L, also known as c-FLIPL, is a critical anti-apoptotic protein that inhibits activation of caspase 8 in mammalian cells. Previous studies have shown that arginine 122 of CFLARL can be mono-methylated. However, the precise role of arginine methyltransferase of CFLARL remains unknown. PRMT5 and PRMT1, which are important members of the PRMT family, catalyze the transfer of methyl groups to the arginine of substrate proteins. PRMT5 can monomethylate or symmetrically dimethylate arginine residues, while PRMT1 can monomethylate or asymmetrically dimethylate arginine residues.CFLARL ubiquitination level was evaluated by proteasomal inhibitor treatment combined with HA-Ub transfection and WB assay. PRMT1 and PRMT5 genes were knocked down by siRNA technique.Lung cancer cells were cultured following the standard protocol and the cell lysates were prepared to detect the given proteins by Western Blot analysis, and the protein interaction was assayed by co-immunoprecipitation (Co-IP) or GST pull-down assay. CFLARL by decreasing the ubiquitination and increasing its protein level. Additionally, PRMT1 down-regulated the protein level of CFLARL by increasing the ubiquitination and degradation. The overexpression of PRMT5 can inhibit the interaction between CFLARL and ITCH, which has been identified as an E3 ubiquitin ligase of CFLARL, while overexpressed PRMT1 enhances the interaction between CFLARL and ITCH. Furthermore, we verified that dead mutations of PRMT5 or PRMT1 have the same effects on CFLARL as the wild-type ones have, suggesting it is the physical interaction between CFLAR and PRMT1/5 that regulates CFLARL degradation other than its enzymatic activity. Finally, we showed that PRMT5 and PRMT1 could suppress or facilitate apoptosis induced by doxorubicin or pemetrexed by affecting CFLARL in NSCLC cells.We show that PRMT5 up-regulated the protein levels of CFLARL degradation by regulating the binding of E3 ligase ITCH in NSCLC cells. This study identifies a cell death mechanism that is fine-tuned by PRMT1/5 that modulate CFLARL degradation in human NSCLC cells.PRMT5 and PRMT1 mediate the distinct effects on CFLARThe online version of this article (10.1186/s13046-019-1064-8) contains supplementary material, which is available to authorized users. Cells were collected for Flow Cytometry analysis. C and D H460 cells were seeded in 6-well plates. PRMT5 siRNA were transfected for 48 h. Cells were treated with pemetrexed [5.0 μM] for 48 h. Cells were collected for Flow Cytometry analysis. E and F A549 cells were seeded in 6-well plates. PRMT1 siRNA were transfected for 48 h. Cells were treated with pemetrexed [5.0 μM] for 48 h. Cells were collected for Flow Cytometry analysis. (DOCX 14 kb)


The recovery rate based on acetone recovered fipronil on eggshells and in egg liquids was 80.13%~87.87%, and 81.34%~88.89%, respectively. The SERS assay was successfully used to monitor fipronil in eggs.A sensitive strategy to rapidly detect fipronil residues in eggs using multibranch gold nanoparticles (AuNPs) as the substrate of surface-enhanced Raman spectroscopy (SERS) was investigated in this study. Under optimized conditions, fipronil molecules preferentially deposited on the multibranch gold nanoparticles with preferential (111) facet-oriented growth due to its low surface energy. This anisotropic growth promoted the increase of SERS “hot spots”, inducing a huge enhancement of Raman signals of the fipronil. An external standard calibration method was employed for quantitative analysis, and the method was validated for linearity, sensitivity, repeatability and recovery. Good linearity were found in the concentration range of 10 ng/L~10 mg/L in fipronil acetone solution ( Since the fipronil egg scandal was exposed in August 2017, eggs containing fipronil residue have been found in more than 45 countries. Millions of eggs are recalled from supermarkets, and many poultry farms have suffered economic losses , 361 cm−1 [δ(N24-C23-C18-C17) + δ(C17-C16-N15-C6) + δ(C18-N19-N15-C6) + ν(C3-C2)], 523 cm−1 [υ(C23-C18) + β(C18-N19-N15)], 729 cm−1 [δ(C18-N19-N15-C6) + γ(C23-C17-N19-C18) + ν(C3-C2)], 856 cm−1 , 1013 cm−1 [ν(C23-C18) + β(C17-C16-N15) + β(C16-N15-N19)], 1285 cm−1 [υ(C9-C3) + β(H7-C2-C3) + β(H8-C4-C5) + ν(C17-C16)], 1385 cm−1 [ν(N19-C18) + β (C18-N19-N15) + β(C16-N15-N19)], 1468 cm−1 [υ(C23-C18) + β(C18-N19-N15) + β(C17-C16-N15) + β(C16-N15-N19)], 1576 cm−1 [υ(N15-C16) + υ(N15-C6)] and 1626 cm−1 [υ(C17-C16) + υ(N20-C16) + β(H22-N20-H21)] ] A. Those 20-H21)] B. Howeve20-H21)] . The enhSERS is the intensity of SERS spectra of the sample, Ibulk is the normal Raman spectra of fipronil, NSERS is the total number of surface adsorbed molecules, and Nbulk is the number of molecular in the laser illumination volume. The mean value of EF for multibranch AuNPs is as high as 9.04 × 105, while for spherical AuNPs is only 572.88. All of the results indicated that multibranch AuNPs performed much more sensitive to detect fipronil than spherical AuNPs. In addition, as expected, multibranch AuNPs offered simplicity and high sensitivity for the real application.In which I−1 was selected for quantitative analysis because of the relatively strong intensity which was able to be observed even when the concentration was very low. The peak intensity had a good linear relationship with logarithm of fipronil concentration (log C) in the wide range of low concentration from 10 ng/L to 10 mg/L (R2 = 0.9916). The calculated limit of detection (LOD0) and the calculated limit of quantity (LOQ0) [Amongst these characteristic fipronil vibration peaks, the SERS peak at 1468 cm 10 mg/L C, with ey (LOQ0)  for the y (LOQ0) ,44 based−1 :The SERS spectra of fipronil with incremental concentrations from 8 × 10ollected A. One ca0 is the original concentrations of fipronil on eggshells, and C1 is the concentration of the recovering solution that could be confirmed according to the calibration curve as shown in 0) levels of 8 × 10−2 mg/m2, 8 × 10−3 mg/m2 and 8 × 10−4 mg/m2, which were 87.87% (RSD = 10.17%), 80.37% (RSD = 7.25%), and 80.13% (RSD = 8.52%), respectively. In Formula (4), C0 and LOQ0 in the standard curve of fipronil acetone solution to the number that is based on the weight of egg liquids, Equations (5) and (6) was used. Theoretical LOD of fipronil in egg liquids was 8.98 ng/kg, and the LOQ was 26.94 ng/kg. In Formulas (5) and (6), LOD0 and LOQ0 are the limit of detection and the limit of quantity, respectively, as calculated by Formulas (2) and (3).To transform the LOD0 would be the original concentrations of fipronil added into egg liquids, and C1 would be the concentration of the recovering solution that could be confirmed according to the calibration curve as shown in 0) levels of 200, 20, and 2 µg/kg was 88.89% (RSD = 8.83%), 85.76% (RSD = 5.00%), and 81.34% (RSD = 5.99%), respectively. These results demonstrated that the SERS method with multibranch AuNPs as the substrate was more compatible with many previous analytical methods for fipronil detecting in eggs . When calculating the recovery rates of egg liquids, C in eggs , and wouEint) was calculated from the difference between the energy of complex (ΔEcom), fipronil molecules (ΔEmol) and gold surface (ΔEsur) with the following Formula (7):For assessing the advantage of crystals with preferential (111) orientation in SERS sensing of fipronil, the molecular dynamic simulations for fipronil on the Au surface were completed . Five laOn the Au(111) surface, the interaction energy was −428.373 kcal/mol, while that of Au(200), Au(220), Au(311), and Au(222) was –417.218, –405.860, –386.327 and −390.041 kcal/mol, respectively . It was With abundant “hot spots”, multibranch AuNPs can effectively enhance fipronil SERS signals. Along with a wide linear response, the LOD for fipronil on eggshells and in egg liquids were better than many analytical methods for fipronil detection, and the recoveries were satisfactory. As a simple and highly sensitive approach for fipronil detecting, the SERS method was successfully applied to detect fipronil from eggshells and egg liquids.


It was determined that the highest polymerization rate of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles was achieved in solution containing 0.75 mg mL−1 of GOx and 0.05 mol L−1 of glucose. The influence of the enzymatic polymerization duration on the formation of PANI/GOx and Ppy/GOx composite nanoparticles was spectrophotometrically investigated. The most optimal duration for the enzymatic synthesis of PANI/GOx and Ppy/GOx composite nanoparticles was in the range of 48–96 h. It was determined that the diameter of formed PANI/GOx and Ppy/GOx composite nanoparticles depends on the duration of polymerization using dynamic light scattering technique (DLS), and it was in the range of 41–167 nm and 65–122 nm, when polymerization lasted from 16 to 120 h.Polyaniline (PANI), polypyrrole (Ppy), and polythiophene (PTh) composite nanoparticles with embedded glucose oxidase (GOx) were formed by enzymatic polymerization of corresponding monomers . The influence of monomers concentration, the pH of solution, and the ratio of enzyme/substrate on the formation of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles were spectrophotometrically investigated. The highest formation rate of PANI-, Ppy-, and PTh-based nanoparticles with embedded GOx was observed in the sodium acetate buffer solution, pH 6.0. The increase of optical absorbance at  Tectively ,6,7,8. Tectively ,12,13, bectively ,15,16.π-systems of proximal polymeric chains [Most of the CPs are insoluble in the common organic solvents, which are explained by favourable interactions between c chains ,17. Suchc chains ,18,19,20c chains ,22, Ppy c chains ,24,25,26c chains  were devc chains .−1) PANI is produced during chemical polymerization in the solution of high acidity (pH < 2.5). The morphology of formed PANI composite depends on the pH value [orto- and para-coupled units with cyclic phenazine fragments [PANI is commonly used in the fabrication of electrochemical biosensors, due to controllable conductivity, good processability, controllable degree of protonation, charge transfer capability, good biocompatibility, and relatively good environmental stability ,20,28. Py pH < 2.. The morpH value . In the pH value . PANI pry pH < 2.. The morragments ,19.One of the most widely investigated CPs is Ppy, which is characterized by a relatively stable electrical conductivity and it can be formed by electrochemical synthesis at low oxidation potentials and in solutions of neutral pH ,20,24,26π-π conjugated sp2 hybridized backbone [Conducting polymer PTh has a great potential for the application in the fields of organic electronics, photovoltaic devices, solar cells, gas, and biological sensors ,32,33. Hbackbone ,35, chembackbone ,34, and backbone  polymeribackbone ,32. Duribackbone . The dimbackbone . Oxidizebackbone ,13, becabackbone . Dependibackbone ,32, PTh backbone ,13,36, tbackbone , or nanoEnzymatic polymerization has been proven by PANI formation while using enzymes  was used for the enzymatic formation of PANI, Ppy, and PTh composite nanoparticles in this research, with embedded GOx , respectively. The effect of pH, the ratio of enzyme/substrate on the formation of these composite nanoparticles and the influence of polymerization duration on the formation of PANI/GOx, Ppy/GOx were investigated by UV/Vis spectrophotometry. The diameter of the PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles was evaluated while using the dynamic light scattering (DLS) technique.Aspergillus niger, 201 units mg−1 protein) and D-(+)-glucose were purchased from Fluka  and Carl Roth GmbH + Co.KG . Glucose solution was allowed to mutarotate for 24 h before all of the investigations, and the equilibrium between α and β optical isomers was established during this time. All other chemicals that were used in present study were either analytically pure or of highest quality. All of the solutions were prepared using deionized water. The solution of sodium acetate (SA) buffer (0.05 mol L−1 CH3COONa·3H2O) with 0.1 mol L−1 KCl was prepared by the mixing of sodium acetate trihydrate and potassium chloride, which both were purchased from Reanal  and Lachema . Alfa alumina powder  was purchased from Electron Microscopy Sciences . Aniline, sodium hydroxide (NaOH), and thiophene were purchased from Merck KGaA , pyrrole — from Acros Organics  and hydrochloric acid (HCl) — from Sigma-Aldrich . The polymers were filtered before measurements through 5 cm column that was filled by Al2O3 powder to remove the coloured components. All of the solutions were stored between measurements at +4 °C.Glucose oxidase  with IEC CL31R Multispeed centrifuge . Subsequently, PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles were washed two times with 0.05 mol L−1 SA buffer, pH 6.0, and they were collected by the centrifugation. Separated and washed polymeric nanoparticles with embedded GOx were resuspended in SA buffer, pH 6.0, and used for further investigations. All of the experiments were performed at +20 ± 2 °C. PANI, Ppy, and PTh composite nanoparticles with embedded GOx were synthesized at room temperature (+20 ± 2 °C) in darkness while using polymerization bulk solution containing 0.05 mol L−1 of glucose, 0.50 mol L−1 of polymerizable monomers , and 0.75 mg mL−1 of GOx was used for 48 h lasting polymerization. Deionized water, 0.1, 0.01, or 0.001 mol L−1 HCl solutions, 0.05 mol L−1 SA buffer, pH 6.0, and 1 × 10−6, 1 × 10−4 or 0.01 mol L−1 NaOH solutions were used to prepare solutions in pH range between 1.0 and 12. During 48 h lasting polymerization, several concentrations of glucose  were tested in the 0.05 mol L−1 SA buffer, pH 6.0, containing 0.75 mg mL−1 of GOx and 0.50 mol L−1 of aniline, pyrrole, or thiophene. The optimal duration of polymerization was determined by varying the duration of polymerization from 5 to 288 h in 0.05 mol L−1 SA buffer, pH 6.0, with 0.05 mol L−1 of glucose, 0.50 mol L−1 of aniline or pyrrole, and 0.75 mg mL−1 of GOx. A UV/Vis spectrophotometer was used to perform the investigations.The efficiency of polymeric nanoparticles formation depends on the pH of solution and the duration of enzymatic polymerization. The optimal pH value for the formation of PANI/GOx, Ppy/GOx and PTh/GOx composite nanoparticles was studied in pH range from pH 1.0 to 12. Polymerization bulk solution containing 0.05 mol LThe UV/Vis spectrophotometer Lambda 25 , which operates in 230–1000 nm wavelength range, was used for the registration of optical absorbance spectra of the formed PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles colloidal solution. The measurements were performed at room temperature in plastic disposable cuvettes of 1 cm optical path length. The optical absorbance of PANI/GOx, Ppy/GOx, and PTh/GOx solutions was registered after the polymerization reaction. UV/Vis measurements were evaluated and presented using SigmaPlot software 12.5 .−1 HCl, 0.05 mol L−1 SA buffer, pH 6.0, and 1 × 10−6 mol L−1 NaOH were used to prepare solutions in pH range between 2.0 and 8.0. Polymerization solutions containing 0.05 mol L−1 of glucose, 0.50 mol L−1 of aniline, pyrrole, or thiophene, and 0.75 mg mL−1 of GOx were monitored during 15–120 h lasting polymerization.The influence of solution’s pH on the diameter and zeta-potential of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles was studied in pH range from 2.0 to 8.0 at +20 ± 2 °C. In this experiment, 0.01 or 0.001 mol L−1 SA buffer, pH 6.0, and collected by the centrifugation , as it was described in chapter 2.2. The diameter and zeta-potential of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles were evaluated by DLS and microelectrophoresis, while using the Zetasizer Nano ZS from Malvern  equipped with a 633 nm He-Ne laser. The obtained data was analysed with Dispersion Technology Software version 6.01 from Malvern. DLS and microelectrophoresis investigations were evaluated and visualized by SigmaPlot software 12.5.Polymeric nanoparticles after enzymatic synthesis were separated, washed two times with 0.05 mol LThe enzymatic polymerization of the PANI/GOx and Ppy/GOx composite nanoparticles was proceeded in darkness at room temperature within 24 h. The procedure of the formation, separation, washing, and collection of nanoparticles was similarly performed, as it was described in chapter 2.2. To reduce the effect of dissolved salts, colloidal solutions of composite nanoparticles were diluted in 50 μL of deionized water and homogenized using an ultrasound Bandelin Sonorex RK 31 H . Afterwards, 6 μL of nanoparticle solution was placed on the surface of graphite rod and dried, this procedure was repeated eight times, and then the surface was characterized by Hitachi SU-70 field emission scanning microscope equipped by energy dispersive X-ray spectrometry EDS and electron backscatter diffraction (EBSD) modules from Hitachi . Turbosputer was applied for the preparation of synthesized CPs for SEM-based imaging.The large number of research papers is dealing with the chemical and electrochemical polymerization of aniline, pyrrole, and thiophene, but only few researches are dealing with the enzymatic formation of these CPs. The enzymatic formation of these CPs can be based on the application of four major compounds: corresponding monomer, glucose oxidase, glucose, and oxygen, which is naturally dissolved in used solution .GOx generates hydrogen peroxide and gluconolactone in the presence of glucose and dissolved oxygen, which is hydrolyzed to gluconic acid in an aqueous solution. Hydrogen peroxide as a strong oxidizer generates radical cation of pyrrole, aniline ,19,25, aPANI and Ppy are very often used for the immobilization of biomolecules . Neutralλmax) at 440 nm, 460 nm, and 450 nm, respectively  was observed after 24 h of polymerization. In the case of the formation of PANI/GOx and Ppy/GOx, the solutions become coloured yellow-brown and black-grey, respectively. It was later observed that the intensity of colour and the value of optical absorbance were dependent on the pH value of the polymerization solution. Preliminary spectrophotometric investigations showed that the enzymatic polymerization of aniline , pyrroleectively A. These λmax = 440 nm . The highest value of optical absorbance (0.690 a.u. at λmax = 440 nm) was registered in the solution of pH 1.0 after 48 h lasting polymerization of aniline. However, the pH 1.0 is not suitable for PANI/GOx formation due to the inactivation of GOx and due to the non-enzymatic polymerization at this extreme pH value. In the research that was performed by another authors, it was presented that, by the increase of pH value, the polaron optical absorbance at λmax = 420 nm and λmax = 823 nm gradually decreases and a strong optical absorbance is observed due to the transition of the quinoid rings, which emerges at λ = 560–600 nm [λmax = 440 nm was observed).0–600 nm . The mosλmax = 460 nm has been observed during the formation of Ppy/GOx composite nanoparticles, which is in an agreement with the results that were obtained during the chemical polymerization of pyrrole [λmax = 460 nm is attributed to a transition from valence bond to the polaron or bipolaron state [λmax = 460 nm was observed at the early stage of pyrrole oxidation, and it was identified that it belongs to the intermediate that formed during the oxidative polymerization of pyrrole. Some authors mentioned the optical absorbance peak at λmax = 360 nm, which is related to π-π* transition [π-electron system of doped Ppy [The relationship between the optical absorbance of colloidal solution of Ppy/GOx composite nanoparticles and the pH values in the range from 2.0 to 12 of polymerization solution is presented in  pyrrole . Opticalon state . The peaansition . During oped Ppy .λmax = 460 nm has increased by 1.60 times  is attributed to the π-π* transition for large conjugated structure [λmax = 890 nm, which is related to the bipolaron state [λmax = 450 nm was in agreement with the investigations related to chemical-oxidative formation of: (i) PTh-based 60–100 nm nanoparticles in water solution containing copper (II) ions and H2O2 as oxidizer [3 as an oxidizer [The main charge carriers in PTh are polarons and bipolarons . Figure tructure ,34. Someon state . The appoxidizer  and (ii)oxidizer .λmax = 450 nm was dramatically changed (4.96 times), from 0.124 to 0.025 a.u. by the increase of pH value from 1.0 to 12. The absorbance of 0.023 a.u. at λmax = 450 nm was registered at pH 6.0 after 48 h of enzymatic polymerization.As presented in λmax = 440 nm, λmax = 460nm, and λmax = 450 nm, respectively, was assessed in SA buffer, pH 6.0, after 48 h lasting polymerization and was 0.329, 0.207, and 0.023 a.u., respectively  monomer’s concentration [The type of oxidizing agent has significant influence on the oxidative polymerization reaction . It was ntration , but furntration ,21.−1 of aniline, pyrrole, or thiophene, 0.75 mg mL−1 of GOx and glucose concentration varying from 0.015 to 0.05 mol L−1. The effect of enzyme/substrate ratio on the rate of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles formation was investigated in the next set of spectrophotometric measurements. The investigations were performed during 48 h in the polymerization solution, pH 6.0, containing 0.50 mol L−1 in the polymerization solution up to 0.05 mol L−1. The absorbance of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles, which were prepared using 0.75 mg mL−1 of GOx and 0.05 mol L−1 of glucose, increased by 3.39, 3.79, and 10.1 times in a comparison to the absorbance that was obtained for polymeric nanoparticles with embedded GOx using 0.015 mol L−1 of glucose. By the increase of glucose concentration up to 0.015 mol L−1, the absorbance of PANI/GOx, Ppy/GOx, and PTh/GOx decreased 1.25, 1.31, and 3.53 times, if compared with the results that were obtained for the absorbance with 0.75 mg mL−1 of GOx and 0.05 mol L−1 of glucose. Thus, the ratio of GOx/Glucose 0.75 mg mL−1/0.05 mol L−1 was selected as the most optimal for polymeric nanoparticle composites formation.The registered absorbance significantly increased by increasing glucose concentration from 0.015 mol LDepending on pH, the negatively or positively charged enzymes can be entrapped within the CPs during oxidation/reduction reactions . EnzymatThe diameter of 48 h enzymatically synthesized PANI-, Ppy-, and PTh-based nanocomposites with embedded GOx in solution of pH 2.0–8.0 was evaluated by the DLS technique. The results of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles diameter and their distribution are presented in N-isopropylacrylamide) as steric stabilizers 50 nm PANI nanoparticles were synthesized [Some of the researchers evaluated the diameter of synthesized polymer nanocomposites by FE-SEM. During the enzymatic polymerization of aniline using chitosan and poly on the formation of PANI/GOx and Ppy/GOx composite nanoparticles was investigated. The polymerization was performed in 0.05 mol Lrization . The amoto 168 h .λmax = 440 nm and λmax = 460 nm has increased by 74.7 and 11.1 times, respectively, in a comparison with the absorbance that was registered after 5 h (λmax = 460 nm was 2.48 times lower than that of PANI/GOx at λmax = 440 nm after the same duration of polymerization. It was determined that the absorbance of PANI/GOx and Ppy/GOx composite nanoparticles has decreased during long-lasting polymerization (more than 168 h). The limited solubility of synthesized polymers and the precipitation of particles or clusters of PANI/GOx and Ppy/GOx from the polymerization solution could explain this phenomenon [λmax = 440 nm and λmax = 460 nm of PANI/GOx and Ppy/GOx after 48 h lasting polymerization was 2.52 and 1.94 times lower than that registered after 168 h lasting polymerization. 48–96 h lasting polymerization seems to be more efficient for PANI/GOx and Ppy/GOx composite nanoparticles formation, because smaller PANI/GOx and Ppy/GOx will be formed.Such an effect indicated the formation of PANI/GOx and Ppy/GOx composite nanoparticles and it is in an agreement with some previous researches ,24. The fter 5 h . After 1enomenon . When thV) was evaluated by formula V = (tgα), where tgα was calculated from −1 h−1, respectively. It is seen that the initial polymerization rate of aniline is 2.86 times slower than that of pyrrole.The initial aniline and pyrrole conversion into polymer reaction rate (The diameter of PANI/GOx and Ppy/GOx composite nanoparticles and their aggregates was investigated by DLS and N-isopropylacrylamide) as steric stabilizers and horseradish peroxidase as enzyme, 50 nm PANI nanoparticles were synthesized [The aggregates of PANI/GOx and Ppy/GOx composite nanoparticles were formed from the 120th hour of enzymatic polymer’s formation. During 168–288 h lasting polymerization, a lot of agglomerates that are characterized by large diameter were formed, which was in an agreement with our previous researches, where 142 nm PANI/GOx composite nanoparticles were formed after 168 h lasting polymerization . For thethesized . HoweverThe electrical charge of particles is usually expressed by zeta-potential. The zeta-potential was determined in SA buffer, pH 6.0, for polymeric nanoparticles formed by 48 h lasting polymerization in our investigations. The zeta-potentials of PANI/GOx, Ppy/GOx, and PTh/GOx nanoparticles were −6.2, −9.4, and −8.6 mV, respectively.λmax = 440 nm, λmax = 460 nm, and λmax = 450 nm, respectively. The formation of PANI/GOx in emeraldine base form was observed. Both factors (i) the pH of polymerization bulk solution and (ii) the duration of polymerization have significant influence on the formation of polymeric nanoparticles. The highest rate and the smallest diameter of formed PANI/GOx, Ppy/GOx, and PTh/GOx were observed in solution of sodium acetate buffer, pH 6.0. The optimal ratio of glucose oxidase/glucose for the formation of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles was determined as 0.75 mg mL−1/0.05 mol L−1. Spherical particles of PANI/GOx and Ppy/GOx were formed during enzymatic polymerization. The most optimal duration for the enzymatic synthesis of PANI/GOx and Ppy/GOx composite nanoparticles was in the range of 48–96 h. The diameter of polymeric nanoparticles, which was determined by DLS, was dependent on the duration of polymerization: 41–167 nm PANI/GOx and 65–122 nm Ppy/GOx particles were formed by polymerization lasting from 16 to 120 h, respectively. The formed PANI/GOx and Ppy/GOx composite nanoparticles were pure from surfactants and other interfering species, which increases the applicability of synthesized polymeric composite nanoparticles for possible biomedical applications.PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles were synthesized by the enzymatic polymerization of aniline, pyrrole, and thiophene, respectively. The PANI/GOx, Ppy/GOx, and PTh/GOx nanoparticles were characterized by optical absorbance maximums at Here, the evaluated PANI/GOx and Ppy/GOx composite nanoparticles formation conditions can be applied in further researches. Some advantages of the application of gold nanoparticles in the formation of conducting polymers-based composite materials have been demonstrated ,39. Ther


Ihh expression, coupled with a dramatic reduction in Mmp13 and RUNX2 expression, in the vertebral growth plate and in regions of the intervertebral disc in the Prmt5 conditional mutant mice. We also demonstrate that PRMT5 has a continuous role in the intervertebral disc and vertebral growth plate in adult mice. Altogether, our results establish PRMT5 as a critical promoter of terminal hypertrophic chondrocyte differentiation and endochondral bone formation during spine development and homeostasis.Idiopathic scoliosis (IS) is the most common type of musculoskeletal defect affecting children worldwide, and is classified by age of onset, location and degree of spine curvature. Although rare, IS with onset during infancy is the more severe and rapidly progressive form of the disease, associated with increased mortality due to significant respiratory compromise. The pathophysiology of IS, in particular for infantile IS, remains elusive. Here, we demonstrate the role of PRMT5 in the infantile IS phenotype in mouse. Conditional genetic ablation of PRMT5 in osteochondral progenitors results in impaired terminal hypertrophic chondrocyte differentiation and asymmetric defects of endochondral bone formation in the perinatal spine. Analysis of these several markers of endochondral ossification revealed increased type X collagen (COLX) and This article has an associated First Person interview with the first author of the paper. Summary: Loss of Prmt5 in osteochondral progenitors impairs terminal hypertrophic chondrocyte differentiation, leading to defects in endochondral bone formation and models infantile idiopathic scoliosis in mouse. Idiopathic scoliosis (IS) is the most common pediatric spinal deformity, characterized by a lateral curvature of the spine >10° and affecting ∼3% of children worldwide . ClinicaGPR126 (ADGRG6), LBX1, CHL1 and SOX9 genes (Adgrg6/Gpr126 (Sox9 (Shp2 (Ptpn11)  , Gdf5/6  . Subsequently, matrix metalloproteinase 13 (Mmp13) is expressed in these cells, which is critical for terminal differentiation of hypertrophic chondrocytes and endochondral bone formation  is required for chondrocyte proliferation and to prevent precocious hypertrophic differentiation , whereaspression , and is pression ,b, 2018.pression . During pression  and RUNXpression .Sox9 expression followed by widespread apoptosis and loss of the cartilage template and skeletal element in the limb  is a critical factor involved in the establishment of chondrocyte progenitors in embryonic limb buds , resultithe limb . Mechanithe limb , and smathe limb , which cthe limb . Given tthe limb . In thisf/fPrmt5 conditional allele . f/fCol2Cre;Prmt5 mutant pups were produced at Mendelian ratios when assessed at embryonic day (E)18.5 . However, mutant animals died of unknown causes prior to weaning  . Two of the mutant mice that survived to P10 (n=4) were much smaller than the control mice . However, at P10, we observed IS-like thoracic scoliosis . Lateral X-rays showed reduced signal attenuation in the distal ribs, indicative of a reduction in mineralized bone in mutant mice . We also observed overall smaller vertebrae and some mild wedging of the vertebra at the apex of the curvature . Sagittal slices of the microCT data at the level of the T6 vertebrae showed a severe reduction in trabecular bone formation in the f/fCol2Cre;Prmt5 mutant mice compared with the controls , which was consistently reduced in f/fCol2Cre;Prmt5 mutant mice at these stages. We did not observe PRMT5 expression in the trabecular bone or cortical bone , suggesting that PRMT5 has a limited role in committed osteoblast lineages.In order to test the role of PRMT5 in the spine, we crossed mice harboring a l allele  to Col2CCre mice . This Cohe spine . At post0, n=62) A. We didrol mice F, wherea mutants D; n=4/4.ant mice F″ relaticontrols I,J. Immucontrols K-L′ and f/fCol2Cre;Prmt5 mutant mice at both P1 . To determine whether these morphological alternations were region specific, we repeated the same analyses in the lumbar spine, observing identical phenotypes in both regions of the spine . Collectively, these data suggest a model in which PRMT5 is critical for several aspects of vertebral body development, including chondrocyte differentiation and endochondral ossification along the entire spine axis.We also observed consistent morphological alterations in some of the IVD tissues and the vertebral growth plate of the mutant mice at P10. In control mice, the IVD endplate contains several layers of flat cells that lightly stain with Alcian Blue  of the mutant mice. However, the expression of proteoglycan 4 (PRG4/Lubricin), a common marker of healthy cartilage and IVD . The expression of SOX9, a key transcriptional regulator of chondrogenesis, was also reduced in the endplate of mutant mice , but was not obviously affected in the proliferative growth plate. Hypertrophic chondrocytes of the vertebral growth plate downregulate Sox9 expression during the process of terminal hypertrophic chondrocyte differentiation ; however, f/fCol2Cre;Prmt5 mutant mice continued to express SOX9 in some of these phenotypically hypertrophic cells of the growth plate . The annulus fibrosus typically expressed low levels of SOX9 ; however, this expression was greatly diminished in mutant mice . Interestingly, the loss of SOX9 expression did not strongly correlate with reduced type II collagen (COLII) expression, which is an established direct target of SOX9 . Together, our results suggest that PRMT5 regulates the differentiation of the cartilaginous tissues of the IVD and growth plate in the spine.We next assayed several established regulators of chondrocyte differentiation  in orderant mice B,B′ in cant mice A,A′. Incant mice B,B′, but and IVD , was rem of SOX9 , in Col2Mmp13 . RUNX2 is also expressed in some cells of the proliferative growth plate and in the majority of hypertrophic growth plate cells, as well as some cells of the trabecular bone, by the age of P10 . Interestingly, we did not observe an obvious loss of RUNX2 expression earlier in development in f/fCol2Cre;Prmt5 mutant mice at P1 . Next, we performed fluorescent in situ hybridization (FISH) using an Mmp13-specific riboprobe on thoracic spine sections. At P10, there is severe reduction in Mmp13 expression in the vertebral growth plate and trabecular bone in f/fCol2Cre;Prmt5 mutant mice . The loss of Mmp13 expression is also observed in the presumptive ossification center of the vertebrae at P1 in f/fCol2Cre;Prmt5 mutant mice . Conversely, at P1, we observed ectopic Mmp13 signal within the nucleus pulposus and the endplate in mutant mice, which was not observed in control mice . The consistent reduction of RUNX2 and its effector, Mmp13, in the vertebral growth plate of f/fCol2Cre;Prmt5 mutant mice demonstrates a mechanistic role for PRMT5 in the regulation of terminal hypertrophic chondrocyte differentiation and endochondral ossification during perinatal development of the spine.We next set out to determine the mechanisms by which PRMT5 regulates hypertrophic chondrocyte differentiation and endochondral bone formation. RUNX2 is a critical driver of terminal differentiation of hypertrophic chondrocytes and osteoblast differentiation in mouse , and actMmp13 , 2018. Ue of P10 A,A′. In ant mice D-D″, comant mice C-C″. We Ihh expression. Ihh signaling is crucial to maintain chondrocyte proliferation and, at the same time, negatively regulate hypertrophic chondrocyte differentiation by regulation of PTHrP expression , which is further supported by typical RUNX2 expression in mutant mice at this stage . However, at P10, we observed a dramatic increase in ectopic Ihh expression in the mutant mice, including ectopic expression of Ihh in the proliferative and hypertrophic growth plate, as well as in some cells of the endplate and annulus fibrosus regions of the IVD , compared with only sparse expression in the prehypertrophic/hypertrophic chondrocytes in control mice . We did not observe upregulation of Bmp4 expression in the growth plate at P1 in either genotype , suggesting that this increased expression accumulates during perinatal development. We also detected a mild increase in cell death in the annulus fibrosus, endplate and growth plate regions of the f/fCol2Cre;Prmt5 mutant mice at both P1 and P10 . Taken together, these data demonstrate that, as in other tissues and organs, PRMT5 is required for the inhibition of Bmp4 expression in the cartilaginous tissues of the spine.Loss of  tissues . Consistf/fCol2Cre;Prmt5 mutant mice, we next sought to determine whether PRMT5 also functions in committed bone-forming lineages. To address this, we utilized an OcCre (BglapCre) transgenic mouse to specifically remove PRMT5 function in mature osteoblasts (n=7). X-ray analysis showed no scoliosis in the f/fOcCre;Prmt5 mutant mice at P10  or at 2 months of age (n=3), and histological analysis at P10 showed no obvious alterations of spine tissues . Furthermore, we did not detect PRMT5 expression in the cortical or trabecular bone of the spine . Taken together, we conclude that PRMT5 functions exclusively in chondrocyte lineages to promote endochondral ossification and does not have an obvious role in mature osteoblast lineages.Given the clear alterations in endochondral bone formation in eoblasts . The conFig. S11). Close examination of this region of the IVD revealed that PRMT5-positive cells reside in various tissues, including the outer layer of the annulus fibrosus, the border of the endplate and within the growth plate .Chondrocytes regulate spine homeostasis by maintaining a dynamic balance between synthesis and degradation of extracellular matrix components in the cartilaginous tissues of the spine. To gain insight into whether PRMT5 may have a continuous role in homeostasis of the adult spine, we next assayed the expression of PRMT5 in wild-type thoracic spine sections at 6 weeks and 4 months of age. We observed low-level PRMT5 expression in a group of cells bordering the junction between the IVD and the vertebral growth plate at both time points  transgenic mouse, which targets committed chondrocyte lineages  . However, we consistently observed increased, ectopic COLX expression in the IVD endplate and vertebral growth plate in f/fATC;Prmt5 mutant mice . Taken together, our data show that loss of PRMT5 in cartilaginous tissues of the adult spine results in reduced expression of normal extracellular matrix component Prg4, coupled with increased expression of the hypertrophic chondrocyte marker COLX and decreased expression of Mmp13. This suggests that PRMT5 has a continuous role in the regulation of hypertrophic chondrocyte turnover in the growth plate in adult mice. Our study demonstrates a novel role for PRMT5 in maintaining homeostasis of cartilaginous tissues in adult spine and defines a limited temporal window during perinatal development for the regulation of spine stability.Because PRMT5 continues to be expressed in adult mice, we tested whether PRMT5 is required for the homeostasis of the spine. To specifically remove PRMT5 function in cartilaginous tissues of the spine after weaning, we crossed lineages , and indlineages A. Beta-g of age) C. At 6 wand Prg4 D, respecstrategy D. Moreovtant IVD F, with tant mice H,H′. We Mmp13 and RUNX2 expression and in negative regulation of Ihh expression. We also demonstrate that loss of PRMT5 in osteochondral progenitors results in the expansion of hypertrophic chondrocytes in the vertebral growth plate; however, ossification of the vertebrae is severely impaired during perinatal development, mimicking an Mmp13 loss-of-function phenotype  but not neonatal stage (P1). Interestingly, these expanded regions of Ihh expression overlap with the regions of ectopic, expanded COLX expression. These results again suggest that PRMT5 is dispensable for chondrocyte proliferation and the early maturation of hypertrophic chondrocyte differentiation; however, it is required to restrict Ihh expression in the IVD and growth plate during perinatal development. Increased Ihh expression may contribute to the increased production and accumulation of COLX-positive cells in the growth plate. We speculate that the misregulation of Mmp13 and Ihh in f/fCol2Cre;Prmt5 mutant mouse spines act synergistically to promote the accumulation of hypertrophic chondrocytes in the growth plate by inhibition of processes driving terminal chondrocyte differentiation, which consequently leads to delays and reductions in endochondral bone formation during perinatal skeletogenesis. The mechanisms of PRMT5-dependent regulation of Mmp13 and Ihh expression remain to be determined.IHH is another important regulator of hypertrophic chondrocyte differentiation. It can directly promote chondrocyte proliferation and inhibit premature hypertrophy of these proliferative chondrocyte via an IHH/PTHrP negative-feedback loop . It can Prmt5 mutant mice is disorganized and shows reduced expression of Col2a1 and Sox9, while the hypertrophic chondrocytes also undergo a blockage in terminal differentiation, highlighted by reduced expression of RUNX2 and Mmp13, and increased expression of Ihh  and catabolic  factors . For ins the IVD . Meanwhi the IVD . Recent ntenance . Here, wThese findings demonstrate that PRMT5 has an important role in the homeostasis of cartilaginous tissues of the spine. It will be important to determine whether ablation of PRMT5 in cartilaginous lineages of mature adult mice can generate susceptibility to the onset of more severe histopathological changes in the IVD and growth plate due to trauma or aging in mice. Interestingly, a study using bromodeoxyuridine labeling demonstrated that some IVD or IVD-adjacent regions, such as the perichondrium and sites of annulus fibrosus ligament anchoring, display label-retaining cells indicative of an adult stem cell niche . Importaf/fCol2Cre;Prmt5 mutant mice phenotypically models infantile-onset IS in humans. This is phenotypically distinct from congenital scoliosis, which is characterized by failure of segmentation, formation or dysplasia in one or more vertebral units , but find a clear thoracic spinal curvature present at P10, coupled with loss of bone formation in the ribcage and perinatal lethality. The natural history of this mouse model partially recapitulates aspects of severe cases of infantile IS in humans. The f/fCol2Cre;Prmt5 mutant mice do not display vertebral dysplasia prior to or at birth. However, we do observe mild wedging of the vertebral body at the apex of the thoracic curvature at P10. Given that bone deposition is highly responsive to mechanical forces, we suggest that the onset of scoliosis is caused by defects in endochondral ossification of the vertebral bodies, which generate anisotropic mechanical loading of the spine, which initiates the onset of scoliosis and vertebral wedging. This model is supported by our observation of a complete lack of scoliosis in conditional f/fATC;Prmt5 mutant mice recombined at 2 or 4 weeks of age, which also display alterations in terminal chondrocyte differentiation, yet exhibit a mature spinal column that has already undergone substantial ossification at the induction of recombination. In conclusion, we suggest a model wherein infantile IS may be induced by subtle defects and/or delays in endochondral ossification during perinatal development, and that severe cases resulting in respiratory stress may reflect delayed endochondral ossification of the floating ribs. Finally, this study further underscores the role of cartilaginous tissues and regulation of chondrocyte differentiation in the pathogenesis of IS, which is a common theme in other genetic mouse models displaying IS Wtsi]  injections by two strategies: (1) starting at 2 weeks of age, once/week (10 mg Dox/kg body weight) for 4 continuous weeks; or (2) starting at 4 weeks of age, once/week (10 mg Dox/kg body weight) for 4 continuous weeks. Mice were harvested at P1, P10, 6 weeks, 2.5 months and 4 months of age for tissue analysis.Animal studies comply with all relevant institutional and national animal welfare laws, guidelines and policies for the care and use of experimental animals, and were approved by the Institutional Animal Care and Use Committee at The University of Texas at Austin (protocol AUP-2018-00276). All mouse strains, including MM)Wtsi] , Col2Cre, as previously described (http://www.ctlab.geo.utexas.edu/). IHC analyses were performed on paraffin sections of thoracic spine after antigen retrieval using 10 mM Tris-HCl and 1 mM EDTA  (PRMT5 and RUNX2), 4 mg/ml pepsin (COLII and COLX), 100 μg/ml hyaluronidase (PRG4) or 10 μg/ml proteinase K (SOX9). Colorimetric development was performed with the following primary antibodies: anti-PRMT5 , anti-COLII , anti-COLX , anti-SOX9 , anti-Lubricin (PRG4)  and anti-RUNX2 . The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) cell death assay was performed on paraffin sections with an In Situ Cell Death Detection Kit, Fluorescein (Roche) according to the manufacturer's instructions. Quantification of TUNEL-positive cells was performed on sections of three control and three mutant mice. Two to four IVDs were analyzed per mouse. Beta-galactosidase staining was performed on frozen sections of thoracic spine as previously described  were isolated in cold PBS, pooled together, snap frozen and pulverized in liquid nitrogen. Three control and three mutant mice were used in each group. Total RNA was isolated using TRlzol Reagent  and cleaned up with a Direct-zol RNA miniprep kit . Reverse transcription was performed using 500 ng total RNA with an iScript cDNA synthesis kit (Bio-Rad). Real-time RT-PCR analyses were performed as previously described (−(ΔΔCt) method. Primer sequences are listed in Table S1.Intervertebral discs and adjacent growth plates from the thoracic spine of the 2.5-month-old escribed . Gene ex


BCLXL and c-IAP1, thereby inhibiting tumor cell apoptosis and sustaining proliferation. Importantly, PRMT5 inhibitor opposed tumor growth and BCLXL and c-IAP1 transcription in the bladder cancer xenograft model. Collectively, the current suggests the crucial role of PRMT5 as a promising therapeutic target in bladder cancers.Protein arginine methyltransferase 5 (PRMT5) has emerged as a key regulator of tumorigenesis. However, how PRMT5 functions in bladder cancer, the most common malignancy of the urological system, is unknown. We described here that PRMT5 is highly expressed in bladder cancer cell lines and primary human bladder cancer tissues. PRMT5 enhances the proliferation and colony formation of bladder cancer cells. PRMT5 knockdown induces bladder cancer cell apoptosis. Mechanistically, PRMT5 enhances NF-kB activation by targeting crucial anti-apoptotic genes such as  Bladder cancer is the most common urological carcinoma with about 430 000 cases and 165 000 deaths worldwide each year  . RadicRMT5 is a member of the protein arginine methyltransferase (PRMT) family and catalyzes arginine methylation by transferring methyl groups to arginine residues . The movitro and in vivo. Mechanistically, PRMT5 promotes NF-κB recruitment on the promoter of anti-apoptotic targets BCLXL and c-IAP1. Our work thus demonstrated PRMT5 is crucial for facilitating bladder cancer growth, and is an important target for cancer therapy. In the present study, we showed for the first time a potential role of PRMT5 in promoting bladder cancer cell proliferation and tumor growth in 2 at 37 °C. The primary human bladder cancer tissues were obtained from patients with invasive transitional carcinoma. 293T cells were transient transfected with human PRMT5 shRNA (Dharmacon) with Lipofectamine and plus reagent (Invitrogen) to produce lentiviral supernatant. T24 and UM-UC-3 cells were infected with shPRMT5 lentivirus to stably overexpress PRMT5 shRNA.T24 and UM-UC-3 were from ATCC and were maintained in RPMI 1640 (Invitrogen) with 10 % fetal bovine serum (Invitrogen), 100 U/ml penicillin, and 100 U/ml streptomycin, in 5 % COWestern blot was carried out as previously described . Antibo6 cells were seeded in 10 cm dishes in RPMI 1640 containing 10 % FBS. Cell numbers were counted in 2 and 4 days. For colony formation assay, 50/well cells were placed in six-well plates in RPMI 1640 containing 10 % FBS for 10 days. Colonies were fixed with methanol and stained with 0.1 % crystal violet in 20 % methanol for 15 min.1 × 10The cells were stained with PE-annexin V/7-AAD according to the manufacturer's instruction (BD Biosciences).siGENOME SMART pool targeting PRMT5 or control random siRNA were from Dharmacon. T24 cells were transfected with 50 nM siPRMT5 by Lipofectamine RNAi MAX reagent (Invitrogen).p5XIP10 [21] was transfected in T24 cells using Lipofectamine and Plus Reagents (Invitrogen). 48 hours later, the NF-кB luciferase activity was monitored .Total RNA was extracted using the RN-easy FFPE Kit (Qiagen) or RNeasy Mini kit (Qiagen). cDNA was prepared by the SuperScript III First-Strand Synthesis Kit (Invitrogen). qPCR was performed using SYBR Green Master Mix (Appliedbiosystems). Primers were designed by Primer3 (v.0.4.0) The truChIP Chromatin Shearing Kit (Covaris) was used to prepare chromatin. Chromatin was sheared into 200- to 700-bp fragments using a Covaris S2 instrument . The IgG and NF-kB p65 antibodies  were used for immunoprecipitation by the Quick Chip Kit  was used for qPCR to quantify precipitated DNA). The primers are from Qiagen EpiTect ChIP qPCR primers.6 T24 cells were injected subcutaneously into C57BL/6J mice  in the right flank. Tumor volume was measured using calipers and calculated as follows: volume = longest tumor diameter × (shortest tumor diameter)2/2. EPZ015666 or control vehicle (PBS) was administered orally twice daily (50 mg/kg) from 14 days after injection. Mice were sacrificed 28 days after injection. 1 × 10t test or one-way ANOVA. A P-value below 0.05 was considered significant.Data were analyzed using GraphPad Prism 7. The statistical difference was calculated by using two-tailed Student PRMT5 transcription was also up-regulated in bladder cancer FFPE samples, as compared to samples from healthy control . In Figc-IAP1 and BCLXL promoters,  was performed using T24 bladder cancer cells treated with EPZ015666 to address critical roles of PRMT5 on NF-кB recruitment. Pharmacological inhibition of PRMT5 by EPZ015666 significantly impairs NF-κB p65 occupancy on Figure 4E, providiTo confirm the essential role of PRMT5 as a therapeutic target in vitro, we investigated the effect of EPZ015666, a selective PRMT5 inhibitor  is a member of the PRMT family of proteins, which symmetrically is dimethylating histones and non-histone protein. It's highlighted that PRMT5 plays central roles in the regulation of diverse cellular signaling and gene expression. PRMT5 protein levels are frequently high in numerous human cancers, such as lymphoma, glioblastoma, esophageal cancer, lung cancer, etc . Nevertin vivo using xenograft tumor models. Our results demonstrate the carcinogenic function of the PRMT5/NF-κB signaling in bladder cancer and the role of PRMT5 inhibitor as a potent agent for the strategic therapy for bladder cancer.The NF-κB transcription family plays substantial roles in tumorigenesis as well as in the immune and inflammatory system. NF-κB signaling pathway consists of the most common heterotrimer of p50 and p65, which bind to its inhibitory protein IκBα. IκBα is the inactive form, masking the nuclear localization signal . The anIn conclusion, our work illustrated that PRMT5 is highly expressed in bladder cancer cell lines and patients. Importantly, PRMT5 inhibition significantly represses bladder cancer cell proliferation and invasive bladder cancer growth by activation of critical NF-кB dependent anti-apoptotic genes. These findings indicated that PRMT5 may be an essential biomarker or drug target of bladder cancer.


Exosomes possess similar functions to their parent cells; however, they are safe and effective cell-free reagents with controllable and predictable outcomes. In this study, we investigated the therapeutic potential and underlying molecular mechanism for human bone mesenchymal stem cells-derived exosomes 4-induced rat liver fibrosis model, after which, we administered hBM-MSCs-Ex in vivo for 4 weeks. The resulting histopathology, liver function, and inflammatory cytokines were analyzed. In addition, we investigated the anti-fibrotic mechanism of hBM-MSCs-Ex in hepatic stellate cells (HSCs) and liver fibrosis tissue, by western blotting for the expression of Wnt/β-catenin signaling pathway-related genes.We established an 8-week CClIn vivo administration of hBM-MSCs-Ex effectively alleviated liver fibrosis, including a reduction in collagen accumulation, enhanced liver functionality, inhibition of inflammation, and increased hepatocyte regeneration. Moreover, based on measurement of the collagen area, Ishak fibrosis score, MDA levels, IL-1, and IL-6, the therapeutic effect of hBM-MSCs-Ex against liver fibrosis was significantly greater than that of hBM-MSCs. In addition, we found that hBM-MSCs-Ex inhibited the expression of Wnt/β-catenin pathway components , α-SMA, and Collagen I, in both HSCs and liver fibrosis tissue.4-induced liver fibrosis via inhibition of HSC activation through the Wnt/β-catenin pathway.These results suggest that hBM-MSCs-Ex treatment could ameliorate CClThe online version of this article (10.1186/s13287-019-1204-2) contains supplementary material, which is available to authorized users. Liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) . Such chExosomes (30–100 nm) are small membrane-bound vesicles derived from multivesicular bodies . They ar4-induced liver fibrosis model in rat. We further analyzed the involvement of Wnt/β-catenin signaling on hBM-MSCs-Ex-induced anti-fibrosis in vitro. Our results provide the first evidence that hBM-MSCs-Ex effectively alleviate liver fibrosis through the Wnt/β-catenin pathway. We believed that hBM-MSCs-Ex could provide a new avenue for the therapeutic treatment of hepatic fibrosis disease.The aim of the present study was to investigate whether transplantation of hBM-MSCs-Ex reduces liver fibrosis in a CCl2. Morphological observation of hBM-MSCs was performed by phase contrast microscopy . Cells at the fifth passage were used for this study [The hBM-MSCs were purchased from the Chinese Academy of Medical Sciences, China. Cells were cultured in DMEM  supplemented with 10% FBS , 500 U/ml penicillin, and 500 μg/ml streptomycin  at 37 °C, with saturated humidity and 5% COis study . Hepatic3), and ultra-centrifuged at 100,000g, 3 h. The exosome-enriched fraction was collected, washed with PBS three times, and centrifuged at 1500g, 30 min with 100-KDa MWCO. Purified exosomes were passed through a 0.22-μm filter and stored at − 80 °C. The protein concentration of the concentrated exosomes was determined using a bicinchoninic acid (BCA) protein assay kit . The presence of the exosomal markers CD9, CD63, CD81, TSG101, and Alix  were determined using western blotting. Purified exosomes were confirmed by transmission electron microscopy.The purification of hBM-MSCs-Ex involves several centrifugation and ultracentrifugation (Himac cp80wx/P70A-980) steps, as described previously . BrieflyThe hBM-MSCs-Ex obtained after differential centrifugation of conditioned cell-culture medium was suspended in PBS. Ten micrograms of exosome suspension was loaded onto formvar carbon-coated 200 mesh copper grids for 10 min at room temperature. Excessive fluid was drained with filter paper. Adsorbed exosomes were negatively stained with 1% phosphotungstic acid for 5 min. Then, the air-dried exosome-containing grids were observed by transmission electron microscope  operating at 100 kV.The NanoSight NS300  was used to determine the concentration and size distribution of purified airway hBM-MSCs-Ex. Polystyrene latex microspheres  of 100 nm diameter were used to calibrate the instrument. Then, hBM-MSCs-Ex were diluted with PBS 1:1000 to make a final volume of 1 ml and loaded into a 1-ml syringe. The syringe was placed on a syringe pump, and hBM-MSCs-Ex were infused at a flow rate of 25 μl/s at room temperature. The camera level was set to 7, gain to 1, and detection threshold to 5. A total of five videos were acquired with a duration of 1 min per video. A minimum of 2000–4000 exosomes/video were processed.4 at a dose of 30% CCl4 3 ml/kg body weight twice weekly in olive oil. Eight weeks later, CCl4 treated rats were randomly assigned into four groups (n = 12): the liver fibrosis group rats were injected with PBS alone; the hBM-MSCs group (positive control) rats were injected with 1 × 106/500 μl hBM-MSCs through the tail vein; the hBM-MSCs-Ex group rats were injected with 250 mg hBM-MSCs-Ex (harvested from 1 × 106 hBM-MSCs) in 500 μL PBS through the tail vein; the sham group rats were treated with PBS alone. After 4 weeks, serum was taken to assay liver function and the livers were collected. Livers were divided into three parts for immediate protein and RNA isolation, preservation in 10% formalin for histological examination, and freezing at − 80 °C for hydroxyproline (Hyp) and malonaldehyde (MDA) assay.Liver fibrosis was induced in Sprague Dawley (SD) rats . All protocols and procedures were approved by the Animal Experiment Ethic Committee of Jilin University . All experimental procedures were in accordance with the Chinese legislation regarding experimental animals. Rats were administered with an intraperitoneal injection of CCln = 12 rats) for quantification of immunohistochemistry (IHC) results. The IHC results were calculated via Image-Pro Plus.Liver tissues were processed for paraffin embedding by slicing into 4-μm sections. The sections were stained with hematoxylin and eosin (H&E), Masson, and Sirius red according to standard protocols. To analyze the extent of liver fibrosis, randomly selected fields of Masson sections were captured from each animal. The collagen stained area was calculated via Image-Pro Plus. The degree of hepatic fibrosis was assessed using the Ishak modified scoring system. The Ishak scoring criteria ranges from 0 to 6 : mild fibrosis  to severe fibrosis  . The livw/v) was prepared by homogenizing the right lobe of liver on ice in 150 mM Tris-HCl buffered saline  using a polytron homogenizer . The levels of hydroxyproline (Hyp) and malondialdehyde (MDA) in liver tissue were measured using kits  according to the manufacturer’s instructions.The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), total bilirubin (TBIL), alkaline phosphatase (ALP), and gamma glutamyl transpeptidase (γ-GT) were assessed using the Automated Biochemical Analyzer . Liver homogenate  according to the manufacturer’s protocol. Then, 1 μg total RNA was reverse-transcribed to give cDNA, which was used as the template, and combined with standard SYBR premix Ex Taq  on the Real-Time PCR Detection System . The following primers were used: GAPDH forward, 5′-agacagccgcatcttcttgt-3′, reverse, 5′-cttgccgtgggtagagtcat − 3′; IL-1 forward, 5′-atttccgccttccagagaat-3′, reverse, 5′-gagtctcatgggggaattga; IL-2 forward, 5′-aaactccccatgatgctcac-3′, reverse, 5′-gaaatttccagcgtcttcca-3′; IL-6 forward, 5′-ccggagaggagacttcacag-3′, reverse, 5′-acagtgcatcatcgctgttc-3′; IL-8 forward, 5′-atgagacactgtggctgtgc-3′, reverse, 5′- actgctggagaccaggaaga-3′; IL-10 forward, 5′-cctgctcttactggctggag-3′, reverse, 5′-tgtccagctggtccttcttt-3′; TNF-α forward, 5′-gtgacgtggagttgggtctt-3′, reverse, 5′-gagtccgtcttggtcagagc-3′. GAPDH served as the internal control, and experiments were conducted in triplicate. All reactions were performed in triplicate and the data were analyzed using the 22O2 for 15 min and blocked with 10% normal goat serum  at 37 °C for 1 h. Sections were then incubated with primary antibodies (1:500 dilution) against hepatocyte nuclear factor-4 alpha , alpha-smooth muscle actin , and Ki-67  at 4 °C for 24 h. Next, sections were incubated with biotinylated goat anti-rabbit IgG antibody , followed by incubation with peroxidase-conjugated biotin-streptavidin complex  for 15 min at room temperature, and lastly stained with diaminobenzidine at room temperature for 2 min and counterstained with hematoxylin at room temperature for 3–5 min. Finally, sections were photographed using an optical microscope . The liver sections were analyzed in ten consecutive optical fields for each rat, and the positive cells were quantified using ImageJ.Immunohistochemistry (IHC) was performed in accordance with the manufacturer’s instructions . Briefly, the liver sections were deparaffinized and then rehydrated in a descending alcohol series. Antigen retrieval was performed by heating the sections for 30 min at 95 °C in 1 mM EDTA buffer (pH 8.0). Thereafter, sections were added to 3% HWhen HSCs reached 60–70% confluency on 24-well plates, they were cultured with hBM-MSCs-Ex (5 ng/ml) for 48 h. Next, HSCs were incubated with 4% paraformaldehyde at room temperature for 10 min and then incubated with 1% bovine serum albumin  for 30 min. Cells were incubated with a primary antibody against α-SMA  for 1 h, followed by incubation with a secondary antibody  for 30 min at room temperature. Rhodamine phalloidin  was stained for cytoskeleton. The nuclei were labeled with DAPI . Fluorescent images were captured using an EVOS Cell Imaging System .HSCs were co-cultured with either SFM, hBM-MSCs, or hBM-MSCs-Ex (5 ng/ml) for 48 h before samples were collected for protein extraction. Liver tissue was collected from each treatment group  for protein extraction. Protein samples were mixed with SDS sample buffer and heated to 95 °C for 10 min, followed by separation on SDS-polyacrylamide gels. Resolved proteins were electro-blotted onto nitrocellulose membrane and probed with antibodies against PPARγ (ab 23673), Wnt3a (ab 248472), Wnt10b (ab70816), β-catenin (ab32572), WISP1 (ab50041), Cyclin D1 (ab16663), α-SMA (ab5694), Collagen I (ab138492), and GAPDH (ab 8245) overnight at 4 °C . Nitrocellulose membranes were then incubated with a secondary antibody, HRP-conjugated goat anti-rabbit IgG (ab15007), at room temperature for 2 h, and visualized by chemiluminescent detection according to the manufacturer’s instructions .p < 0.05 was considered to be statistically significant.Statistical analysis was performed using GraphPad Prism Version 6. One-way ANOVA with Dunnett’s multiple comparisons test was used to test for statistically significant differences. All quantitative data are expressed as mean ± SD. hBM-MSCs-Ex were successfully isolated from hBM-MSCs using the differential centrifugation method Fig. a, b. We 4-induced liver fibrosis was assessed in a rat model. Visual inspection showed that the liver from sham control rats had a smooth, uniform surface and soft texture . The Ishak fibrosis score showed a statistically significant decrease in the hBM-MSCs and hBM-MSCs-Ex-treated groups compared to the untreated liver fibrosis group. Again, the hBM-MSCs-Ex treatment group had a lower score than the hBM-MSCs treatment group .Histopathological examination using H&E staining, Masson’s trichrome, and Sirius red staining was performed to quantify the degree of liver fibrosis Fig. b. The arp < 0.01, p < 0.001). These results suggest that hBM-MSCs-Ex-treatment significantly alleviates liver fibrosis in CCl4-induced rats and that the therapeutic effect of hBM-MSCs-Ex-treatment was greater than that of hBM-MSCs treatment for some indicators.Next, we analyzed the levels of hydroxyproline (Hyp) and malondialdehyde (MDA) which indicate the presence of liver collagen fibers and lipid peroxidation changes. Hyp is a non-essential amino acid and a main component in collagen tissue. MDA is an end product of membrane lipid peroxidation, and its level can be used as a marker for oxidative stress and liver cell injury . Comparep < 0.05). In addition, the serum level of TP in hBM-MSCs and hBM-MSCs-Ex-treated groups was higher than that in the untreated liver fibrosis group . These results suggest enhanced liver function in the two hBM-MSCs and hBM-MSCs-Ex treatment groups.Biochemical analyses were performed to assess the restoration of liver function after hBM-MSCs and hBM-MSCs-Ex treatment. The serum levels of ALT, AST, TBIL, ALP, and γ-GT were significantly suppressed in the hBM-MSCs and hBM-MSCs-Ex-treated groups . Interestingly, we found that the expression of IL-1and IL-6 in hBM-MSCs-Ex treatment group was significantly decreased when compared to the hBM-MSCs treatment group . Therefore, both hBM-MSCs and hBM-MSCs-Ex treatment had an anti-fibrosis effect, as evidenced by decreased liver collagen, together with improvement of liver function and reduction of inflammation.Next, we detected the expression of inflammatory factors in liver tissue by qRT-PCR. Compared to the untreated liver fibrosis group, the expression of inflammatory factors including IL-1, IL-2, IL-6, IL-8, IL-10, and TNF-α were significantly decreased in the hBM-MSCs and hBM-MSCs-Ex treatment groups  and hBM-MSCs-Ex (71.28%) treatment groups was increased significantly when compared to the untreated liver fibrosis group . HNF-4α was a key nuclear receptor protein required for liver development [+ cells in hBM-MSCs-Ex (45.18%) group was increased significantly when compared with the liver fibrosis (28.54%) and hBM-MSCs group . To further investigate the mechanism underlying the hBM-MSCs and hBM-MSCs-Ex-mediated rescue of CCl4-damaged liver, we examined the expression level of α-SMA, which is a key cytokine involved in the development of liver fibrosis and HSC activation  and hBM-MSCs-Ex (13.84%) treatment groups when compared with the untreated liver fibrosis group . The above results indicate that hBM-MSCs-Ex promote anti-fibrosis by stimulating hepatocyte regeneration and inhibiting α-SMA expression on liver fibrosis.To evaluate whether hBM-MSCs-Ex treatment could enhance hepatocyte proliferation in cirrhotic liver, Ki-67 expression levels were assessed by IHC Fig. a, b. In elopment . The perMSCs 55.1% and hBMMSCs 55.1% and hBMp < 0.005, p < 0.001). Interestingly, the expression of Collagen I on hBM-MSCs-Ex treatment was significantly decreased compared to hBM-MSCs treatment, in both HSCs and liver fibrosis tissues . In addition, the expression of Cyclin D1 on hBM-MSCs-Extreatment was significantly decreased compared to that in HSCs .To further explore the association between the anti-fibrosis effect of hBM-MSCs-Ex treatment and the Wnt signaling pathway in liver fibrosis, we examined the expression levels of proteins involved in the Wnt/β-catenin signaling pathway, both in HSCs and in liver fibrosis tissues, through western blotting Fig. a, c. BotIn this study, we have demonstrated that hBM-MSCs-Ex have the ability to alleviate liver fibrosis, including the recovery of markers associated with improved liver function, inhibition of inflammation, and increased hepatocyte regeneration. Moreover, the therapeutic effect of hBM-MSCs-Ex appears enhanced over the effect of the parent hBM-MSCs. Furthermore, we found that, both in vitro and in vivo, the hBM-MSCs-Ex inhibit HSC activation via inhibition of Wnt/β-catenin signaling.4-induced liver fibrosis model demonstrated that administration of hBM-MSCs-Ex effectively alleviated liver fibrosis. Furthermore, we found that hBM-MSCs-Ex could aid in the recovery of liver functionality and increase hepatocyte regeneration. This is consistent with reports previously showing that human umbilical cord MSC-derived exosomes (hucMSC-Ex) could ameliorate CCl4-induced liver fibrosis and provide a protective effect on hepatocytes [MSCs-derived exosomes have been reported to have therapeutic properties on hepatic disease . Recentlatocytes . In addiatocytes . Our stup < 0.05). These results indicate that the therapeutic effect of hBM-MSCs-Ex against liver fibrosis was greater than that of hBM-MSCs.Although both hBM-MSCs and hBM-MSCs-Ex groups can reduce liver fibrosis in the study, exosomes have several special characteristic compared to their parent cells. The reasons that exosomes may offer advantages over their parental cells may be as follows: (1) they are smaller in size and less complex than their parent cells and, thus, easier to produce and store ; 2) the the2 and4-induced liver fibrosis [It has been reported that the inhibition of Wnt/β-catenin signaling results in the downregulation of HSC activation and an eventual reduction in CClfibrosis . In our fibrosis . In addifibrosis , 37. We Altogether, our results clearly demonstrate that hBM-MSCs-Ex treatment alleviates liver fibrosis in vivo. Moreover, administration of hBM-MSCs-Ex reduces liver fibrosis via inhibition of Wnt/β-catenin signaling to prevent HSC activation. Therefore, the use of hBM-MSCs-Ex presents a new and promising therapeutic strategy for hepatic disease in the clinical setting.Additional file 1:Figure S1. The rat body weight change in CCl4-induced liver fibrosis, n = 12. (DOCX 175 kb)Additional file 2:Figure S2. The rat organ index of liver, kidney and spleen in CCl4 -induced liver fibrosis, **p < 0.01, n = 12. (DOCX 114 kb)


Neurocan is a chondroitin sulfate proteoglycan present in perineuronal nets, which are associated with closure of the critical period of synaptic plasticity. During postnatal development of the neocortex dendritic spines on pyramidal neurons are initially overproduced; later they are pruned to achieve an appropriate balance of excitatory to inhibitory synapses. Little is understood about how spine pruning is terminated upon maturation. NrCAM  was found to mediate spine pruning as a subunit of the receptor complex for the repellent ligand Semaphorin 3F (Sema3F). As shown here in the postnatal mouse frontal and visual neocortex, Neurocan was localized at both light and electron microscopic level to the cell surface of cortical pyramidal neurons and was adjacent to neuronal processes and dendritic spines. Sema3F-induced spine elimination was inhibited by Neurocan in cortical neuron cultures. Neurocan also blocked Sema3F-induced morphological retraction in COS-7 cells, which was mediated through NrCAM and other subunits of the Sema3F holoreceptor, Neuropilin-2, and PlexinA3. Cell binding and ELISA assays demonstrated an association of Neurocan with NrCAM. Glycosaminoglycan chain interactions of Neurocan were required for inhibition of Sema3F-induced spine elimination, but the C-terminal sushi domain was dispensable. These results describe a novel mechanism wherein Neurocan inhibits NrCAM/Sema3F-induced spine elimination. Dendritic spines of cortical pyramidal neurons are the principal site of excitatory synapse formation. Spines undergo dynamic changes during development, including rapid spinogenesis in early postnatal life, followed by substantial pruning during adolescence . Spines Mechanisms governing the active phase of spine remodeling in adolescence and transition to more stable spines in adult brain are not well-defined. Perineuronal nets (PNNs) are mesh-like structures that surround neurons and arise postnatally around the time when synaptic contacts are stabilized . PNNs arNeurocan is a CSPG that is a prominent organizer of PNNs in the neocortex. Genetic studies in humans have identified Neurocan as a potential risk factor for schizophrenia, bipolar disorder , and dysad libitum availability of chow diet and water. For labeling postmitotic pyramidal neurons in the cerebral cortex, Nex1-CreERT2 mice were crossed with the Ai9 reporter strain (both in C57Bl/6) to generate a tamoxifen-inducible reporter line of mice expressing tdTomato in postmitotic pyramidal neurons under the control of Nex1-Cre as previously characterized (Wild type (WT) and NrCAM null mutant mice in the C57BL/6 genetic background were maintained in a 12-h day and night cycle environment with cterized . Recombicterized . All aniLysates of mouse cortex  and cell cultures were prepared in lysis buffer . Lysates (50 μg) were subjected to Western blotting with the following antibodies: anti-NrCAM , anti-Neurocan , anti-Sema3F , and anti-β actin . Blots were developed with HRP-tagged secondary antibodies  using Western Bright ECL Substrate (Advansta) and bands quantified by densitometry.For immunostaining, neuronal cultures transfected with pCAGGS-IRES-mEGFP were fixed at DIV14 in 4% paraformaldehyde (PFA), permeabilized with Triton X-100, blocked in 10% horse or donkey serum, and labeled with chicken anti-GFP (Abcam). Secondary anti-chicken Alexa Fluor 488 antibodies (1:500) were added for 1 h before mounting and confocal imaging. For Neurocan localization, 100 μm coronal brain sections were prepared on vibratome from Nex1-CreERT2:Ai9 mice (P18 and P80) expressing tdTomato in pyramidal neurons. Serial 100 μm vibratome sections from P18 and P80 brain were matched for level based on rostrocaudal distance from the anterior end of the brain. Samples were blocked in PBS, 10% donkey serum, 0.3% Triton X100, then incubated with Neurocan antibodies  for 24 h at 4°C, then with anti-sheep Alexa Fluor 488 secondary antibody (1:500). After washing, sections were mounted with Prolong Gold anti-fade reagent (Invitrogen) and imaged using a Zeiss LSM 700 confocal microscope. All images were captured using identical microscope settings, we kept the total z thickness (7.35 μm) as well as thickness of single optical sections (0.35 μm) same for all samples. tdTomato (red) fluorescence was excluded from analysis. The intensity of total Neurocan fluorescence observed in the green channel was quantified for each image after auto-thresholding without regard to tdTomato fluorescence. Quantification of pixel intensity was performed blindly using ImageJ software (NIH).C57BL/6 WT mice (P18 and P80) were anesthetized and perfused transcardially with phosphate buffer  and postfixed in 4% PFA, 0.1% glutaraldehyde in PBS. Coronal vibratome sections (50 μm) were subjected to pre-embedding immunogold labeling with silver enhancement using Neurocan antibodies and streptavidin-nanogold (Nanoprobes 2016), as we previously described . Sectiont-test . To test the effect of GAG chain digestion, Neurocan was incubated in solution with 0.1 units/μg chondroitinase ABC  at 37°C for 1.5 h, followed by heat inactivation of enzyme at 100°C for 10 min. Efficacy of GAG digestion was assessed by immunoblotting.Cortical neuronal cultures were prepared from mouse embryos (E15.5) and plated on laminin, poly-D-lysine coated chamber slides. Neurons were maintained in neurobasal medium (Gibco) supplemented with B27 and antibiotics. Cells were transfected with plasmid pCAGGS-IRES-mEGFP at DIV11 using Lipofectamine 2000 (Thermo Fisher Scientific). As described in , transfe2 were classified as retracted (collapsed), and those greater than 1000 μm2 were classified as non-retracted. The mean percent of collapsed cells relative to the total GFP-positive cells was calculated from three experiments, 10 images per condition, and compared by the t-test for significant differences . Preliminary dose response experiments showed that Neurocan (4–40 nM) effectively inhibited the Sema3F-Fc-induced COS-7 cell retraction response; we selected 8 nM for subsequent studies.COS-7 cells were plated on eight well chamber slides  coated with poly-D-lysine. The following day cells were transfected with plasmids expressing NrCAM (pCMV6), Npn2 (pCOS), and/or PlexA3-EGFP (pCAGGS-PlexA3-IRES-mEGFP). At 48 h after transfection cells were treated with 3 nM Sema3F-Fc or Fc for 30 min, subjected to immunofluorescence staining for GFP and imaged on confocal microscope. The particle tool of ImageJ was used after auto-thresholding to measure the area of individual labeled cells. The software auto-detects the boundary of cells in thresholded images. Cells between 500 and 1000 μmCOS-7 cells were transfected with NrCAM in pCMV6 or vector alone, together with pCAGGS-IRES-mEGFP. Cortical neurons from NrCAM homozygous null mice were transfected on DIV11 with pCAGGS-NrCAM-IRES-mEGFP or vector alone. At 48 h post-transfection, cells were treated with 20 nM recombinant Neurocan at 37°C for 30 min. To assay Neurocan binding to the cell surface, cultures were fixed and subjected to indirect immunofluorescence staining with antibodies to Neurocan and secondary Alexa Fluor-conjugated antibodies. Single optical section images were taken on the confocal microscope using the same settings for each condition, and the fluorescence intensity of staining measured using Image J.ELISA was performed to measure the binding between NrCAM-Fc (R&D) and alkaline-phosphatase-tagged Neurocan (Neurocan-AP). The Neurocan-AP fusion protein construct was generated by PCR amplification of full-length mouse Neurocan cDNA (Accession: BC065118), followed by subcloning into the APtag5 vector so that the alkaline phosphatase (AP) tag was inserted at the N-terminus of Neurocan . NeurocaTo assess direct binding of Neurocan with semaphorins, 1 μg of full length recombinant Neurocan (R&D) was incubated with 1 μg of either control Fc, Sema3A-Fc, or Sema3F-Fc in Tris-buffered saline for 1 h at 37°C. Protein A/G Sepharose beads were used to pull down Fc proteins for detection of bound Neurocan by immunoblotting with Neurocan antibodies. Blots were reprobed with anti-Fc antibodies.Figure 1A). Quantification showed that Neurocan expression in the cortex increased markedly (∼8 fold) from P7 to P14, declined at P30, and persisted in adulthood . The level of Sema3F (90 kDa) in the developing cortex paralleled that of Neurocan with a ∼6 fold increase from P7 to P14 then a decrease with maturation . To determine if Neurocan was localized to cortical pyramidal neurons at adolescent (P18) and adult (P80) stages, immunofluorescence staining for Neurocan was performed in the medial frontal cortex (MFC) and primary visual cortex (V1). These cortical regions have been shown to be sites of spine density regulation on apical dendrites of pyramidal neurons by Sema3F and NrCAM/Npn2/PlexA3 . Nonimmune IgG (IgG) staining was negligible. The intensity of Neurocan immunofluorescence  was quantified in coronal sections matched for level from confocal images obtained under identical settings. Neurocan immunofluorescence levels throughout the cortical layers were significantly greater at P18 than P80 in both the MFC and V1 . At higher magnification Neurocan labeling of pyramidal neurons at P18 in the MFC appeared to be diffusely localized around soma, dendrites, and extracellular space . Neurocan was also evident as a diffuse net at or near the membrane of dendritic branches, including in the vicinity of dendritic spines .To evaluate the postnatal expression of Neurocan in developing mouse brain, cortical lysates were analyzed at P7, P14, P30, and adult by Western blotting. Neurocan was detected as a broad band from 150 to 250 kDa reflecting GAG-modification  but it was not found directly within synaptic junctions. Neurocan was also evident at the membrane or extracellular space, and could be seen near presynaptic axon terminals (AT), which harbored synaptic vesicles . Neurocan labeling was frequently clustered at or near dendritic membranes as well as the extracellular space . At P80, Neurocan was observed at the plasma membrane near spines, and sometimes adjacent to asymmetric, excitatory synapses . At both stages labeling was rarely present within the cytoplasm. The appropriate dilution of Neurocan antibodies and specificity was pre-assessed by immunoperoxidase staining of COS-7 cells transfected with plasmids expressing Neurocan-AP or control AP protein. Cells expressing Neurocan-AP were clearly stained with Neurocan antibodies, whereas cells expressing AP elicited minimal staining, as did omission of primary antibody . In summary, Neurocan was expressed prominently in the postnatal MFC and V1, declining with maturation, and was localized near the plasma membrane of pyramidal cell processes and adjacent to dendritic spines.The subcellular localization of Neurocan was investigated in greater detail in the MFC by immunogold labeling at the electron microscope level. At P18, labeled Neurocan was observed near the neuronal membrane adjacent to excitatory, asymmetric synapses of dendritic spines . Similar results in neuronal cultures with a lower spine density were obtained using a lower concentration of Neurocan (8 nM) .To assess the potential of Neurocan to terminate spine remodeling during postnatal maturation, we tested the ability of Neurocan to inhibit Sema3F-induced spine retraction in cortical neurons in culture. In this assay , dissociFigures 3D,E). Cells expressing only Npn2 and PlexA3 also retracted upon Sema3F-Fc treatment but required NrCAM for maximal response . Significant retraction was not observed in COS-7 cells expressing Npn2 or PlexA3 alone, or in cells doubly expressing NrCAM/Npn2 or NrCAM/PlexA3 . Cells expressing only EGFP also did not respond to Sema3F-Fc . A basal level of retracted cells was observed in each condition (25–30%), which may be due to changes in shape as a result of mitosis, migration, or cell heterogeneity. To determine if Neurocan negatively regulated Sema3F-induced cell retraction, COS-7 cells expressing NrCAM/Npn2/PlexA3 were pre-treated with 8 nM Neurocan, then stimulated with 3 nM Sema3F-Fc or Fc. Neurocan effectively blocked NrCAM/Npn2/PlexA3-dependent retraction to Sema3F-Fc . These results demonstrated that Neurocan negatively regulates Sema3F-induced retraction through a mechanism involving the NrCAM/Npn2/PlexA3 receptor complex.To evaluate the molecules involved, we used a heterologous assay in which morphological retraction of COS-7 cells can be induced by Sema3A-Fc or Sema4D-Fc . COS-7 cFigures 4A,B). The low level of fluorescence seen in untreated cells was likely nonspecific. Immunoblotting for Neurocan and GAPDH (loading control) in lysates of similarly treated cells, showed that Neurocan was detected in cells transfected with the NrCAM plasmid but not in control cells without NrCAM .We hypothesized that Neurocan negatively regulates Sema3F-mediated retraction by binding to the extracellular region of NrCAM at the cell surface. To test this, we examined the ability of Neurocan to bind to COS-7 cells transfected with NrCAM or vector alone, together with EGFP. COS-7 cells were treated 48 h post-transfection with or without 20 nM Neurocan for 30 min. Cells were washed extensively, fixed without permeabilization, and subjected to immunofluorescence staining with Neurocan antibodies. The amount of surface-bound Neurocan on EGFP-labeled cells was quantified by measuring the fluorescence intensity of Neurocan staining in single optical sections. Results showed that Neurocan bound at greater levels to the surface of cells that expressed NrCAM compared to cells without NrCAM transfected with vector alone . The lower level of Neurocan fluorescence on NrCAM-minus neurons may be due to interaction with other binding proteins on the cell surface.To determine if Neurocan bound to neuronal cells expressing NrCAM, cortical neuron cultures from homozygous NrCAM null mutant embryos (E15.5) were transfected at DIV11 with pCAGGS-IRES-mEGFP or pCAGGS-NrCAM-IRES-mEGFP, and treated at DIV14 with 20 nM Neurocan for 30 min. After washing and fixation without permeabilization, neurons were immunostained to detect Neurocan on the cell surface. EGFP-labeled neurons expressing NrCAM exhibited a greater amount of surface-bound Neurocan than NrCAM-minus neurons, and quantitation showed that this difference was significant . Similar levels of Neurocan-AP binding to NCAM-Fc were observed. Taken together, these results support the conclusion that Neurocan binds to the extracellular region of NrCAM and inhibits Sema3F- mediated spine elimination in maturing pyramidal neurons of the mouse neocortex.Results of the cell binding assays showed that expression of NrCAM leads to increased binding of Neurocan to the cell surface. To assess a direct interaction of Neurocan with the NrCAM extracellular region, we developed an ELISA using purified, full-length mouse Neurocan fused to AP expressed in HEK293T cells. NrCAM-Fc protein was adsorbed to ELISA plates that were pre-coated with Protein A, then incubated with Neurocan-AP or AP control protein for 1.5 h. Binding was quantified by colorimetric detection of bound AP using p-nitrophenylphosphate. As a positive control, Neurocan-AP was also assayed for binding to NCAM, a different cell adhesion molecule known to engage Neurocan at its extracellular Ig2 domain . Neurocain vitro. Fc-containing protein complexes were pulled down with Protein A/G-Sepharose and subjected to Western blotting to detect Neurocan present in the Protein A/G pull-downs. Results showed that Neurocan bound to Sema3A-Fc, but there was no detectable association with Sema3F-Fc or Fc alone . Reprobing blots with anti-Fc antibodies confirmed that pull-downs contained approximately equivalent amounts of Fc-containing protein.Sema3A is a class 3 semaphorin that has been reported to bind to chondroitin sulfates in PNNs but has not been shown to bind Neurocan . The posFigures 5A,B). GAG removal from Neurocan was confirmed on Western blots by a shift in GAG-modified Neurocan to lower molecular weight and band narrowing .CSPGs have been shown to restrict plasticity of cortical and hippocampal neurons, and digestion of their associated GAG chains with chondroitinase ABC (chABC) increases spine dynamics and density . TherefoFigure 5D).Neurocan binds to the related adhesion molecule L1 through the C-terminal sushi domain, which contains about half of the GAG chains present in Neurocan . To asseFigure 5E) in which NrCAM/Npn2/PlexA3 functions as a holoreceptor complex for Sema3F that prunes excess dendritic spines from pyramidal neuron subpopulations during postnatal stages of active spine remodeling in the cerebral cortex. As PNNs arise during subsequent maturation, Neurocan within the PNN meshwork interacts with NrCAM on dendritic spines to terminate Sema3F-mediated spine pruning.We report here that Neurocan, one of the first CSPGs to be expressed in the maturing neocortex , is exprIn postnatally developing areas of the mouse neocortex (MFC and V1), Neurocan was localized around processes, spines, and soma of Nex1-expressing postmitotic pyramidal neurons, then declined to lower levels in adulthood. As shown by immuno-electron microscopy of the frontal cortex (P18), Neurocan was prominent at plasma membranes and extracellular space, often in proximity to spines, axon terminals, and postsynaptic densities. Previously, we showed that Neurocan was also localized to perisomatic axon terminals and soma of inhibitory interneurons in the MFC . The preCell binding and ELISA assays showed that Neurocan was able to bind NrCAM within its extracellular region, which engages in homophilic and heterophilic interactions. chABC perturbed the ability of Neurocan to block Sema3F-mediated spine pruning in cultured neurons, indicating that GAG chains mediated this inhibition. Our binding results with recombinant proteins support radioimmunoassay studies showing binding of full length NrCAM purified from mouse brain to Neurocan . Our expA limitation of the present work is that it is not known how NrCAM association with Neurocan inhibits spine remodeling, such as by altering upstream interactions or downstream signaling. Our results clearly showed that Neurocan did not interact with the upstream ligand Sema3F. Unlike Sema3A, which associates with the CSPGs versican and aggrecan in the brain extracellular matrix , Sema3F-Neurocan and other CSPGs in PNNs have been reported to associate with Ig-class adhesion molecules, including NCAM, L1, NgCAM, Neurofascin, and TAG1, as well as integrins . Thus, Nin vitro studies, might serve to protect a subpopulation of cortical pyramidal neurons from over-pruning of dendritic spines. Other mechanisms of synaptic elimination involve astrocyte-mediated phagocytosis through engulfment receptors (MerTK and MEGF10), microglia-mediated pruning    and auto CX3CR1) . The inv CX3CR1)  where de CX3CR1) , and Neu CX3CR1) .In conclusion, we provide evidence that the PNN protein Neurocan acts as a developmental brake for spine remodeling mediated by Sema3F and NrCAM in the maturing mouse neocortex.VM conducted most of the experiments, analyzed the results, and contributed to writing the manuscript. EW performed the pull-down experiments and assisted in immunostaining. IG and SD performed the ELISA. RW and KP performed the immuno-EM experiments. BD assisted in neuronal cultures. AT assisted with the Neurocan binding experiments. PM designed the study, supervised the research, analyzed the EM data, and contributed to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.