Xanthomonascampestris pv campestris (Xcc) biofilm. We used nuclear magnetic resonance (NMR)-based metabolomics to investigate the molecular determinants related to the lytic action, aiming at identifying the metabolic pathways dysregulated by phage treatment. Furthermore, we identified specific markers  which are involved in the control of biofilm stability. Our data show that Xccφ1, alone or in combination with HA and C20:0, interferes with the metabolic pathways involved in biofilm formation. The approach described here might be extended to other biofilm-producing bacteria.Bacterial biofilm provides bacteria with resistance and protection against conventional antimicrobial agents and the host immune system. Bacteriophages are known to move across the biofilm to make it permeable to antimicrobials. Mineral hydroxyapatite (HA) can improve the lytic activity of bacteriophages, and, together with eicosanoic acid (C20:0), can destroy the biofilm structure. Here, we demonstrate the efficacy of the combined use of phage, HA and C20:0 against  The concern about bacterial resistance to antibiotics and microbial biofilm production is rapidly increasing. The latest data collected by the European Centre for Disease Prevention and Control highlight a persistent increase of antibiotic-resistant bacteria in the clinical area, as well as in the food industry and agriculture .Biofilm production is the response of bacteria to adverse environmental conditions , such asBacteriophages (phages) are viruses infecting bacteria, and in contrast to many antibiotics , phages Xanthomonas campestris pv. campestris (Xcc) biofilm. Xcc is a Gram-negative bacterium distinguished into several pathovars with specific host range. Xcc is the causal agent of crucifer  black rot disease, causing yield loss in agricultural production world-wide [Xcc infection in plants (submitted by Papaianni). Here, we demonstrated that the simultaneous use of HA, C20:0 and Xccφ1 destroys the Xcc biofilm structure, identifying specific biomarkers involved in the control of biofilm stability.In this paper, by using NMR-based metabolomics, we investigated the molecular determinants related to the action of the phage against the rld-wide . As withrld-wide . The exorld-wide . In partrld-wide . The eicrld-wide ,22,23, aBrassica oleracea plants with black rot symptoms (characteristic of Xcc infection) were suspended in 15 mL of nutrient broth  and shacked for 30 min at 24 °C. Soil sediment was removed by centrifugation (5000 rpm for 10 min), and individual supernatants (15 mL) were transferred into sterile flasks. Forty milliliters of 106 colony-forming units (CFU) per ml of Xcc bacteria in exponential growth phase were added to each flask. Flasks were incubated overnight at 24 °C. Cultures were treated with chloroform, clarified by centrifugation, and filtered through Millipore 0.22 µm-pore-size membrane filters . Filtrates were tested for the presence of Xcc-specific phages as described [Ten grams of rhizospheric soil from escribed .Xcc bacteria (106 colony-forming units per mL). To facilitate bacterial attachment, the plates were incubated for 72 h at 24 °C without shaking. C20:0 was then added , and again incubated for 8 h. After treatment, planktonic cells were gently removed, and the wells washed three times with water. For NMR studies, C20:0 was used at the lowest concentration.The eicosanoic acid (C20:0) activity was tested by the crystal violet staining test . IndividXcc bacterial suspension was distributed in Erlenmeyer flasks (50 mL/flask) and incubated for 72 h at 24 °C under a static condition. Next, 5 mL of phages (108 plaque forming-units (PFU)/mL), acid (30 µg/mL) or Xccφ1+HA+C20:0  were added to each flask. After 3 h incubation at 24 °C, the cultures were collected, centrifuged  and the supernatants stored at +4 °C for NMR analysis.To facilitate biofilm formation, the TM fitted with a gradient along the Z-axis, at a probe temperature of 27 °C. One-dimensional (1D) proton spectra were acquired at 600 MHz by using the excitation sculpting sequence [1H-13C heteronuclear single-quantum coherence (HSQC) spectra were recorded at 150.90 MHz for 13C using pre-saturation for water suppression [1H and 93.10 ppm for 13C.NMR spectra were recorded on a Bruker Avance III-600 MHz spectrometer , equipped with a TCI CryoProbesequence . Two-dimsequence ,27 were pression . HSQC sp2 and Q2 parameters, which represent the goodness-of-fit and the goodness-of-prediction, measuring how well the model fits the data, and how well the model predicts new data, respectively. For R2 and Q2, acceptable values must have been ≥0.5, with |R2 - Q2| < 0.2–0.3. Normality test and ANOVA test with Bonferroni correction were performed with the OriginPro 9.1 software package .The 0.50–9.50 ppm spectral region of each spectrum was automatically binned into 0.02 ppm width regions (buckets) and integrated using the AMIX 3.9.7 package . The residual water resonance (4.40–5.60 ppm) was removed from the analyzed spectral area, and the integrated sections were normalized to the total spectrum area. To discriminate samples according to their metabolic variations, NMR profiles were studied using the Soft Independent Modeling of Class Analogy (SIMCA)14 package . Principal component analysis (PCA) and Orthogonal Projection to Latent Structures Discriminant Analysis (OPLS–DA)  were perPathway topology and biomarker analysis were carried out using Metaboanalyst 4.0 . MetabolIn bacterial infections, hydroxyapatite (HA) nanocrystals help in bacteriophage delivery and are reported to improve some of the bacteriophage biological properties . In addiIt has been reported that C20:0 does not show a significant decrease in biofilm formation, especially at low concentration . HoweverTransmission electron microscopy (TEM) examination identified Xccφ1 as a member of the Myoviridae family because of the contractile, long and relatively thick tail, with a central core separated from the head by the neck .Xcc biofilm  and exerts its enhancing action [The action of HA on the biofilm was also tested. From crystal violet measurements, we found that HA has no effect on g action   were included in the analysis.The stability of the Xcc, HA, and C20:0 classes were located at positive values. For such a model, we obtained good quality parameters , indicating that this was statistically significant. In particular, the scores plot data indicate that the first component highlights the effects of the phage , while the second one the effects of the C20:0 and HA on the biofilm [We next applied supervised OPLS-DA to uncover metabolic differences between classes. In the scores plot of p(corr)| greater than 0.7. With respect to the untreated biofilm, Xccφ1 induced the production of ethanol, galactomannan and glutamate and downregulated 2-aminoadipate, arginine, betaine, glycine, 3-methylhystidine, isobutyrate, isoleucine, lactate, leucine, lysine, methionine, phenylalanine, propionate, pyroglutamate, saturated fatty acids (SFAs), tyrosine and valine. With respect to Xcc biofilm, C20:0 presented an upregulation of arginine, dimethylamine, isobutyrate, lysine, 3-methylhystidine, pyroglutamate and tyrosine and downregulation of 2-aminoadipate, betaine, glutamate, glycine, isoleucine, leucine, methionine, phenylalanine, SFAs and valine. In comparison with Xcc biofilm, HA brought about an increase of dimethylamine, isobutyrate, lysine, 3-methylhystidine, and tyrosine, with a parallel reduction of betaine, glutamine, glycine, leucine, phenylalanine, and valine. Xccφ1+C20:0 amplified ethanol, galactomannan and glutamate; and reduces arginine, glycine, 3-methylhystidine, isobutyrate, lactate, leucine, lysine, methionine, phenylalanine, propionate, SFAs, tyrosine and valine. Compared to Xcc biofilm, the Xccφ1+HA+C20:0 class showed an increase of ethanol, dimethylamine, galactomannan and SFAs and a decrease of 2-aminoadipate, betaine, glutamate, glycine, isobutyrate, lactate, leucine, lysine, methionine, phenylalanine, propionate, pyroglutamate and valine.The discriminating metabolites were identified in the associated loadings plot of Interestingly, in the phage groups , the dysregulated metabolites showed the same trend, with an increasing tendency towards the Xccφ1+HA+C20:0 class.p(corr)| > 0.7 were used to identify the main metabolic pathways dysregulated between sample classes. Among the found pathways, the statistically significant examples were phenylalanine metabolism .In the present study, by using NMR-based metabolomics, we investigated the metabolic changes brought about by HA, C20:0, Xccφ1, Xccφ1+C20:0, and Xccφ1+HA+C20:0 on the The pathway analysis identified the following dysregulated metabolic pathways involving amino acids: glycine, serine and threonine metabolism; arginine biosynthesis; glutamate and glutamine metabolism; arginine and proline metabolism; and glutathione metabolism . InteresXcc, the C20:0 class, which does not include the phage, displays high levels of arginine, lysine, 3-methylhistidine, pyroglutamate and tyrosine; and low levels of glutamate, glycine, isoleucine, leucine, methionine, phenylalanine and valine. HA increases dimethylamine, isobutyrate, lysine, 3-methylhystidine, and tyrosine, with a parallel reduction of betaine, glutamine, glycine, leucine, phenylalanine, and valine. Xccφ1 shows higher glutamate, and lower arginine, glycine, 3-methylhystidine, isoleucine, leucine, lysine, methionine, phenylalanine, pyroglutamate, tyrosine and valine. Compared to Xcc, Xccφ1+C20:0 amplifies ethanol, galactomannan and glutamate; and reduces arginine, glycine, 3-methylhystidine, isobutyrate, lactate, leucine, lysine, methionine, phenylalanine, propionate, SFAs, tyrosine and valine. Finally, the Xccφ1+HA+C20:0 class is characterized by a decrease of glutamate, glycine, isobutyrate, lactate, leucine, lysine, methionine, phenylalanine, pyroglutamate and valine. Even though all classes affect the film, the metabolic responses involve amino acids at different levels, implying that the lytic action is exerted in different way. For example, although the C20:0 and HA do not exert a bactericidal action, they modify the microbial biofilm structure by altering the permeability of the constituent cells [In particular, with respect to nt cells ,22,31,32Xcc, lactate was downregulated at comparable levels in both Xccφ1, Xccφ1+C20:0 and Xccφ1+HA+C20:0 classes. Lactate contributes to biofilm production [The pattern of lactate is also interesting. We described here a mature (72 h old) biofilm, potentially marked by reduced levels of oxygen—a condition promoting anaerobic glycolysis and the inhibition of the TCA cycle . With reoduction , and addoduction . Therefooduction ,41.High levels of SFAs are observed in the classes treated with phage. Since bacteria in the biofilm state increase their membrane stability and rigidity by incorporating exogenous fatty acids into the membrane , the obsX. campestris [The phage classes also show high levels of galactomannan. It has been reported that xanthan and galactomannan synergistically increase the biofilm viscosity of mpestris . Althougmpestris . Thus, tTaken together, the above results highlight the ability of the phage to dysregulate the amino acids’ metabolic pathways responsible for the formation and maturation of the bacterial biofilm, to reduce the lactate that favors biofilm production, and to upregulate the production of galactomannan that weakens the biofilm.Xcc bacterial biofilm identifying specific metabolic pathways that are dysregulated by the lytic action. Our data demonstrate that Xccφ1 alone or combined with HA and C20:0 interferes with the metabolic pathways involved in biofilm formation. The altered pathways may become the possible targets for the treatment of bacterial biofilm, as well as providing an indication of the efficacy of the treatment. The approach might be extended to the study of other biofilm-producing bacteria, such as Escherichia coli and Pseudomonas aeruginosa, in which Pf4 bacteriophage (filamentous bacteriophage) inhibits the metabolic activity of Aspergillus fumigatus biofilms [In conclusion, we have described here the action of Xccφ1, Xccφ1+C20:0 and Xccφ1-HA-C20:0 against biofilms , and NMR


Synthetic materials using epoxy resin and woven Kevlar fiber nanocomposites were fabricated in the presence of functionalized multiwalled carbon nanotubes (F-MWCNTs). Kevlar-reinforced epoxy nanocomposites were designed to manufacture a small blade of vertical axis wind turbines (VAWT). It is important to estimate the deflection of the versatile composite turbine blades to forestall the blades from breakage. This paper investigates the effect of F-MWCNTs on mechanics and deflection of reinforced epoxy composites. The outcomes show that the mixing of F-MWCNTs with epoxy resin using a sonication process has a significant influence on the mechanical properties. Substantial improvement on the deflections was determined based on finite element analysis (FEA). The vortices from the vertical axis wind turbines (VAWTs) blades have a negative impact on power efficiency, since small blades are shown to be effective in reducing tip vortexes within the aerospace field. To support the theoretical movement of the VAWT blade, modeling calculations and analyzes were performed with the ANSYS code package to achieve insight into the sustainability of epoxy nanocomposites for turbine blade applications below aerodynamic, gravitational, and centrifugal loads. The results showed that the addition of F-MWCNTs to epoxy and Kevlar has a significant effect on the bias estimated by finite element analysis. ANSYS analysis results showed lower deflection on the blade using epoxy with an additional of 0.50 wt.% of MWCNTs-COOH at tip speed ratios of 2.1, 2.6, and 3.1. Today, the extent of consumption of nonrenewable fuel-related energy sources is very high. Solar energy, wind energy, hydroenergy, tidal energy, geothermal energy, and biomass energy are the foremost necessary kinds of renewable energy obtainable . Wind geSeveral synthetic polymer composites are used for wind turbine blade fabrication. glass, Kevlar, and carbon-reinforced matrix are commonly used. Combining Kevlar with epoxy matrix creates a composite that is durable, light, and dimensionally stable. Dielectric strength of Kevlar improves epoxy lightning damage resistance. Composite materials can provide higher stiffness in many cases and reduce the weight of finished parts. These materials may require unique manufacturing techniques to meet design requirements. Thermosetting and thermoplastics polymers can be used for such techniques. Thermoplastics are more viscous when compared to thermosets so impregnation is difficult in thermoplastics. Increasing temperature lowers viscosity, but in some polymers decomposition can occur before very low viscosity is obtained. Epoxy and vinyl ester are the most common matrices that are used to fabricate wind turbine blades. Various functionalized carbon nanotubes (CNTs) such as amino and carboxylated functionalized multiwalled carbon nanotubes (F-MWCNTs) are used to improve the mechanical and physical properties of epoxy ,7. The v2 and Al2O3, especially for the utilization of wind turbine blades. The composites were made by a hand-applied procedure that changes the share of nanomaterials. Results showed that the nanocomposite with 1% Al2O3 confirmed extraordinarily good tensile strength and fatigue strength with most excellent hardness. The results of the ANSYS evaluation showed that the nanocomposite with 1% Al2O3 worked properly under edge and flap loads, which is very acceptable for a long lifetime of wind turbine blades.Muhammed et al.  examinesThe assessment of the structural integrity and harm of the lengthy wind blade under excessive load conditions was studied . The blaA horizontal axis five Megawatts (MW) wind turbine blade was designed and a calculation analysis was carried out. Ansys Fluent was used for the analysis and finishing. It was observed that the utmost momentum was formed at three different entry velocities: 3.5, 12, and 24 m/s at 80° of blade attack angle (Amjith and Bavanish ).As wind turbines expand, suitable regulations are necessary to assure reliable operation and maintenance . To makeThe aerostructure multidisciplinary optimization system was performed for the SERI-8 wing using the Q blade for 2D aerodynamic evaluation. Therefore, the ANSYS bench for 3D aerodynamic and structural evaluation was carried out . A new mSong et al. studied aerodynamic contour layout and overall performance evaluation of wind turbine blades as a critical part of wind turbine layout and application . AccordiMixing MWCNTs with epoxy resin using sonication technique was applied to improve the dispersibility of CNTs. Synthetic fiber-epoxy composites were manufactured using vacuum infusion process. Surface morphology of fracture surface was investigated using scanning electron microscopy (SEM) to study the matrix-fiber interfacial strength. Tensile properties were measured to determine the ultimate tensile strength. Computational fluid dynamics simulation was carried out based on mechanical testing results.Five samples of unmodified epoxy and F-MWCNTs mixtures were synthesized for nanocomposite fabrication. Nanocomposite materials behave differently depending on the work plane, and in order to get valid simulation results, the properties of the materials must be known. Samples comprising F-MWCNT/epoxy/Kevlar laminates were prepared. Unmodified and functionalized (—COOH) grade multiwalled carbon nanotubes were used. Carbon nanotubes were received from Grafen Co., Ankara, Turkey. The specifications of the MWCNTs are given in Bisphenol-A diglycidyl ether-based epoxy is mixed with a hardener in a 100:29 ratio based on the supplier data sheet. Neat epoxy has a tensile strength of 48.1 MPa and modulus of elasticity 2.7 GPa if cured at room temperature for 24 h. Glass transition temperature after complete curing is 45 °C. Specifications of matrix and synthetic fiber are listed in p-phenylene terephtalamide). Fibers are produced in a solution extruder where fiber strength can be controlled. The phenyl groups of the terephthalic segment and p-phenylene segment make angles of −30° and 38° with respect to the chain backbone. Two different models have been proposed for the supermolecular structure of Kevlar fibers. The first model proposes that the molecule’s hydrogen bond together to form sheet along the (100) crystal plane, as shown in Kevlar fibers are high modulus fibers with applications in polymers reinforcements, as shown in Carbon nanotubes were pretreated with acetone in an ultrasonic bath at room temperature for 30 min to separate the nanotube bundles to ensure greater dispersion. Acetone was then evaporated from the blends by holding the samples for 1 h in a vacuum oven at 70 °C. To improve the dispersibility of MWCNTs in epoxy resin, the temperature of the resin was raised to 50 °C for 2 h to reduce the resin viscosity. The unmodified and F-MWCNTs were added at different percentages (0.1 and 0.5 wt.%) and mixed using ultrasonic technique. A titanium alloy probe was immersed in the epoxy/MWCNTs mixture.In functionalized MWCNTs, carboxyl groups serve as proton donors in the epoxide ring opening reaction, leading to enhanced polymerization near the surface of nanotubes and increased interfacial bonding, as shown in The ultrasonic generator transforms the electric current into ultrasonic energy of 20 Hz. Elastic distortion was induced by transducer as alternate voltage transduces mechanical vibration where cavitation and a multitude of microbubbles are formed. This technique allows the epoxy/MWCNTs mixture to undergo intense shaking and vibration, as shown in 2 sheet. Seven layers of Kevlar fiber were placed on a clean glass plate. Care was taken during fiber cutting due to the sensitivity of the material. Fibers could be very easily detached or disoriented from their original places, which would affect the flow of the resin during infusion and might undermine the mechanical properties of the final laminate. All holes were sealed using tacky tape. The vacuum pump was then switched on to start the evacuation process. It was allowed to evacuate for a while before resin infusion to ensure complete evacuation of the cavity and to increase the compaction of the fiber sheets, as shown in Kevlar-reinforced epoxy CNTs were prepared using VIP technique. The pressure from the vacuum pump provides both the driving force for the epoxy/MWCNTs mixture to flow inside the cavity and the compression force to compact the preform to the Kevlar volume fraction. The vacuum was left for 30 min until the resin had completely gelled. The Kevlar/epoxy/MWCNTs composites were cured at room temperature for one day. Five samples  were prepared using a matrix made of neat epoxy, unmodified MWCNTs 0.1% and 0.5%, and COOH-functionalized MWCNTs 0.1% and 0.50%, as listed in Mechanical testing and surface morphology were carried out to study the effect of unmodified and F-MWCNTs on Kevlar-reinforced epoxy.Mechanical tests were carried out on Kevlar/epoxy/F-MWCNTs nanocomposites to measure the static properties. Multidirectional Kevlar-reinforced epoxy specimens were fabricated according to ISO 527-4 and tested at a tensile speed of 2 mm/min. For each type of composite, the number of specimens used for tensile tests was five. Average values of tensile strength were reported and addressed for Kevlar epoxy composite materials.The morphology of the fracture surface of Kevlar-reinforced epoxy nanocomposites were investigated using a field emission scanning electron microscope . Accelerating voltages of 5 and 20 kV were applied. A gold sputter coating of several nanometers was applied on tensile fracture surfaces.The VAWT geometry and its operating conditions were selected based on an optimization study. The optimized VAWT was a three-bladed VAWT with a 1.8 m diameter. Each blade followed the NACA20015 airfoil profile and had a span of 3.6 m. This gave an aspect ratio of two based on the ratio between the turbine height and diameter. At the optimal conditions, the turbine rotated at 20.222 rad/s when the wind speed was 7 m/s. The main parameters of the optimized turbine are presented in r is radius (m), and The tip speed ratio (TSR), which defines the relative velocity of the wind and the velocity of the blade, is presented in Equation (1):The FEA modeling of the optimized VAWT with composite blades was carried out using the ANSYS Static Structural module , which iThe geometry of the modeled VAWT includes the shaft, three blades, and six supporting struts. The geometry was created in Autodesk’s AutoCAD software  based on the information provided in Reference . The shaIn the FEA model of the proposed VAWT, five different types of composite materials were considered. More details about the materials used and their properties are presented in A structured mesh topology was utilized for the meshing of the turbine blades. The VAWTs were subjected to several loads and these included the aerodynamic, gravitational, and centrifugal loads. The current study focused mainly on the effect of the different structure materials and their properties on the maximum deflections of the blades at different centrifugal loads. These centrifugal loads corresponded to three different tip speed ratios (TSRs), including 2.1, 2.6 , and 3.1, corresponding to 16.33, 20.22, 24.11 rad/s. These centrifugal loads were applied to the VAWT rotor in ANSYS Static Structure by imposing the relevant rotational velocity. In addition, a cylindrical support was applied to the turbine shaft.ANSYS Static Structure software  was utilized for the solution and postprocessing of the results. This software can perform different structural analyses, including static analysis and modal analysis. Several results can be extracted, such as the blade deformation and stress distribution. In the current study, the blade deformation was extracted from the static structure simulations.Tensile strength of Kevlar-reinforced epoxy improved by 4% and 19% in the presence of F-MWCNTs (1%) and (5%), respectively. Based on the method discussed in In this study, unmodified MWCNTs and functionalized MWCNTs-COOH were incorporated into epoxy in the presence of Kevlar fiber. The effect of F-MWCNTs and Kevlar fibers on the mechanical and physical properties of epoxy was studied in detail. Failure mechanisms of wind turbine blades depend upon materials or structural defects, and manufacturing process. Many factors like deflection of the wind turbine blades can cause delamination damage that can lead to blade failure. Tensile strength and surface morphology analyses showed that good dispersibility of F-MWCNTs (0.5 wt.%) was achieved using sonication process. Poor distribution and low mechanical strength of Kevlar/epoxy composites were observed in the presence of high CNTs load of unmodified MWCNTs (0.5 wt.%). The Ansys study showed that maximum deflection of Kevlar-reinforced epoxy was successfully decreased to 14% in the presence of F-MWCNTs (0.5 wt.%) at TSR 3.1.


Diabetic nephropathy (DN), a complication of diabetes, is the result of high glucose-induced pathological changes in podocytes, such as epithelial-mesenchymal transition (EMT). Autophagy is an important mechanism of podocyte repair. Ginsenoside Rg1, the active ingredient of ginseng extract, has antifibrotic and proautophagic effects. Therefore, we hypothesized that ginsenoside Rg1 can reverse podocyte EMT via autophagy and alleviate DN. β/β/ This study aimed to investigate the effect of ginsenoside Rg1 on DN rats and high glucose-induced podocyte EMT by regulating the AKT/GSK3in vitro by exposing podocytes to high glucose levels and treated with ginsenoside Rg1. The expression of EMT and autophagy-related markers was analyzed in vivo and in vitro by exposing podocytes to high glucose levels and treated with ginsenoside Rg1. The expression of EMT and autophagy-related markers was analyzed  Diabetic rats induced by STZ injection were treated with 50 mg/kg ginsenoside Rg1 for 8 weeks, and the renal functional, metabolic, and histopathological indices were evaluated. DN was simulated β/β/ Ginsenoside Rg1 significantly alleviated renal fibrosis and podocyte EMT in diabetic rats, and podocytes exposed to high glucose levels, which was abolished by the autophagy inhibitor 3-MA. Furthermore, ginsenoside Rg1 regulated the AKT/GSK3 β/β-catenin pathway-mediated autophagy, indicating its therapeutic potential for DN and other glomerular diseases.β/β/ Ginsenoside Rg1 alleviated podocyte EMT by enhancing AKT/GSK3 It is ain vitro and in vivo models of DN.Ginseng has been used in traditional Chinese medicine formulations for more than 2,000 years and has shown anti-transdifferentiation, antioxidant, antiapoptotic, and autophagic effects –16. The 42H72O14, molecular weight = 801.01, purity by high-performance liquid chromatography (HPLC) ≥ 98%) was purchased from Solarbio. Rapamycin and 3-MA were bought from Selleck Chemicals and STZ from Sigma.Ginsenoside Rg1  were purchased from the Beijing Vital River Laboratory Animal Technology Co. Ltd. The animals were housed in the Laboratory Animal Center of Capital Medical University at 24 ± 1°C and a 12 h light/dark cycle. All experiments were conducted in accordance with the guidelines for the care and use of laboratory animals of the National Institutes of Health and approved by the Animal Welfare Committee of the Animal Laboratory of Capital Medical University.n = 8). Three days after injection, tail vein blood was collected and serum glucose was measured, and fasting glucose level ≥16.7 mM for 3 consecutive days was indicative of diabetes. The diabetic rats were fed with high-fat diet , while the control rats were given normal food. Four weeks after STZ injection, the diabetic rats were randomly divided into the (untreated) DN group (n = 8) and ginsenoside Rg1-treated group (n = 8). The latter was administered 50 mg/kg ginsenoside Rg1 once daily by intraperitoneal injection for 8 weeks, and the other groups received the vehicle for the same duration. At the end of the regimen, urine creatinine (UCr), urinary microalbumin , blood urea nitrogen (BUN), and serum creatinine (SCr) were measured. The rats were sacrificed, and their renal cortices were collected for further analyses.Diabetes was induced by intraperitoneally injecting the rats with 50 mg/kg STZ (streptozocin), and 8 rats were injected with an equal volume of the vehicle  as the placebo/normal control , normal glucose containing mannitol , high glucose , and high glucose with ginsenoside Rg1  for 48 h.The conditionally immortalized mouse podocyte line MPC5 was purchased from the National Platform of Experimental Cell Resources for Sci-Tech. The cells were cultured at 33°C in DMEM/low glucose medium supplemented with 10% fetal bovine serum (FBS) and recombinant IFN-μm thick sections. After dewaxing with xylene and rehydrating through an alcohol gradient, the sections were stained with hematoxylin and eosin, periodic acid Schiff (PAS), and Masson's stain according to standard protocols. For electron microscopy, the cortex tissues were immobilized with 2% glutaraldehyde in 0.1 M phosphate buffer at 4°C for 2 h. After gradient dehydration, the tissues were osmotically embedded and cut into ultrathin sections that were then double stained with 4% uranyl acetate and lead citrate.Kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 3 α-smooth muscle actin (α-SMA), beclin-1, p62, AKT, P-Akt, GSK3β, p-GSK3β , and LC3-II (Sigma). The blots were washed and incubated with the HRP-conjugated secondary antibody and developed using chemiluminescence reagents.The podocytes and kidney tissues were lysed, and equivalent amount of protein (20 g) per sample was separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skimmed milk in the TBST buffer, the membranes were incubated with antibodies against nephrin, Total RNA was isolated from the cells/tissues using TRIzol® reagent according to the manufacturer's instructions and reverse transcribed using the SuperScript RT kit. The SYBR Green kit was used for qRT-PCR, and the 2ΔCT method was used to calculate the relative gene expression levels. The sequence of primers is shown in The tissue sections were processed as described above and, after antigen retrieval, blocked with sheep serum for 1 hour. The sections were incubated overnight at 4°C with the primary antibodies. After counterstaining with DAPI for 5 minutes, the sections were observed under a fluorescence microscope.P < 0.05 was considered statistically significant.SPSS software  was used for statistical analysis. All data were expressed as the average (±SD) of at least three independent experiments, and analyzed by single factor variance. Compared to the control animals, all indices of renal function-renal weight/body weight ratio and the levels of serum creatinine, urea nitrogen, urinary creatinine, and urinary microalbumin were significantly increased in the DN group. Ginsenoside Rg1 improved the above parameters in the DN rats (see Figures α-SMA levels compared to controls, which was strongly indicative of EMT in the podocytes, and was reversed by Ginsenoside Rg1 (see Figures The renal cortex of the DN rats showed a significant decrease in nephrin levels and increased β/β-catenin pathway is frequently implicated in EMT, we also analyzed the expression levels of its components in in vivo model of DN. Hyperglycemic conditions resulted in a significant increase in β-catenin and decrease in p-GSK3β and p-AKT in the podocytes and renal cortices compared to the controls, which were restored by ginsenoside Rg1 treatment (see Figures β/β-catenin pathway.Since the AKT/GSK3in situ levels of LC3-II and beclin-1 protein and mRNA, along with decreased p62 levels. Furthermore, ginsenoside Rg1 also upregulated the autophagic markers in podocytes cultured under hyperglycemic conditions. Taken together, ginsenoside Rg1 induced autophagy in the podocytes both in vivo (see Figures in vitro (see Figures To investigate the effect of ginsenoside Rg1 on podocyte autophagy in DN, we analyzed the expression levels of LC3-II, beclin-1, and p62 in the renal cortices of the diabetic rats. Compared to the untreated DN rats, those treated with ginsenoside Rg1 showed a significant increase in the α-SMA under hyperglycemic conditions, inhibition of autophagy by 3-MA reversed the effects of ginsenoside Rg1 (see Figures β activity and β-catenin expression along with ginsenoside Rg1, while 3-MA inhibited the AKT/GSK3β/β-catenin pathway (see Figures β-catenin pathway.To further explore the role of autophagy in ginsenoside Rg1 action in the podocytes, we treated the cells additionally with either the autophagy activator rapamycin or inhibitor 3-MA. While ginsenoside Rg1 and rapamycin increased nephrin levels and decreased that of β1/Smads pathway [Ginsenoside Rg1 is the main active ingredient of the ginseng rhizome extract and has antiapoptotic, antioxidant, anti-inflammatory, and neuroprotective functions –19. In a pathway . In thisα-SMA is a marking factor of interstitial formation as well as a marker of mesenchymal fibroblasts [α-SMA, which was reversed by ginsenoside Rg1. Therefore, the Rg1 ginsenosides suppressed hyperglycemia-induced podocyte EMT, which alleviated the DN symptoms.Hyperglycemic conditions can result in abnormal renal structure and function such as tubulointerstitial fibrosis and glomerulosclerosis in the early stage of diabetes mellitus , 25. Theroblasts . The expα-SMA was decreased, indicating that autophagy can protect podocyte injury. Treatment with ginsenoside Rg1 restored autophagy in the podocytes, which is the likely mechanism underlying inhibition of EMT. The autophagy inhibitor 3-MA abolished the protective effects of ginsenoside Rg1, thereby validating our hypothesis.Autophagy is a highly conserved eukaryotic cell cycle process, which plays an important role in cell survival and maintenance. In mammalian cells, there are three different types of autophagy: microautophagy, macroautophagy, and partner-mediated autophagy. Although the morphology of these three autophagy modes are different, the final form of autophagy is to deliver materials. The substance is transported to lysosome for degradation and recovery . In receβ [β-catenin pathway [β-catenin in diabetic patients and mouse models plays a key role in inducing podocyte EMT [β-catenin pathway may be a key node in the treatment of diabetic nephropathy. Glycogen synthase kinase 3β is a widely expressed serine/threonine protein kinase, as an important substrate of AKT, originally identified as an enzyme needed to regulate glycogen metabolism, and is now found to be a multifunctional kinase that plays an important role in many cell processes and diseases [β is negatively regulated by AKT, which inhibits glycogen synthesis and lowers blood glucose by inhibiting GSK3β phosphorylation [β plays an important role in cell growth and development by phosphorylating a variety of endogenous substrates. Decreased activity of AKT could inhibit the phosphorylation of GSK3β and then leads to the accumulation of β-catenin in the cytoplasm of podocytes. β-catenin eventually enters the nucleus and triggers EMT [β/β-catenin pathway, which weakens the invasion and migration of hepatocellular carcinoma cells [β/β-catenin pathway is still unclear. According to the results of this study, the activation of autophagy in hyperglycemic conditions will promote Akt phosphorylation and then activate the phosphorylation of GSK3β, which is the key protein of Wnt/β-catenin pathway. This will lead to the decrease in accumulation of β-catenin and expression of α-SMA and ultimately alleviate podocyte EMT. The amelioration of podocyte EMT after ginsenoside Rg1 intervention may be correlated to the regulation of the AKT/GSK3β/β-catenin pathway by ginsenoside Rg1-enhanced autophagic activity.Podocyte EMT involves various signaling cascades, such as Wnt and TGF-β . Hypergl pathway . Increascyte EMT . Therefodiseases –42. GSK3rylation . In addigers EMT . Astragama cells . Autophaβ/β-catenin pathway, providing a reasonable basis for its therapeutic use in DN.In conclusion, ginsenoside Rg1 inhibits podocyte EMT by inducing autophagy via regulation of the AKT/GSK3β/β-catenin pathway. Meanwhile, ginsenoside Rg1 improves albuminuria and the renal function in DN rats. These findings point to the possible therapeutic use of ginsenoside Rg1 in DN. However, additional studies are needed to comprehensively understand the role of ginsenoside Rg1 in ameliorating DN.The data obtained in the present study indicate that ginsenoside Rg1 induces autophagy in renal tissues and podocytes exposed to high glucose via activation of the AKT/GSK3


NF2-mutant meningiomas) account for ~40% of the sporadic meningiomas. However, there is still no effective drug treatment for the disease.Meningiomas with Neurofibromin 2 gene mutations (n = 346). A 20-agent library covering a wide range of meningioma relevant targets was tested using meningioma cell lines IOMM-Lee (NF2 wildtype) and CH157-MN (NF2 deficient). Therapeutic effects and biological mechanisms of the identified compound, ICG-001, in NF2-mutant meningiomas were further characterized in vitro and in patient-derived xenograft (PDX) models.Expression profile of Merlin protein was explored through immunohistochemistry in a meningioma patient cohort -binding protein (CBP) inhibitor, selectively suppressed tumor growth of cells with low Merlin expression. Besides, ICG-001 mediated CH157-MN and IOMM-Lee growth inhibition primarily through robust induction of the G1 cell-cycle arrest. Treatment with ICG-001 alone significantly reduced the growth of NF2-mutant meningioma.This study highlights the importance of ligand-mediated Wnt/β-catenin signaling as well as its drugable potency in  Low Merlin expression was associated with meningioma proliferation and poor clinical outcomes.Low Merlin expression sensitizes meningiomas to ICG-001 treatment, at least partly, by attenuating the FOXM1-mediated Wnt/β-catenin signaling.NF2-mutant meningiomas. We conducted the largest cohort study showing the expression and clinical significance of the Merlin protein, the NF2 gene translated product, in meningioma patients. Based on drug testing, the Wnt/β-catenin pathway, which is blocked by ICG-001, is important for the growth and survival of NF2-mutant meningiomas. Our study highlights the importance of ligand-mediated Wnt/β-catenin signaling as well as its targetable potency in NF2-mutant meningiomas.Some ongoing clinical trials are looking for new methods to treat refractory meningiomas in order to obtain better prognosis. However, there are currently no FDA-approved drug therapies for the treatment of 2 The WHO grade II–III group comprises ~20% of all meningiomas characterized by aggressive biology with increased proliferation and a strong tendency to recur, resulting in substantial morbidity and increased mortality.2 Although most benign meningiomas can be treated with surgical resection, high-grade meningiomas that exhausted all surgical or radiotherapeutic options still need novel therapies to improve outcomes.3 Recent high-throughput studies have defined novel driver mutations that strongly associate with clinicopathologic features.5 Loss of the Neurofibromin 2 (NF2) gene, located on chromosome 22, is one of the main genomic alterations found in high-grade meningiomas.6 Splice site, nonsense, and frameshift mutations are the most common disruptors of NF2, resulting in a truncated and nonfunctional protein.6 Moreover, NF2-mutant cases often present multiple and malignant tendency, inducing severe neurologic morbidity and mortality.6 The lack of effective systemic therapy for meningiomas represents a significant challenge in the clinical management of patients with NF2 mutations.Meningiomas are the most common primary intracranial tumors in adults and ~80% are classified by the World Health Organization as WHO grade I lesions.NF2 gene translated product, is a cytoskeletal protein that provides a functional link between the cell membrane and the actin cytoskeleton.7 A number of studies demonstrated that Merlin is a versatile tumor suppressor that can inhibit cancer cell proliferation and motility by modulating a wide range of signaling pathways. For instance, active Merlin suppresses Rac-PAK signaling,8 restrains activation of mTORC1 independently of Akt,9 inhibits PI3K-Akt and FAK-Src signaling,11 and negatively regulates the EGFR-Ras-ERK pathway.12 Merlin has also been reported regulating the Hippo tumor-suppressor pathway in meningiomas.13 Besides, the loss of Merlin results in aberrant activation of the Wnt/β-catenin signaling in brain tumors.14 Forkhead box M1 (FOXM1) is a key mediator of the Wnt/β-catenin signaling, which binds to β-catenin, thus enhancing β-catenin nuclear localization and transcriptional activity.16 Recently, FOXM1/Wnt signaling was found to be associated with mitotic gene expression in aggressive meningioma.17 Thus, we are interested in potential functional connections between these two intensively studied oncogenic pathways.Merlin, the www.synapse.org/SynodosNF2).18 Yet despite these advances, targetable pathways associated with poor meningioma outcomes remain elusive. A number of drugs including hydroxycarbamide, interferon-alfa, megestrol acetate, medroxyprogesterone acetate, octreotide, pasireotide (long-acting release), trabectedin, imatinib, erlotinib, gefitinib, vatalanib, sunitinib, and bevacizumab, as well as cyclophosphamide-doxorubicin-vincristine chemotherapy, have been evaluated.20 Unfortunately, most of the small and uncontrolled studies and case series suggest that systemic therapies have only minimal activity against aggressive meningiomas.21–24 The high failure rate of preclinical agents in clinical trials clearly demonstrates that even when targetable genomic alterations are discovered, patients do not always respond to therapy.Prospective clinical trials are evaluating novel drugs based on the further understanding of the pathobiology of treatment-resistant meningiomas. The ongoing preclinical studies also presented several targets for systemic therapies, seeking treatments for neurofibromatosis 2–related meningioma -binding protein inhibitor, both in vitro and in vivo. The effect of ICG-001 is at least partly through attenuating the FOXM1-mediated Wnt/β-catenin signaling. Our results thus advocate ICG-001 as a promising agent for further preclinical evaluation in NF2-mutant meningiomas.Strategies to confirm therapeutic efficacy or identify additional options would be beneficial to both clinicians and patients. To address this need, we screened 10 patient-derived primary tumor cell lines and two high-grade meningioma cell lines with a panel of agents under clinical and preclinical investigation. A focused drug screen showed that We recruited 355 adults with primary meningioma who underwent surgery from 2008 to 2017 in the Department of Neurosurgery, Huashan Hospital of Fudan University, Shanghai, China  and S2. NF2 mutation based on diagnoses from referring pathologists. Patients with a family history of neurofibromatosis type 2-related meningioma history were not recruited for this study. The tissue microarray (TMA) constructions were sectioned (5 μm) according to standard protocols for hematoxylin and eosin (HE) staining. The TMA immunohistochemical analysis was conducted with anti-Merlin, anti-FOXM1, β-catenin, and anti-Ki-67  antibodies as described previously.25 The staining TMAs were scanned  and analyzed by Quant center software. Histoscore (H-SCORE) was calculated according to the following formula：H-SCORE = ∑（PI × I）=（percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + percentage of cells of strong intensity × 3).26Tumor specimens for drug testing were classified as wildtype or associated with the CH157-MN and IOMM-Lee meningioma cell lines were a courtesy of Prof. Wan’s lab  and cultured in DMEM/F12 medium  with 10% Fetal Bovine Serum . Primary meningioma cells were separated from high-grade meningioma patient tissue and cultured in DMEM/F12 medium containing 2% B27, 1% N2, bFGF, and EGF (20 ng/ml each) supplement (Invitrogen). Spheres from three independent experiments (day 7) were plated in standard culture medium with 10% FBS to allow cells to adhere. Tumor cells were identified by Vimentin  and epithelial membrane antigen (EMA) staining  before drug screening.50 and the maximum effect for each compound were calculated in the dose–response screens.CH157-MN, IOMM-Lee, and primary cells were cultured for 12 hours before the compounds were added. To perform dose–response screening, agents were tested in a nine-concentration semilog serial dilution ranging from 0 to 20 µM with eight replicates for each concentration. ATP levels were analyzed following compound treatment for 72 hours using the Cell Counting Kit 8 assay  according to the manufacturer’s recommendations. The percentage of viable cells for each agent was calculated by normalizing the raw data to the mean of DMSO control wells (100% viability). The absolute half-maximum effective concentration ICP-value < .05. For functional analysis, gene ontology enrichment was tested accounting for gene length via R-package goseq. GO-annotation analyses were performed using DAVID Bioinformatics Resources 6.8 .28 RNA-seq data can be accessed from the BPbrowse database (http://122.112.248.194/GMOD/BP_yun/?data=sample_data/json/BPdata/Project_1711SZSC03B/data).RNA was extracted from three biologic replicates of CH157-MN and IOMM-Lee cells 8 and 24 hours after treatment with 5 μM ICG-001 or DMSO. The RNA quality and integrity were analyzed by Qubit 2.0  and Bioanalyzer 2100 . The sequencing library was qualified by Qubit 2.0  and Bioanalyzer 2100  and then sequenced on Illumina Hiseq X with 2 × 150 bp paired-end sequencing, which were controlled by Hiseq Control Software (HCS). Candidate genes were filtered to a minimum of 2× fold changes and FDR-corrected For cell-cycle analysis, cells were treated for 2 hours prior analyses. Cells were washed in PBS and then trypsinized. All the material was collected, combined, centrifuged at 15,000 rpm for 5 minutes, and washed with PBS. Pellets were resuspended in 500 µl binding buffer and stained with propridium iodide (PI). Cell-cycle analysis was performed using Summit 5.2 software. Following designated treatment, cells were pulsed with 5-ethynyl-2-deoxyuridine , for 2 hours before fixation in 4% paraformaldehyde and subsequent EdU detection as per the manufacturer’s protocol. Coverslips were mounted on slides and imaged using a confocal microscope. Quantification of EdU+ tumor cells was carried out using the cell counter plugin from the Image J software (d 1.47 version). The percentage of EdU+ for each field of view captured was recorded and analyzed.25 using antibodies specific for β-catenin , CBP , Merlin , FOXM1 , p21 , survivin , and β-actin . For immunoprecipitations, cell lysates and indicated antibodies (1 µg) were subjected to overnight rotation at 4°C. Protein A-Sepharose beads  were added for 3 hours, followed by centrifugation, washed four times, and boiled with loading buffer for immunoblotting analysis.Immunoblotting analysis was performed as described previously29 Patient tumor material was collected in culture medium and kept on wet ice for engraftment within 12 hours after resection. The patient-derived xenograft model was established by direct implantation of 20–30 mg of surgically resected tumor tissue into the flank region of female BALB/c nude mice  followed by a second round of serial propagation in nude mice. Intraperitoneal drug injections were administered once the tumor volume reached about 30 mm3: ICG-001  and vehicle alone . The treatment was extended for a total of 8 weeks. Tumor size and animal body weight were measured on designated days.All animal studies were conducted in accordance with the guidelines as published in the Guide for the Care and Use of Laboratory Animals.The plasmid pcDNA3.0-Flag-tagged Merlin was the full-length Merlin isoform I and was obtained from Addgene. Mutated Merlin-S518A was generated via overlapping PCR and inserted into pcDNA3.0 (pM-S518A) and pcDNA3.0-HA (HA-M-S518A) vectors. Targeting FOXM1 (siFOXM1) consisted of a pool of three target-specific 20- to 25-nt siRNAs designed to knockdown FOXM1 expression and was obtained from Santa Cruz Biotechnology . After transfection, cell proliferation was measured with the CCK8 assay according to the manufacturer’s instructions. Cells were collected on day 3 to check for knockdown efficiency.t test, unless otherwise noted in the figure legend. Survival curves were generated using the Kaplan–Meier method; differences were assessed by log-rank tests. Data were considered to be significant when P < .05.All statistical comparisons between two groups were performed using GraphPad Prism software 6.0 and a two-tailed unpaired P < .05) between groups of different mitotic activity in meningioma  (P = .0244) and decreased overall survival   and a mean age of 52.29 ± 15.7 years (range 19–86 y) were included in this study. Thirty-three patients (9.54%) did not attend follow-ups and were excluded from the final analysis. For the remaining 313 patients, the median follow-up was 76.5 months (range 1–142 mo). Clinicopathological characteristics of patients with low or high Merlin expression are summarized in ningioma , as defi < .001) . No sign = .014)  and 1D.NF2 wildtype), CH157-MN (NF2-deficient)  , and a 2ficient) . We asseficient) . BesidesNF2-mutant meningiomas, we performed another testing with ICG-001 in ten high-grade patient-derived cell lines , whereas among the less responding cell lines , most of them expressed high Merlin levels  in RNA-seq microarray analysis  and G1/S transition of the mitotic cell cycle  biological process in CH157-MN  and cellular response to oxidative stress , which means IOMM-Lee resisted damage, whereas CH157-MN was more vulnerable to ICG-001 treatment  (Both CH157-MN and IOMM-Lee cells treated with ICG-001 resulted in a significantly decrease in proliferation as evidenced by reduced EdU incorporation . The expICG-001) . NoticeaICG-001) .NF2-mutant meningioma (M8 and M9) using PDX models as well as safety considerations measuring by animal weight. ICG-001 significantly inhibited xenografts growth compared with vehicle alone as determined by tumor volume , brain invasion, and/or at least three other aggressive morphologic features.2 The degree of mitotic activity is an indicator of the proliferative potential of the tumor. MIB-1 expression in tumor lesions directly correlates with tumors’ proliferation rates and thus may aid in predicting meningioma recurrence.35Over the past decades, our knowledge of meningioma genetics has expended exponentially; NF2-mutant meningiomas. Specifically, we described 10 high-grade meningioma cases that underwent surgical excision together with focused drug screen using a comprehensive library of up-to-date targeted agents as well as chemotherapeutics. Interestingly, for the NF2-mutant cases, ICG-001, a CBP inhibitor of the Wnt/β-catenin signaling pathway, showed the optimal treatment effect both in vitro and PDX models. Merlin acts as a tumor suppressor and is capable of modulating a wide range of signaling pathways, including the Ras/Raf/MEK, PI3K/AKT/mTOR, Rac/PAK/JNK, and Wnt/β-catenin pathways.9–12 Numerous studies have documented the amplification or overexpression of positive regulators of the Wnt pathway in meningiomas.37 Moreover, recent studies have suggested that Merlin can inhibit Wnt/β-catenin signaling through inhibiting β-catenin phosphorylation, thus blocking the translocation of β-catenin from the membrane to the nucleus by inhibiting its dissociation from the adhesion junction.38 The fact that Merlin-negative cells were especially sensitive to ICG-001 suggests that the Merlin/Wnt/β-catenin axis may play a role within meningioma tumor cells.Based on these results, we look further to find more therapeutic avenues in 40 The central interaction between β-catenin and T cell factor (TCF) is critical to its Wnt activation and represents an attractive target for the general inhibition of canonical Wnt signaling. ICG-001 disrupts the interaction of CBP with β-catenin, specifically downregulating the expression of a subset of β-catenin/TCF-responsive genes.31CDKN1A encodes the cyclin-dependent kinase inhibitor p21 that opposes the function of multiple cyclin-CDK complexes mediating G1 and S cell-cycle progression.41 ICG-001 increased CBP occupancy at CDKN1A promoter site in tumor cells by increasing the availability of CBP to different subsets of transcription factors mediating the CDKN1A expression.30 Mutagenesis experiments demonstrated the importance of TCF-binding elements in the survivin promoter.42 We believe that the observed decrease in survivin expression is also responsible for the induction of apoptosis of the meningioma cells, leading to tumor reduction in vivo. Together, we anticipate that aberrant activation of β-catenin/CBP transcription is a critical event in the development of meningiomas.Besides, ICG-001 strongly induced G1 cell-cycle arrest in meningioma cell lines and inhibited the expression of certain genes linked to downstream of the Wnt/β-catenin pathway. Multiple interactions have been described between β-catenin and components of the Wnt enhancer on promoters of Wnt target genes.16 Several studies suggested that TCF/LEF and β-catenin coactivators, such as Pygopus/Bcl9, mediate β-catenin nuclear import or retention/anchoring.43 In the present study, we systematically analyzed the Merlin expression patterns in meningioma cell lines and tumor specimens. Our results revealed for the first time that the overexpression of FOXM1 attenuated the suppressive effect of Merlin on Wnt/β-catenin signaling, thus sensitizing NF2-mutant meningioma cells to ICG-001. To some extent, Merlin may indirectly regulate modification of FOXM1 protein, which decreases the stability of FOXM1.32 Although the detailed mechanisms underlying the regulation of FOXM1 protein stability by Merlin require further elucidation, this novel finding regarding Merlin/FOXM1/β-catenin signaling is critical to identifying the molecular mechanisms underlying NF2-associated meningioma pathogenesis.FOXM1 is an unexpected but key mediator of the Wnt/β-catenin signaling as it binds to β-catenin, thus enhancing β-catenin nuclear localization and transcriptional activity.NF2-associated meningiomas are especially sensitive to ICG-001 in vitro and in vivo partly because of the attenuation of the FOXM1-mediated Wnt/β-catenin signaling. Our findings also provide the rationale for the future development of novel mechanism-based therapies for NF2-mutant meningiomas.In summary, we have demonstrated for the first time that NF2-mutant meningiomas will certainly be required in the future.The present study has several limitations. First, the clinical data set presented here is limited by the selection bias of retrospective nature of this study. Second, about the intratumor heterogeneity of meningiomas, a subset of tumor Merlin-positive tumors might be scored as negative in TMAs. However, this potential misclassification of specimens due to heterogeneity would be considered as randomized distribution, and hence, would most likely have yielded null results. Lastly, the number of targets interrogated with currently available drugs was limited. Additional studies exploring the biology and treatment of vdz055_suppl_Supplementary_Figure_S1Click here for additional data file.vdz055_suppl_Supplementary_Figure_S2Click here for additional data file.vdz055_suppl_Supplementary_Table_S1Click here for additional data file.vdz055_suppl_Supplementary_Table_S2Click here for additional data file.vdz055_suppl_Supplementary_Table_S3Click here for additional data file.vdz055_suppl_Supplementary_Table_S4Click here for additional data file.vdz055_suppl_Supplementary_Figure_LegendClick here for additional data file.


Background: Since numerical calorie labels have limited effects on less-calorie food ordering, an alternative called physical activity calorie equivalent (PACE) labels, which exhibit calories using visible symbols and the minutes of exercise to burn off the calories, may be more effective in reducing calories ordered. Methods: By using a choice experiment (CE) approach, the aims of this study were to estimate the effects of PACE labels on consumer preferences for healthy and unhealth food. Red date walnuts and potato chips were used as the representatives of healthy and unhealthy foods respectively in this study. Moreover, future time perspective (FTP) is an individual trait variable of consumers, which has been recognized as a significant driver of healthy behaviors. We also included FTP into the interaction with PACE labels. Results: Firstly, the results were opposite between the healthy and unhealthy food groups. Respondents showed significantly more positive attitudes toward red date walnuts  with PACE labels, while they showed significantly more negative preferences for chips  with PACE labels. Secondly, people with higher FTP are preferred red date walnuts with PACE labels, while PACE labels on chips could undermine the preferences of respondents with higher FTP. Thirdly, we found that women (vs. men) were less inclined to choose healthy food with standard calorie labels and labels showing the minutes of running to burn off the calories, as well as that the elderly (vs. younger) people in the healthy food group preferred the labels showing the minutes of running to burn off the calories. People with a higher body mass index (BMI) were reluctant to purchase walnuts with the information about the minutes of walking. Conclusions: Results from this study showed that PACE labels have significant effects on consumers’ preferences for food products. The global obesity epidemic has gained a lot of attention in recent decades due to its links with some chronic diseases, such as type 2 diabetes, dyslipidemia, cardiovascular disease, and periodontal disease ,2,3. TheTwo major causes of obesity across all populations are unhealthy eating and inadequate exercise . GovernmPhysical activity calorie equivalent (PACE) labels, which were proposed by the Royal Society for Public Health , which eSeyedhamzeh et al.  mentioneWhen it comes to research on healthier food purchasing, the future time perspective (FTP) is an individual trait variable of consumers that might play a significant role in healthy behaviors . AccordiThe aims of this study were triple: (a) to estimate the effects of different forms of labeling  of an unhealthy product and a healthy alternative ; (b) to determine the FTP of consumers and the interaction effects between FTP and PACE labels; (c) to measure the interaction effects of socio-demographic indicators  and PACE labels on consumers’ preferences for foods.In previous studies on consumer preferences for PACE labels, participants were assigned to different label information conditions and were asked to select the provided foods, furthermore, researchers estimated the differences in evaluations of provided foods or calorie ordering between groups under different label conditions ,44,45. IThe efficiency and accuracy of the CE approach were determined according to the chosen attributes , which mAccording to the review by Thow et al. , the effThe accuracy of a CE depends on the structure of choice sets, which means that the designed choice sets should explain the maximum variance of the attributes while minimizing the random errors . Many no2 = 256 choice sets. It was impossible for the respondents to evaluate all of the choice sets. A fractional design was determined to be appropriate for this study, as it can maintain the efficiency of profiles while minimizing the number of tested choice sets [A full factorial design in the Negene 1.1 software  was based on the two selected attributes and four levels of each attribute, and as for the two options we designed, which generated (4 × 4)ice sets . FinallyThe FTP of consumers was measured with an improved Zimbardo Time Perspective Inventory derived from a study by He et al. . In partIn order to confirm whether consumers could distinguish between the health levels of red date walnuts and potato chips. We tested this question with two items, which referred to the study by Naylor et al. : “The foBefore we conducted the experiment, a pre-test (N = 56) was conducted in the School of Business in Renmin University of China. We sent designed stimuli  to 56 undergraduates through E-mail, and asked them to reply whether they could clearly distinguish the differences of PACE labels with running or walking. Finally, we got 53 answers that expressed they can understand the meaning of PACE labels, and other 3 students didn’t reply to us. Results indicated that respondents can correct understanding of the stimuli in this study.We conducted the experiment through a professional online survey company, Credamo, as  pointed We set a “attention check” in the questionnaire to identify the careless respondents . The queThe CE was based on the Lancaster consumer theory , which sn’s utility from alternative i is expressed as nitU. The consumer utility consists of the observable representatives nitV and the unobservable random error nitε [An individual ror εnit .In discrete choice modeling, different hypotheses of the random error distribution and heterogeneity will lead to different models. In this study, we assumed that all respondents share a homogeneous preference for the attributes of the product , and thuWhen estimating the main effects of the attributes, consumer utility can be expressed with Equation (2). In this study, the attribute “Label” is a nominal variable, as it represents the different forms of labels. Dummy variables were generated with the baseline of not being labeled. The price is the metric variable, and was designed with the four levels shown in nit of each variable indicates the attributes for which individual n chooses an alternative i in the choice set n. β1 to β4n are the parameter vectors of the attributes to be estimated.The nFTP is the mean score of individual n’s answers to the five questions. β1 to β7n are the parameter vectors of the attributes to be estimated:As for the interaction effects between the attributes and FTP, we put the FTP variable into Equation (3). FTP is a metric variable, and n’s WTP for attributes x is estimated as:nxβ is the coefficient of the non-price attribute x and npβ is the coefficient of the price attribute np.Individual After dropping the questionnaires containing careless answers to the “attention check”, there were 285 questionnaires left in the red date walnut group and 243 in the potato chip group. All of the data are calculated with Stata 15.0. Similar distributions could be found in the two groups. Female respondents slightly exceeded men in both groups, making up 56.49% and 53.50%, respectively. Among all of the respondents, ages between 25 and 34 had the highest percentages in both groups (69.93% and 67.90%), followed by people under 24 years old and 35–44 years old. However, respondents over 45 years old were rare in this study. We also asked respondents to fill in their weight and height to calculate the body mass index (BMI) of the respondents. The mean BMI scores were 21.32 in the red date walnut group and 20.90 in the potato chip group .p < 0.001), which indicated that the two items concerning the health level were significantly correlated and thus reliable to form a health level index. The mean scores for the health levels demonstrated an obvious difference between the two groups. The mean value was 4.04 in the red date walnut group, which means that the consumers recognized red date walnuts as a healthy food. Meanwhile, the score for potato chips was 2.78 , which indicated that people perceived that potato chips contain few nutrients. These results proved that the foods we chose in this study were able to represent an unhealthy and a healthy alternative.All item scores for the FTP are shown in The main effects of the standard calorie and PACE labels are illustrated in Taking the FTP into consideration, the results of the main effect including the interaction are shown in the Results of interaction effects between labels and socio-demographics were shown in In this study, the effects of four labels  on consumers’ preferences for two products  were explored with a CE approach, and we also estimated the interaction effects between FTP and socio-demographic variables. Based on the results of this study, three important research conclusions and their theoretical contributions are summarized.Firstly, the effects of the PACE labels, standard calorie label, and lack of label on consumers’ preferences were investigated. Results showed that PACE labels have statistically significant effects on consumers’ preferences for both products in this study. It has also been demonstrated that the public prefer PACE labels over other types of food labels . The sigResults of this study illustrated that the coefficients of minutes to walk label (0.54) and minutes to run label (0.54) were higher than the standard calorie label (0.37) in red date walnut group, which means PACE labels can most effectively improve consumers’ preferences for healthy food. However, the effects of those three labels were changed in potato chips group. Minutes to walk label (−0.25) has a more negative effect on consumers’ preferences for unhealthy food than the calorie label (−0.20), while minutes to run label (−0.18) was inferior to the calorie label. In previous studies, investigators showed the effects of PACE labels from multiple perspectives, such as by researching the utility of PACE labels from the perspective of consumers’ sensory and emotional cognitions , studyinSecondly, the results showed that compared with the no-label condition, the positive or negative effects of the PACE and calorie labels on consumers’ preferences depending on the food type. In this study, for the healthy alternative , compared with the lack of a label, the PACE and calorie labels significantly improved preferences of consumers, and consumers in this group preferred PACE label with minutes to walk most. For the unhealthy food (potato chips), the main effects of the labels were the absolute opposite, which means that people were reluctant to see any information about the calories contained in the potato chips, especially with the label showing the minutes of walking needed to burn off the calories. In this group, the PACE and calorie labels significantly reduced preferences of consumers. The contrary results were in line with previous literature that effects of FoP labels on consumers preferences were inconsistent between healthy and unhealthy foods ,32,33,69Thirdly, the future time perspective (FTP)  was intrFourthly, the socio-demographic of respondents  were contained in relative literature about PACE labels ,36. We fThis study has two important theoretical contributions: The first theoretical contribution of this study is to demonstrate the difference in the effects of PACE labels on different food types of varying healthiness, but also tried to explain the current literature conflict in the study of effects of PACE labels. Meanwhile, because PACE labels can improve consumers’ preferences for healthy food, but inhibit consumers’ preferences for unhealthy food . In realThe second contribution is to determine the interaction effects between FTP and PACE labels. No previous study has analyzed the relation between FTP and food choices in the context of PACE labels. FTP in this study not only improved the applicability of the interaction effect in terms of socio-demographics, but also had an insight into the potential effects of PACE and calorie labels on consumers’ preferences for different type of foods. In future research, efforts should not only put on the interactive analysis of labels and socio-demographics, but also introduce a psychological perspective  to analyze consumers’ preferences for foods.The results of this study can also give some practical implications to the government, health organizations and food enterprises. As a visual reminder of calorie information, PACE labels can be used as a nudge tool to effectively improve consumer behavior ,10. And The limitations and future research directions of this study are as follow. Firstly, in this study, a CE was used to explore the main effects of PACE labels, with no labeling and calorie labeling, and the moderating effect of healthy and unhealthy food types on this main effect was also explored. But what psychological factors of consumers ultimately lead to the main and moderating effect? This is not explored in this article. Although in the discussion, we tried to explain it through consumers’ anticipatory guilt and justification reasons, in the future research, we still need to analyze it through more rigorous behavioral experiments and explore the stabilizing role of the potential psychological mechanism in the main and moderating effect.Secondly, we choose age, gender and BIM as the basic socio-demographics to analyze the main effects of PACE label and calorie label, and obtained a certain result of interaction effect in this study. At the same time, we also introduced FTP as a consumer characteristic to analyze the interaction effect. However, there are also many consumer characteristics that deserve to be studied, such as time pressure and sensitivity , which mThis study demonstrates that PACE labels have different influences on consumers’ preferences for difference food types. In terms of red date walnuts, which were the representative healthy food in this study, people showed positive preferences for products with PACE labels. In other words, healthy foods with PACE labels are more likely to be purchased by consumers. On the other hand, consumers showed a negative preference for potato chips with kcal and PACE labels, which means that people are reluctant to buy unhealthy foods with those labels, especially with PACE labels that show information on the minutes of walking needed to burn off the calories. FTP was shown to enhance the preferences for healthy food with PACE labels, while people with higher FTP scores showed negative preferences for unhealthy food with PACE labels. This study indicates that PACE labeling may be an effective strategy for promoting purchase of healthy foods, as well as a tool for decreasing purchase intentions for unhealthy food.