugs,’ which can produce toxic reactive oxygen species to inhibit the growth of orthotopic brain tumor.The online version of this article (10.1007/s40820-020-00490-6) contains supplementary material, which is available to authorized users. Glioblastoma (GBM) remains a formidable challenge in oncology. Chemodynamic therapy (CDT) that triggers tumor cell death by reactive oxygen species (ROS) could open up a new door for GBM treatment. Herein, we report a novel CDT nanoagent. Hemoglobin (Hb) and glucose oxidase (GOx) were employed as powerful CDT catalysts. Instead of encapsulating the proteins in drug delivery nanocarriers, we formulate multimeric superstructures as self-delivery entities by crosslinking techniques. Red blood cell (RBC) membranes are camouflaged on the protein superstructures to promote the delivery across blood–brain barrier. The as-prepared RBC@Hb@GOx nanoparticles (NPs) offer superior biocompatibility, simplified structure, and high accumulation at the tumor site. We successfully demonstrated that the NPs could efficiently produce toxic ROS to kill U87MG cancer cells in vitro and inhibit the growth of GBM tumor in vivo, suggesting that the new CDT nanoagent holds great promise for treating GBM.The online version of this article (10.1007/s40820-020-00490-6) contains supplementary material, which is available to authorized users. Glioblastoma multiforme (GBM) is the most malignant cancer in our central nervous system (CNS) . It has 2O2) into highly cytotoxic hydroxyl radial (·OH), triggering the death of cancer cells through the apoptosis or necrosis processes [Recently, chemodynamic therapy (CDT) has emerged as a new reactive oxygen species (ROS)-mediated therapeutic strategy. It employs Fenton-type reactions to convert intracellular hydrogen peroxide  , 27. The2+, Cu2+, Mn2+, and Co2+. Examples include Hb [2O2, such as GOx [In this study, to tackle the major obstacles in CDT, we constructed a new type of CDT nanoagent Fig. a. The poclude Hb  nitrite clude Hb  and ceruclude Hb . In addih as GOx  and NADPh as GOx . In thisTo co-deliver the two proteins-Hb and GOx, instead of loading them into the nanocarriers, we proposed a new concept of ‘self-delivery.’ With proper assembling and crosslinking techniques, the two protein monomers were formulated into multimeric protein superstructures (denoted as Hb@GOx NPs), thus eliminating the need of the nanocarrier component in conventional drug delivery systems. The protein superstructures present a unique way to deliver multiple proteins. They can be regarded as ‘pure particles of drugs,’ which greatly simplifies the structure and the synthesis steps and offers extremely high drug loading yields. In addition, the stability of the protein nanoparticles is much more enhanced compared to free proteins loosely encapsulated in the carriers.To facilitate the delivery of Hb@GOx NPs to the GBM microenvironment, we employed a passive targeting strategy to cross BBB/BBTB. It has been shown that for disorders such as GBM, the integrity of the BBB is partially compromised. Hence, passive targeting is possible if the size of NPs is sufficiently small and the circulation time is long. We camouflaged the protein superstructures with red blood cell (RBC) membranes (denoted as RBC@Hb@GOx NPs). The CD 47 protein and glycosyl groups on the membrane surface endow the nanoparticles with prolonged systematic retention time, less reticuloendothelial system uptake, and reduced immuno-recognition , 40. Com2O2 causes cell membrane destruction, which increases the access of Hb@GOx protein superstructures. The two electron oxidation of Fe(II)–Hb by H2O2 produces the Fe(IV)-ferrylHb that can react with an additional molecule of H2O2, resulting in the degradation of heme groups and the release of free ions. The ions catalyze Fenton reactions, converting H2O2 to toxic ·OH and O2·−  and GOx (15 mg) powder were added to MQ water (6 mL) and stirred overnight at 500 rpm using a magnetic stirrer for complete hydration. Afterward, 10 µL of 2% glutaraldehyde solution was mixed to induce intra-particle crosslinking. The solution was stirred continuously at 500 rpm at room temperature for 24 h.Hb powder (25 mg), GOx (15 mg), and 6 mg ICG powder (6 mg) were added to MQ water (6 mL) and then were stirred overnight at 500 rpm using a magnetic stirrer for complete hydration. The following process was the same as mentioned above.Briefly, the blood was taken from the cattle and diluted by PBS (pH 7.4). After centrifuging for 5 min , the collected RBCs were washed 3 times (pH 7.4), re-dispersed in the water and placed in the ice bath for 50 min. Finally, the solution was centrifuged  and then washed with PBS (pH 7.4) until the supernatant became colorless. The obtained RBCs membrane was re-dispersed in PBS (pH 7.4), sonicated, and filtered through a 0.45 μm and 0.22 μm filter before use.100 µL of RBC membrane was added to 400 µL of MQ water. The solution was sonicated for 10 s before being mixed with 500 µL of Hb@GOx NPs or ICG-loaded Hb@GOx NPs. The mixture solution was sonicated for 60 s to complete the membrane coating. The excess RBC membrane was removed by centrifugation, and RBCs@Hb@GOx NPs or ICG-RBCs@Hb@GOx NPs were re-dispersed in MQ water for further use.2O2 generates a red fluorescent product (λex = 540/λem = 590 nm). At each time point , the production of H2O2 was calculated according to the fluorescence standard curve obtained from a fluorescent microplate reader. In addition, the UV–Vis absorbance of the mixed solution at 425 nm was measured to further investigate the degradation of RBCs@Hb@GOx NPs at room temperature.The catalytic performance of RBCs@Hb@GOx NPs in the presence of glucose (10 mM) was conducted using fluorimetric hydrogen peroxide assay kit. The produced H2O2 solution was set as the control group.The 1,3-diphenylisobenzonfuran , as a hydroxyl radical indicator, was applied to investigate the generation of hydroxyl radicals via measuring the absorption intensity at wavelength of 410 nm in UV–Vis absorption spectrum. Firstly, the test solution (RBCs@Hb@GOx NPs mixed with glucose) was prepared, and samples were collected at reaction time points of 0, 10, 15, 20, and 25 min. After that, DPBF (100 μL) in methanol (20 mM) was added into the sample solutions (100 μL). The absorption intensity of DPBF was recorded. At same time, the group of DPBF solution treated with Hb plus H−1) in PBS, Hb@GOx NPs, and RBCs@Hb@GOx NPs mixed with glucose (10 mM) or without glucose (as control group) were prepared. ESR spectra were recorded after incubation of 6 min. For detecting O2·−, BMPO  in PBS, Hb@GOx NPs and RBCs@Hb@GOx NPs mixed with glucose (10 mM) or without glucose (as control group) were prepared. ESR spectra were recorded after incubation of 6 min.For detecting ·OH detection, BMPO . All cell lines were stored in a 100% humidity atmosphere of 37 °C and 5% CO−1) at 100% humidity atmosphere at 37 °C and 5% CO2 for 24 h. Then, 20 μL of MTT solution was added into each well, placed in CO2 incubator for further incubation for 4 h. DMSO (150 μL) was added, and the absorbance at 490 nm was obtained to calculate cell survival rate by using MTT assay kit.MTT assay was applied to evaluate the cytotoxicity of RBCs@Hb@GOx NPs. Firstly, NIH3T3 or U87MG cells were randomly seeded into 96-well plates and incubated with RBCs@Hb@GOx NPs at different concentrations . After being stationary in PBS (negative controls) and deionized water (positive controls) for 1 h, the supernatant was obtained after centrifuging and was further accessed to calculate the hemolysis ratio.Firstly, the obtained cattle blood red cells were washed with PBS by centrifuging. Next, the properly diluted blood samples were added into the RBCs@Hb@GOx NPs solution with different sample concentrations (500 − 2500 μg mL4 cells per well) were seeded into a 96-well and incubated with PBS, Hb, GOx, Hb@GOx NPs, RBCs@Hb@GOx NPs, Hb and H2O2, GOx and H2O2, Hb@GOx NPs and H2O2, RBC@Hb@GOx NPs and H2O2, Hb and glucose, GOx and glucose, Hb@GOx NPs and glucose, RBC@Hb@GOx NPs and glucose at 100% humidity atmosphere of 37 °C and 5% CO2 for 24 h, respectively. Next, 20 μL of MTT solution was added into each well and placed in CO2 incubator for further incubating for 4 h. DMSO (150 μL) was added, and the absorbance at 490 nm was obtained to calculate cell survival rate using the MTT assay kit. Moreover, in order to investigate the chemodynamic efficacy of RBC@Hb@GOx NPs on U87MG cells in vitro, Calcein-AM (100 μL) and PI solution (100 μL) were incubated with U87MG cells for 30 min. Living cells were stained with calcein-AM (green fluorescence) and dead cells with PI (red fluorescence).U87MG cells , adjusting the cell concentration to 1 × 106 mL−1. DCFH-DA was diluted with serum-free culture solution at 1:1000 to achieve a final concentration of 10 μM. Then, the collected cells were suspended in diluted DCFH-DA solution and further incubated at 37 °C for 20 min. Besides, the incubation solution was shaken to make the probe fully contact with the cell. Finally, the cells were washed with serum-free cell culture medium 3 times to fully remove DCFH-DA that did not enter into cells. The fluorescence intensity of cells was determined by confocal fluorescence microscope.The U87MG cells were randomly seeded into 6-well plates and then incubated with different groups under DMEM at a 100% humidity atmosphere at 37 °C and 5% COQuantitative analyses for ROS production were analyzed by flow cytometric  experiments. The U87MG cells after being treated with different groups were incubated with 10 μM of DCFH-DA for 20 min. Then, the incubation solutions were analyzed by flow cytometry with excitation at 488 nm and emission at 530 nm.2 for 24 h. The logarithmic growth cells were seeded into six-well plate. When the cells grew adhering to the wall after 24 incubation, the corresponding drug-containing medium was added according to the group setting, and a negative control group was set up. Then, the cells were washed with PBS (centrifuged at 2000 rpm for 5 min), adjusting the cell concentration to 1 × 106 mL−1. Next, the 100 μL of 10 × buffer solution and 900 μL of sterilized deionized water were diluted into 1 × Buffer, mixed and preheated to 37 °C. JC-1 working solution was formed via absorbing 500 μL of incubation buffer (1 × Buffer) into 1 μL of JC-1 solution. After that, 500 μL of JC-1 working solution was suspended with cells and incubated them at 37 °C and 5% CO2 incubator for 15–20 min. Finally, cells treated with different groups were collected by centrifugation at room temperature  and washed twice in 1 × Buffer, and further analyzed by confocal fluorescence microscope and flow cytometry.The U87MG cells were randomly seeded into 6-well plates and then incubated with different groups under 90% DMEM plus 10% FBS at a 100% humidity atmosphere of 37 °C and 5% COU87MG cells were seeded into in 6-well plates. After 24-h treatment with different groups, dead cells were removed by centrifugation and the supernatants were concentrated to 100 μL. Released HMGB1 in supernatants were tested via western blot.® imaging  equipped with LivingImage™ software (Xenogen).U87MG human glioma cells were purchased from American Type Culture Collection (ATCC) and maintained in DMEM media (Gibco) with 10% FBS (Hyclone). Six-week-old NCR nude female mice were used to generate intracranial orthotopic U87MG gliomas. Briefly, mice were anesthetized using 2% isoflurane and their heads were immobilized in a stereotactic headframe using atraumatic ear bars. A burr hole was made using a steel drill bit  1.4 mm right of the sagittal and 1 mm anterior to the lambdoid suture. Tumors were allowed to grow for 14 days prior to treatment. Intracranial tumor growth was monitored in vivo using bioluminescence IVIS® imaging equipped with LivingImage™ software (Xenogen) at 0, 6, 12, 36, 48, and 72 h post-injection. In addition, the main organs from the treated mice, including heart, liver, spleen, lung, kidney, and brain, were collected after 72 h injection for imaging and biodistribution analysis.ICG-loaded Hb@GOx NPs or RBC@Hb@GOx NPs were employed to investigate the transportability across the BBB based on the ICG fluorescence at tumor site. The orthotopic U87MG gliomas (without self-fluorescence) bearing mice were intravenously injected with ICG-Hb@GOx NPs and ICG-RBC@Hb@GOx NPs (250 μL). The fluorescence signal at brain was detected in vivo using bioluminescence IVIS−1); (3) Hb@GOx NPs ; and (4) RBC@Hb@GOx NPs . Treatment began from day 21 after tumor implantation, and all treatments were intravenously administered. They were administered once every 10 days. After treatment with above groups, tumor response to treatment was tracked using IVIS imaging. Signal intensity was quantified within a region of interest using Living Image™ software.The orthotopic U87MG gliomas (with self-fluorescence) bearing mice were randomly divided into four groups: (1) PBS ; (2) GOx .TUNEL Apoptosis Assay was used for determining tumor cells apoptosis. The orthotopic U87MG gliomas bearing mice brain tissue were stained with TUNEL Apoptosis Assay Kit after treatment. For histology, the frozen tissue sections of 4 ~ 5μm were immersed in 1% acetone fixation solution at room temperature for 5 min. Afterwards the tissue sections were dried, washed with PBS (3 times) for 3 min, and further stained with H&E and Congo Red. Finally, the collected sections were analyzed by an Olympus BX43 microscope (Japan). All staining experiments were conducted following the standard protocol.t test. Differences were considered statistically significant at p < 0.05.The statistical analyses were conducted by Student’s All experiments involving animals were performed in compliance with relevant ethical regulations in adherence with the Nanjing University and Nanjing Normal University for the Care and Use of Laboratory Animals, Nanjing, China.The key technical challenge here is to develop an appropriate method to construct the protein superstructures, so that their biological activities can be retained. There have been crosslinked protein nanoparticles derived from gelatin , albumin−1 belonging to amide I was attributed to C=O stretching vibration of peptide linkages in the protein’s backbone. The absorption band of 1620–1500 cm−1 for amide II was due to a combination of N–H bending and C–N stretching. Normally, the absorption bands of amide I and amide II are eliminated when the structure of Hb is denatured. Figure −1, respectively, which were nearly the same as those obtained for the native protein (1650.01 and 1536.79 cm−1) [The Fourier transform infrared (FT-IR) spectrum is shown in Fig. 79 cm−1)  The abso2+ and heme. After incubation with glucose solution, Fenton reactions occurred. The peaks belonging to Fe 2p and Fe 3p were detected . Besides, the presence of peaks of Fe 2p, Fe 3p, Fe 2s, and Fe 3s in the high-resolution XPS further verified the existence of Fe2+ and Fe3+ in the Fenton reaction . The zeta potential was measured after each synthesis procedure, with a value of 4.79 ± 1.05 mV for Hb, −5.70 ± 2.09 mV for GOx, −4.59 ± 0.1 mV for Hb@GOx, −7.22 ± 0.75 mV for RBCs, and −14.867 ± 0.33 mV for RBC@Hb@GOx NPs  was applied to analyze the existing elements in the as-prepared NPs. No obvious Fe peaks were detected from the samples without adding glucose solution , indicating the tight combination between the Fe2O2 with fluorescence intensity changes, was applied to measure the generated H2O2 during the catalytic process. As presented in Fig. 2O2. Almost no H2O2 were observed in control groups  was employed to quantitatively analyze ·OH [The Fenton catalytic performance of the RBC@Hb@GOx NPs was evaluated , 46. Theups Fig. d, suggeslyze ·OH . The gen2·−. As shown in Fig. 2·−. Thus, as-fabricated NPs had excellent performance to produce ·OH and O2·−.Since electron spin resonance (ESR) spectrum has been considered as the most effective assay to detect the generation of ROS . We sele2O2. This problem was perfectly overcome by Hb, which is an oxygen-carrying protein. The oxygen-carrying capability of Hb was investigated in Fig. S9, showing that the dissolved oxygen in RBC@Hb@GOx solution increased gradually with time. In short, our results suggested that the RBC@Hb@GOx NPs could be utilized as a powerful CDT nanoagent.We then studied the change in pH values during the catalytic reaction. As shown in Fig. −1 . Moreover, the degradation of RBC@Hb@GOx NPs was also investigated via UV–Vis absorption spectrum . The NPs completely degraded after 30 min in glucose solution. These results indicated that the nanoparticles possess excellent biocompatibility and biodegradability. When the glucose was added to a mixture of cells and RBC@Hb@GOx NPs, the NPs were activated. High toxicity to the cells was observed as a result of ROS-caused oxidative damage  assays were conducted . U87MG a−1 Fig. a. RBC@Hbage Fig. b. In comTo quantify the intracellular ROS generated by the RBC@Hb@GOx NPs, 2′-7′-dichlorofuorescin diacetate (DCFH-DA) assay was used  which coc), and caspase-activating protein. In the cell apoptosis progress, the loss of mitochondrial membrane potential is normally a signal of early mitochondrial damage, which can be investigated by JC-1 staining assay. It has been proved that JC-1 is an ideal fluorescent probe to detect mitochondrial membrane potential (∆Ψm). When the mitochondrial membrane potential is high , JC-1 dyes aggregate in the mitochondrial matrix to form polymers called JC-1 aggregates, producing red fluorescence. While JC-1 aggregates are release from mitochondria matrix to cytoplasm to form JC-1 monomer, corresponding fluorescence transits from red to green, indicating that the mitochondrial membrane potential is low (damaged mitochondria) . Thus, it is very convenient to detect mitochondrial membrane potential through observing the changes of fluorescent color and the relative ratio of red and green fluorescence [We further investigated the mechanisms of ROS-mediated cell death. Mitochondria are central organelles and play a critical role in apoptosis in reacting to stress. Many factors induce cell apoptosis, including loss of mitochondrial membrane potential, release of cytochrome c  is rich in endoplasmic reticulum. Normally, ROS-based endoplasmic reticulum stress would induce the CRT transfer to the surface of plasma membrane . Besides, high mobility group protein B1 (HMGB1), as a highly conserved nuclear protein, is also released from the nuclear while the cells are dying . TherefoOrthotopic U87MG gliomas (without fluorescence) bearing mice were constructed as animal models. ICG-loaded RBC@Hb@GOx NPs were injected intravenously to investigate their ability to cross the BBB Figs. a and S14To further investigate the role of RBC membrane in crossing the BBB, we synthesized ICG-loaded RBC@Hb@GOx NPs and Hb@GOx NPs to compare their accumulation in tumors Fig. e. From F® imaging system was used to track the tumor response to treatment. Firstly, we did a short-term survival experiment in mice . PBS (control group), GOx, and RBC@Hb@GOx NPs were administrated by tail vein injection to evaluate the therapeutic performances, respectively. As shown in Fig. S19b, during 15-day treatment, the fluorescence intensity in control and GOx groups showed significant increase, indicating that no therapeutic effect on the brain tumor. In contrast, for the group treated with RBC@Hb@GOx NPs, fluorescence intensity increased slowly and the area with fluorescence was much smaller compared to the other two groups, which implied that the RBC@Hb@GOx NPs was able to suppress the tumor growth. The therapeutic effect was further confirmed by H&E and Congo red staining . These staining images indicated an extensive cell apoptosis in GBM in the RBC@Hb@GOx NPs-treated group. Besides, the whole brain was also sliced from mice after 15 days . It was apparent that the satisfactory tumor suppression effects came from the highly toxic hydroxyl radicals produced by the sequential biological/chemical-catalytic reactions by the RBC@Hb@GOx NPs. The as-produced toxic hydroxyl radicals then killed the cancer cells in a mitochondria-mediated apoptosis pathway.Encouraged by the satisfactory BBB transportability in vivo, we further investigated the therapeutic efficacy of RBC@Hb@GOx NPs. The mice bearing orthotopic GBM tumor with self-fluorescence were constructed. IVISA long-term survival experiment was conducted in mice to compare the therapeutic effect of Hb@GOx NPs and RBC@Hb@GOx NPs as outlined in Fig. Besides, the blood biochemical data, including red blood cells (RBC), white blood cells (WBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), and mean corpuscular hemoglobin (MCH), liver and kidney function test including alanine transaminase (ALT), aspartate transaminase (AST), albumin (ALB), creatinine (CREA), blood urea nitrogen (BU), and uric acid (UA) were applied to evaluate the in vivo toxicity of RBC@Hb@GOx NPs . There was no apparent discrepancy can be observed from all groups, what is more, no significant pathological changes of the major organs from all experimental groups were observed by H&E stained organ slices , demonstrating the excellent biocompatibility of RBC@Hb@GOx NPs. These results showed that the RBC@Hb@GOx NPs as a powerful CDT nanoagent could efficiently inhibit the growth GBM and elongate the survival time.In summary, we developed a new type of CDT nanoagent for GBM treatment. The natural Hb and GOx were employed as highly efficient Fenton catalysts. The excellent biocompatibility and biodegradability greatly reduced the toxicity of the nanomaterials and improved the safety of CDT. With facile assembling and crosslinking techniques, Hb and GOx were formulated into protein superstructures. They could be utilized as self-delivery entities, thereby eliminating the need of conventional drug carriers. RBC membranes were coated on the protein superstructures to reduce the immunogenicity and enhance the circulation time, which facilitated the delivery of the NPs to cross BBB/BTB. We showed that the RBC@Hb@GOx NPs with the size of 51.55 nm could accumulate and retain at the GBM tumor site. More importantly, we successfully demonstrated in both in vitro and in vivo studies that the NPs could elicit powerful anti-tumor efficacy by generating ROS to induce mitochondria-mediated apoptosis. The RBC@Hb@GOx NPs thus presented a promising therapeutic direction for GBM treatment. We envision that the strategy could also be useful for other primary and metastatic brain tumors.Supplementary material 1 (PDF 2142 kb)Below is the link to the electronic supplementary material.