Cure Index (CI) was used for qualitative analysis of epoxy crosslinking in the presence of Mica and Mica-IM, while qualitative cure behavior and kinetics were studied by using isoconversional methods. The results indicated that both Mica and Mica-IM improved the curability of epoxy system from a Poor to Good state when varying the heating rate in the interval of 5–15 °C min−1. The isoconversional methods suggested a lower activation energy for epoxy nanocomposites with respect to the blank epoxy; thus, Mica and Mica-IM improved crosslinking of epoxy. The higher order of autocatalytic reaction for epoxy/Mica-IM was indicative of the role of liquid crystals in the epoxide ring opening. The glass transition temperature for nanocomposites containing Mica and Mica-IM was also lower than the neat epoxy. This means that nanoparticles participated the reaction because of being reactive, which decelerated segmental motion of the epoxy chains. The kinetics of the thermal decomposition were evaluated for the neat and mica incorporated epoxy nanocomposites epoxy with varying Mica and Mica-IM amounts in the system  and heating rates. The epoxy/Mica-IM at 2.0 wt.% of nanoparticle showed the highest thermal stability, featured by the maximum value of activation energy devoted to the assigned system. The kinetics of the network formation and network degradation were correlated to demonstrate how molecular-level transformations can be viewed semi-experimentally.We synthesized pristine mica (Mica) and N-octadecyl-N’-octadecyl imidazolium iodide (IM) modified mica (Mica-IM), characterized it, and applied it at 0.1–5.0 wt.% loading to prepare epoxy nanocomposites. Dynamic differential scanning calorimetry (DSC) was carried out for the analysis of the cure potential and kinetics of epoxy/Mica and epoxy/Mica-IM curing reaction with amine curing agents at low loading of 0.1 wt.% to avoid particle aggregation. The dimensionless  Epoxy resin is the main material in diverse fields of technology for developing painting materials, electronic devices, adhesives, surface coatings, automotive exterior parts, shipbuilding and aerospace; thus, mechanical and thermal properties of epoxy have been widely studied ,2,3,4. EThere are quite a wide range of synthetic and natural nanoparticles for the modification of polymers, depending on the performance ,21, amongT) of the system according to isothermal investigations. However, reports solely address the degree of conversion [Many publications have addressed properties of epoxy composites containing mica nanoclay. The presence of mica in epoxy composites delays thermoelectric aging of epoxy insulation and improves the thermal properties of the epoxy . Moreovenversion . TherefoCure Index (CI) was employed for the qualitative analysis of the state of cure of the systems, while detailed kinetics analysis was performed applying isoconversional approaches, to name Kissinger–Akahira–Sunose (KAS) and Friedman. Accordingly, we visualized the contributions of autocatalytic and non-catalytic reactions to epoxy crosslinking in the presence of Mica and Mica-IM. Dynamic mechanical analysis (DMA) was also applied, which allowed for taking the signature of viscoelastic behavior of epoxy nanocomposites. Finally, thermal decomposition mechanism of composites was characterized by the aid of thermogravimetric analysis (TGA).In the light of above, exploring the correlation between network formation and network degradation is of crucial importance from a practical point of view. In the present work, highly intercalated mica based on imidazolium which is an ionic liquid (IM) was employed for the sake of appropriate dispersion throughout the epoxy resin. Cure kinetics analyses were carried out applying nonisothermal differential scanning calorimetry (DSC) with a varying heating cycle. The effects of pristine mica and mica-IM, hereafter referred to as Mica and Mica-IM, on crosslinking kinetics of epoxy were thereby compared. The dimensionless The materials used together with the methodology applied in the synthesis of imidazolium ionic liquid and its organic modification, prepation of epoxy nanocomposites and characetrization of nanocomposites for cure kinetics and DMA together with TGA are provided in detail in the We used X-ray diffraction (XRD), in order to confirm surface treatments of mica with imidazolium salt (formation of Mica-IM). As shown in Based on previous studies , the proAlthough modification of nanoparticles was confirmed through the XRD method, TEM gives a close-up of the morphology of mica in the epoxy nanocomposites. In −1 we can see a broad peak, indicative of stretching vibration bonds present in hydroxyl groups. Moreover, symmetrical stretching methyl group (CH3) bonds appeared at 2920 cm−1, while asymmetrical ones formed at 2870 cm−1. The stretching vibration and absorption of the ring were also detected at 825 and 1510 cm−1, respectively. At around 1030 and 1234 cm−1 we can see two explicit adsorptions assigned to C–O–C and C–N stretching, one-to-one. The absorption bands in the 1610 cm−1 indicate C=C bond of aromatic rings can be seen for EP/Mica and EP/Mica-IM [−1 for EP/Mica-IM sample [−1 decreased for EP/Mica and EP/Mica-IM due to the interaction during epoxy ring opening [We compared the FTIR spectra of neat epoxy with epoxy nanocomposites containing Mica and Mica-IM to evaluate the changes in chemical functionality, as shown in /Mica-IM ,43. The M sample . Evident opening .Nonisothermal cure analysis was performed varying the heating rates in DSC for the blank epoxy resin and also epoxy with 0.1 wt % Mica or Mica-IM, Cure Index (CI) [CI values:To qualitatively analyze curing we used dex (CI) . EquatioH* and ΔT*, respectively:Equations (2) and (3) can be used to calculate the quantities of ΔRefH and ΔCH in these formula are the total values of enthalpy of cure dedicated to the neat epoxy and epoxy nanocomposites, respectively. Furthermore, the cure temperature ranges for EP/Mica, EP/Mica-IM and EP are appeared into ΔCT and ΔRefT, respectively. Detailed analysis is available in Δβ from 5 to 15 °C min−1 increased both PT and ΔH, as expected from previous investigations [onsetT, which is a signature of the facilitation of the cure, except for the first rate of heating, 5 °C min−1. The same behavior was observed for the epoxy systems containing Mica-IM. The facilitated cure of epoxy caused by mica can be confirmed by higher ΔH∞ values obtained for epoxy nanocomposites whatever the heating rate. Nevertheless, for both Ep/Mica and Ep/Mica-IM systems cured at 15 °C min−1 the system was highly energetic and chains have no adequate time for interaction with Mica or Mica-IM with epoxy, as previously seen for different nanoparticles [H∞ for the assigned nanocomposites at 15 °C min−1 are lower compared to 10 °C min−1, but still higher than that for the reference epoxy. Similar results were reported in our previous works varying the heating rate [igations . The prearticles ,50. Corring rate ,52.H∞ values. However, at higher heating rates more kinetic energy was imposed to the system, such that the surface chemistry of Mica and Mica-IM contributed to the epoxy ring opening, reflected in ΔH∞  cases ,56. Accosslinked ,58. Onceod state .TH by ΔH∞ [The conversion of cure reaction, α can be simply obtained at each temperature by dividing ΔT by ΔH∞ :(4)α=ΔHTα values increases gradually at the early times of the curing process, while the slope of curves rose significantly in the middle of the cure reaction due to the occurrence of the gelation. Eventually, the cure is decelerated at late stages by leveling the curves off [β = 5 °C min−1), Mica and Mica-IM could not easily diffuse in the epoxy dense network, because of the lack of required energy. Therefore, the effect of nanoparticles was not pronounced at the low heating rate and resulted in the same curing rate with the neat epoxy. However, both Mica and Mica-IM played the role of cure promoter after vitrification (β = 10 °C min−1). Because of higher kinetic energy and adequate time in the system at 10 °C min−1, Mica and Mica-IM could possibly diffuse in the in epoxy network and stay until they react [β = 15 °C min−1), the curing moieties had a shorter time for reaction, such that nanoparticles did not accelerate the curing reaction [The sigmoidal shape of the curves in rves off . When cuey react . At the reaction .αE) two different approaches were considered; Friedman as differential approach in contrast to Kissinger–Akahira–Sunose (KAS) representative of integral methods [Kinetic data of epoxy nanocomposites are derived from the nonisothermal DSC based on isoconversional method. To achieve the amount of activation energy .f(α) and k(T) are the reaction model and reaction rate constant, respectively, where:In the above equation, R, T and A are represented universal gas constant and absolute temperature and frequency factor, respectively. The following equation as a curing rate is derived from replacing Equation (6) in Equation (5):In Equation (6), Friedman and KAS methods for isoconversional model are described as Friedman and KAS methods as mentioned in Friedman and KAS models as a function of α, respectively. All plots follow an almost ascending trend until the middle of the reaction. This might be related to the shift in crosslinking state from chemical- to diffusion-control mechanism. The hindrance effect imposed by Mica and probably its improper dispersion into the epoxy matrix leads to obvious decline in activation energy in comparison with neat epoxy. However, the modification of nanoparticles with imidazolium is facilitated cure reaction and led to decrease the activation energy. Furthermore, significant drop in EP/Mica and EP/Mica-IM curves at higher conversion are because of possible esterification reaction between epoxy and hydroxyl groups [According to the assumptions mentioned above, the differential l groups .Freidman and Malek methods as described by The autocatalytic and non-catalytic mechanisms are studied by the use of Friedman method approves having a nth-order cure mechanism, α ranging in the interval 0.2–0.4, characteristic of autocatalytic crosslinking.The Malek method (α) plots obviously shows a maximum by p∞α in the same range. However, y(α) for mα has peaked around α equal to 0.1, demonstrating nth-order non-catalytic mechanism. Thus, an autocatalytic reaction model can explain curing whatever the system [According to the k method , the z(αe system .n) and non-autocatalytic (m) reaction, together with frequency factor (A) were calculated, derived from Friedman and KAS methods (m + n), represents an overall order of network formation. From m + n) are more than one, indicating the complexity of curing reaction in the epoxy/amine systems. This is the characteristic of the autocatalytic nature of reaction taking place between the epoxy resin and amine curing agent. Furthermore, the autocatalytic reaction order (m) increased with incorporation of Mica and Mica-IM into epoxy resin, notifying the improved contribution of autocatalytic reaction to crosslinking. Friedman and KAS approaches fitted the experimental data on dα/dT per temperature.By using the  methods . The resgT , a plasticizing effect induced crosslinking [The peaks in Tan δ curve are indicative of Tg . Tg valumobility . Neverthslinking . For EP/5T) and also the peak temperature (PT) (ure (TP) . The amo5 is increased for nanocomposites as a whole, which was expected. The value of 5T is higher for EP/Mica-IM compared to the EP/Mica because of the stronger interaction of imidazole groups of Mica-IM with epoxy rings of resin. Furthermore, T5 in the high proportion of nanofillers with 5% showed less thermal resistance than that with amounts of 0.5 and 2 wt % due to the high proportion of nanoparticles preventing complete curing of the coating and thus reducing the density of the cross-linking reaction. Accordingly, the samples containing mica-IM 0.5 and 2 wt % have the highest 5T, respectively, which means the presence of modified nanofillers facilitated complete curing and enhance crosslinking density. As a result, the thermal resistance of the coating is improved. Nevertheless, pT remained stable with negligible change for all samples compared to blank resin.We can obviously see from α) was calculated as [For kinetics analyses, the conversion of thermal decomposition reaction (lated as :(8)α=W0−W0, tW and fW stand for the mass of sample before TGA test, the mass of polymer at time t of experiment, and final mass of the polymer remained at the end of thermal degradation.In the above equation, α variation for the studied samples  are obtained as a function of temperature, as shown in β increased, degradation took place at higher temperatures in correspondence with the increase of the α value. It means that a faster pyrolysis would be expectable in a shorter time available to the samples [The  samples .Friedman, Flynn–Wall–Ozawa (FWO), KAS and modified Coats–Redfern (m-CR) to take the signature of the network degradation of the systems in terms of αE  revealed large difference with that of the blank epoxy, which is characteristic of hindered network degradation. The highest αE value of αE was obtained for EP/Mica-IM-2 due to the presence of well-dispersed thermally stable Mica nanoplates with imidazolium-based IL on their surface. The imidazole of IL on the surface of Mica can interact with epoxy resin and results in denser network which makes the decomposition reaction more difficult and leads to higher activation energy.Similar to studying the cure kinetics , we used isoconversional approaches of nd in Eα . On the f(α) function, which is defined as follows:Sestak and Berggren proposed a semi-empirical kinetics model that was used for finding the m) and non-catalytic (by order of n) reactions contributing to thermal degradation of the networks. The overall degree of degradation reaction was taken as (m + n), which increased for epoxy after nanoparticles were added.Poor cure state was identified in EP/Mica and EP/Mica-IM at low β of 5 °C min−1 caused by hindrance of reaction arising from incorporation of the Mica or Mica-IM. By contrast, the Good cure state was obtained at higher β, demonstrating the facilitation of crosslinking, as detected by the higher ΔH values assigned to the system comprising Mica-IM. The αE values obtained by Freidman and KAS isoconversional approaches were obviously lower for nanocomposites compared to the blank epoxy. Thus, network formation was supported by nanoparticle incorporation, particularly in the case of Mica-IM. On the other hand, a higher thermal stability was reflected in the kinetics analysis of network degradation based on Friedman, FWO, KAS and m-CR isoconversional methods. In this case, the higher αE values for the nanocomposites indicated the formation of a denser network after nanoparticle incorporation into the epoxy resin. The TEM micrographs witnessed the obvious formation of the Mica-IM particles with smaller sizes compared to the Mica within the epoxy matrix, which can be considered as a clue for the interpretation of higher properties of Mica-IM based nanocomposites.Mica surface modified with imidazoline ionic liquid (Mica-IM) was analyzed by the use of XRD and TGA techniques. Then, epoxy nanocomposites with 0.1 wt % Mica or Mica-IM were prepared and analyzed nonisothermally by DSC to investigate the qualitative and quantitative cure parameters of the systems. The


Scanning electron microscope (SEM), UV–VIS spectrophotometer (UV-vis), X-ray diffraction (XRD), and attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR) were applied to evaluate the properties of the synthetic microparticles. This material could lead to the release of corrosion inhibitor under different pH conditions of the aqueous media. However, the extent of release of the corrosion inhibitor in the acidic media was higher, leading to enhanced shielding effect of the Q235 steel. These microparticles can serve as anti-corrosion additive for epoxy resin-coated Q235 steel. Electrochemical experiments were used to assess the anti-corrosive ability of the epoxy coatings in simulated concrete pore (SCP) solution, confirming the superior corrosion inhibition of the epoxy coating via incorporation of 5 wt % calcium carbonate microparticles loaded with SLS (SLS/CaCO3). The physical properties of coating specimens were characterized by water absorption, contact angle, adhesion, and pencil hardness mechanical tests.In this study, calcium carbonate (CaCO Among the various building materials used in civil engineering, concrete is considered as one of the most critical materials. Life span of the concrete mainly depends on the steel bars along with different other properties of concrete. Steel corrosion is one of the key factors reducing the life span of the concrete . The corTherefore, efficient prevention and repair of the corrosion damage of the reinforced concrete structures are necessary. Various methods have been proposed for mitigating the corrosion damage including admixing of suitable corrosion inhibitors ,7,8, selEpoxy resins have extensive applications in the protection of metallic corrosion due to their low costs and excellent anti-corrosive ability. The matrix of the metal is prevented from getting destroyed by the corrosive ions by the coating. However, most of the coatings are vulnerable against mechanical damage. Therefore, some researchers mixed corrosion inhibitors to the coating for enhancing the effective corrosion inhibition and mechanical properties of the coating ,16. Howe3) can be divided into three kinds of crystal polymorphs according to the crystalline form having similar composition but different structures, i.e., calcite, aragonite, and vaterite [3 is mostly present as calcite or aragonite. The thermodynamics of vaterite is unstable and quick transforms vaterite into calcite or aragonite in an aqueous solution. Vaterite is rare in the nature. In recent times, CaCO3 microspheres have received great attention thanks to their better mechanical and thermal stability, biocompatibility, and biodegradability [3 can be transformed into various structures and morphologies using modifiers, and it is possible to easily load them with the corrosion inhibitors [3 powders have been used as filler in coating and as a carrier material to control the release rate [3 microspheres loaded with corrosion inhibitors are embedded in the coating, slight modification of the coating is required and the modification step is a simple one.Calcium carbonate  and polyethylenimine (PEI) in epoxy coating and found that the coating pore resistance of the modified coating presented about four order of magnitude higher compared to the neat epoxy coating [Earlier reports in the literature have shown the application of epoxy coatings modified by CaCO coating . These cbstrates . Roma Ra coating .Sodium lignosulfonate (SLS) is a natural polymer and is being used as an anionic surfactant . As a by3 particles in the epoxy coatings applied on Q235 steel. Various physicochemical analytical methods were used to characterize the microspheres with or without SLS loading. After immersion in the SCP solutions containing 3.5 wt % sodium chloride, the inhibition protection of the coated Q235 coupons was evaluated by electrochemical measurements and physical properties tests. This study explores the anti-corrosion properties of a novel composite prepared by the combination of SLS and pH-sensitive CaCO3 microparticles were synthesized via the co-precipitation method. In the first step, 0.5 M CaCl2 (50 mL) , ≥99.9%) was poured rapidly into an equal volume of 0.5 M Na2CO3  under vigorous stirring . The produced suspension was evacuated applying vacuum filtration with filter paper of 0.22 μm pore size and rinsed with deionized water and ethanol. Then, the synthesized microparticles were dried for 24 h at 50 °C. Dried calcium carbonate particles were soaked into a 15 wt% SLS solution (50 mL) (purchased from Macklin) in vacuum for 1 day. The suspension was separated using circulating water vacuum pump, followed by washing with Millipore ultrapure water and subsequent drying in an oven at 50 °C.CaCO3 microparticles with or without SLS loading was applied onto the prepared specimens. CaCO3 particles with SLS (3 wt% and 5 wt% of the gross weight of the hardener and epoxy resin) were added into ethanol under ultrasonic dispersing for 1 h. After that, the dispersion of CaCO3 particles with or without SLS loading was stirred with the epoxy resin for 10 min. Subsequently, the mixture of the hardener and epoxy resin keeping a weight ratio of 1:1 was stirred for 15 min before applying the coating mixture. Then, various coating formulations were applied on steel bar by dipping coating with the withdrawal rate of 18 cm/min. Finally, the coated steels were cured for 7 days.The Epoxy resin (E44) and polyamide hardener (650) were provided by Zhenjiang Danbao Resin Co., Ltd. . Epoxy resin with CaCOBefore coating, the Q235 carbon steel specimen  was abraded with metallographic paper , then sonicated in acetone for 5 min, followed by the subsequent rinsing with ethanol and deionized water . The epo3 microbeads were stuck with conductive glue on the aluminum block, gilded, and then put into the sample chamber. In addition, SEM presented the fracture surface morphologies of the coatings after broken in liquid nitrogen.SEM  was applied to observe the micrographs of the blank and SLS-loaded microspheres (accelerating voltage of 5 kV). Appropriate amount of CaCO3 particles before and after the loading with SLS. The data was collected between the angular range of 10° ≤ 2θ ≤ 80° employing a scanning rate of 2°/min with a 2θ step of 0.013°.X-Ray diffraction (XRD) study  using Cu-Kα radiation source was carried out to confirm the phase identification of CaCO3 microparticles employing wavelength range scanning from 4000 to 400 cm−1.The attenuated total reflection-Fourier-transformed infrared spectroscopy  was employed to analyze the structure of the CaCO3 particles. The absorption peak at about 280 nm belongs to the benzene ring in the sodium lignosulfonate. The SLS concentration and its absorbance peak at 280 nm show good linear correlation. Standard curve was obtained by measuring the absorbance of known concentration of SLS in the aqueous solution and a linear plot was obtained. The loaded microspheres were dispersed in aqueous solution with varying pHs  after different immersion times .UV–VIS spectrophotometer  was employed to measure the concentration of the released inhibitor in the aqueous solution from SLS/CaCOThe steel was immersed in SCP test solutions for 15 days at ambient temperature. After stabilizing the open-circuit potential (OCP) for at least 30 min, inhibition properties of the coated-Q235 steel were measured using electrochemical impedance spectroscopy (EIS) spectra within the frequency range of 100 kHz to 10 mHz and oscillation amplitude of 20 mV from peak to peak utilizing Princeton 263A electrochemical workstation. Potentiodynamic polarization measurements also applied in the electrochemical workstation. The experimental potential range was in the range of −1.0 to +1.0 V (vs. OCP) and the scan rate was 0.8 mV/s. Reproducibility of the results was confirmed by carrying out each experiment at least thrice.0 and mt, respectively. Prior to weighing, the coating specimens were wiped with filtration paper. The coated samples were weighed after various immersion periods. Then, the water absorption Qt was calculated by Equation (1) [Water absorption tests were used according to the ASTM D570 2005 standard in order to characterize the anti-corrosive ability of the coating. The weight of each coated samples was recorded before and after the immersion in deionized water at room temperature and was indicated as mtion (1) (1)Qt= was carried out to analyze the wettability of the coating. Prior to the measurement, 3 μL distilled water was dropped on the surface of the coating steel. Each sample was measured three times.The adhesion property of the coating is an effective criterion to evaluate the inhibition performance of the coatings. Adhesion test was performed according to ISO-2409: 2013 Standard. A coating surface was drawn out 100 grids and swept the surface with a brush. Then, a transparent adhesive tape was firmly stuck on the scratched surface and tore off with an angle of about 60° within 0.5–1.0 s. The spalling area of the coating was observed by magnifying glass. The adhesion level of the coating without the detached area can be classified as level 0, whereas coating surface with the lowest adhesion belong to level 5 .The pencil hardness test was performed on the coatings according to ASTM D3363. The hardness of the wood pencils scale ranges from 6B to 6H. The test started from the pencil with minimum hardness (6B). Until the pencil scratched or dug the coating, the coating hardness was equated to the previous pencil. Gouge hardness and scratch hardness determined the hardness value. The first one presents the resistance of the coating to being cut, the second one indicates the mechanical resistance of the coating to being scratch [3 microbeads with or without SLS loading were investigated by SEM , (104), (006), (110), (113), (202), (024), (018), (116), (211), (122), (214), (119), (125), (300), (0012) of vaterite and the peaks at 2θ = 20.880, 24.883, 26.973, 32.711, 42.613, 43.767, 49.098, 49.946, and 55.768° correspond to the respective planes of (002), (100), (101), (102), (004), (110), (112), (104), and (202) for calcite. We estimated that the vaterite and calcite relative mole fractions in the CaCO3 microparticles can be correlated to the X-ray diffraction intensity from the following expression [C and V are the calcite and vaterite mole fractions, respectively, CI is the X-ray diffraction intensity of the corresponding planes of calcite, and IV is the X-ray diffraction intensity of planes of vaterite. Crystalline structure of the synthesized CaCO3  demonstrpression :(2)Xv= I3 microparticles without SLS were found to be ~60.64% and 39.36%, respectively, while the corresponding values for the SLS-loaded CaCO3 microparticles were 55.95% and 44.05%, respectively.The mole fractions of vaterite and calcite for the CaCO3 microparticles from the vaterite to calcite. The dissolution of vaterite would lead to quick transformation of vaterite to calcite, so the addition of the SLS induced increasing rate of dissolution rate of the vaterite [Increasing pH of the synthetic solution due to the presence of SLS leads to the phase transformation of the synthesized CaCOvaterite . 2 = 0.9906 where A, C and R2 are the absorbance, the concentration of SLS and the correlation coefficient, respectively). Under different pHs of the aqueous solution, the release of SLS/CaCO3 micro-containers were also studied employing UV–vis spectrophotometer. 3 micro-containers under various pH of the aqueous solution over 72 h . These solutions were prepared with deionized water, 0.1 M acetic acid, and 0.1 M sodium hydroxides (NaOH). 3 micro-containers for 72 h of immersion in various pH solutions. At all immersion times, the highest absorbance intensity was presented at pH 4. The released SLS concentration decreased with increasing pH values. In acidic and neutral media, the SLS released from the CaCO3 micro-containers rapidly in early time. However, in alkaline media, the concentration of SLS increased slowly as the immersion time increases. Release behavior of SLS from the CaCO3 under various pH of the aqueous solution showed higher release in the acidic medium in comparison to the alkaline medium. Therefore, the SLS-loaded CaCO3 micro-containers have promising applications in the reinforced concrete.The absorbance of the aqueous solution with varying concentration of SLS was monitored by UV–VIS spectrophotometer. Linearity of the standard plot with eight different concentrations of SLS  is depicted in 3 micro-containers loaded with SLS were obtained by ATR-FTIR analysis. The absorption peaks at 713 and 876 cm−1 shown in −1 are originated from the presence of vaterite. The broad areas of absorption in 1455–1490 cm−1 correspond to the amorphous calcium carbonate. The typical peaks attributed to calcite and vaterite remained unchanged in the spectra for the CaCO3 particles in absence or in presence of SLS. The presence of two weak peaks at 874–877 and 743–746 cm−1 corresponding to the out-of-plane and in-plane stretching of carbonate ions, respectively, confirm the presence of CaCO3. The carbonate ion is confirmed by the asymmetric stretching at 1446 cm−1 and the symmetric stretching at 1086 cm−1. The structure of SLS was also investigated by ATR-FTIR , which corresponds to the chemical structure of SLS [−1, respectively [−1 could be a sign of C-H bond because of syringyl lignin [−1 represents the S=O stretching vibration of sulfonate groups [3 particles could also be indirectly inferred from the broad spectrum between the range of 3390–3450 and 2940–2990 cm−1 because of the stretching vibration of O-H bonds and C-H bonds, respectively [3 particles. The bands in the range of 670–2050 cm−1 are from the bending vibrations of the carbonate ions. These overlap with the C-H, S=O, and C-S stretching, respectively, which are related to the presence of SLS.Further details regarding the chemical composition of the CaCOATR-FTIR . The abse of SLS . The strectively . The ban3 microcontainers-containing epoxy coatings had a higher OCP value than blank epoxy coating at the initial stage of the immersion. After the prolonging immersion of the coating system, all specimens presented the gradually dropping OCP values. For instance, the OCP value for blank epoxy specimen dropped remarkably from the initial −0.344 to −0.545 V after immersion for 15 days. The open circuit potential of epoxy-CaCO3 coating increased slightly on the tenth day, which may be attributed to the corrosion products block penetration path of corrosive medium. Meanwhile, for the epoxy coating modified with SLS/CaCO3 microcontainers, the OCP value declined slowly. The OCP value for the epoxy coating containing 3 wt% SLS/CaCO3 and 5 wt% SLS/CaCO3 presented a decreasing trend . This confirmed that the inhibitor SLS could release from CaCO3 particles and optimized the protective properties of epoxy coating.0.01 Hz) of the coatings containing CaCO3 particles with SLS are higher than that of blank epoxy coating during the early stage of immersion period. Especially, the epoxy-5 wt% SLS/CaCO3 coating exhibited that the |Z|0.01 Hz value reached 7.22 × 1010 Ω·cm2, being five order of magnitude higher than that of the blank epoxy coating. The Bode curves of the specimens after 15-day immersion are presented in 0.01 Hz value dropped from 3.99 × 105 to 4.05 × 104 Ω·cm2 after 15 days, which was slightly lower than that of the epoxy coating containing CaCO3 particles in the absence of SLS (from 4.20 × 105 to 2.04× 105 Ω·cm2) after 15 days of immersion in the SCP solution. The |Z|0.01 Hz value decreased from 4.52 × 1010 to 3.88 × 1010 Ω·cm2 for the epoxy-3wt%SLS/CaCO3 coating. In addition, the coating modified by the 5 wt% CaCO3 particles loaded with SLS exhibited the higher |Z|0.01 Hz values at the early stage, and then, the values presented a slow descending trend, reaching values approximately 3.73 × 1010 Ω·cm2.−2–1 Hz (low frequency region), owing to the corrosion activity at interface of the metallic surface and the coating; slight defects or pores on the coatings are reflected by the peak exhibiting within the range of 1 Hz to 103 Hz (intermediate frequency region); the shielding behavior of the coating can be observed by the peak at 104–105 Hz (high frequency region) [3 exhibited pronounced peak under all immersion durations, suggesting that a better anticorrosion property than the other coatings. The phase angle of the epoxy-5wt% SLS/CaCO3 coating at high frequency was relatively higher than that of the epoxy coating with 3 wt% SLS/CaCO3 particles. For the blank coating, the peak appeared at the intermediate frequency region after 15 days of immersion, revealing the existence of tiny defect on the coating.The protective performance of the coating system was illustrated by the Bode phase angle. The peaks appear at the range of 10 region) . In the The presence of one-time constant corresponding to the coatings at the initial stage of the soaking were fitted by equivalent electrical circuit a to modes), capacitance of the coating (Qc), resistance of the coating (Rc), charge transfer resistance (Rct), and double-layer capacitance (Qdl) [Those EIS data were modeled applying equivalent circuit, as shown in ce (Qdl) . The valce (Qdl) .c value. The higher value of Rc indicates better corrosion protection of the coating. Specific changes exist in the Rc values with the increase in soaking time. For all the coatings, the values of Rc declined because of the invasion of the electrolyte and the accumulation of chloride ions between the coating and the steel surface. Thanks to the CaCO3 particles as fillers in the coating, the epoxy-CaCO3 coating Rc value (9.53 × 104 Ω·cm2) was relatively higher than that of pure epoxy coating. The coatings modified by 3 wt% and 5 wt% SLS/CaCO3 micro-containers had higher values of Rc (5.16 × 1010 and 4.54 × 1010Ω·cm2), while the Rc value of blank epoxy coating was 1.96 × 104 Ω·cm2 over the 15 days of testing period. Observation demonstrates that the tortuous penetration path of the aggressive medium thorough the coating can be increased by these micro-containers. This trend evidenced that the addition of SLS/CaCO3 microparticles significantly enhanced the corrosion resistance of the epoxy coating.The anticorrosive performance of the coating can be evaluated by the Rct value is related to the corrosion protection of the coating [ct value of epoxy-SLS/CaCO3 coating was much higher than that of blank epoxy coating after immersion for 15 days, implying that its better corrosion-protective ability compared to the pure epoxy specimen.The evolution of the R coating . The Rctdl reflects the delamination of the interface of metal and coating. Therefore, the lower Qdl represents that the excellent barrier effect of coating [3 particles exhibited lower Qdl value than neat epoxy coating. Thus, the metal-coating interface reaches stabilization because of the presence of CaCO3 particles.The variation of the Q coating . The epoc means the water absorption of coating. The penetration of corrosive electrolytes accompanied by an increase in Qc. The Qc values for all the coatings are stable during 15 days of immersion, revealing all the coatings still have barrier ability.Qc and Qdl represents the prefactor of a constant phase element (CPE)to replace the pure capacitance because of the heterogeneities of surface. The pure capacitance can be calculated as follows [xC, xQ, and xn represents the capacitance, the constant-phase element, and the empirical exponent, respectively, Rx is either the pore resistance of the coating (Rc) or charge transfer resistance (Rct). The Q follows (4)Cx=, the corrosion current density (corri), and the inhibition efficiency (IE%). The inhibition efficiency was defined using Equation (5)The polarization curves of the coated steel after 5 and 15 days of immersion in the SCP solutions are illustrated in 3 micro-containers shifted toward more positive potentials and exhibited a decreased icorr. This suggests that the addition of SLS/CaCO3 particles can further enhance the inhibition performance of the epoxy coating. In corr of 3 wt% SLS/CaCO3 epoxy coating and 5 wt% SLS/CaCO3 epoxy coating moved to more positive position of 0.042 and 0.059 V, respectively, after 5 days of immersion. Moreover, it was noticed that the IE% of 5 wt% SLS/CaCO3 epoxy coating reached 90.83%. After immersion period of 15 days, the corri values of the coating system showed increasing trend. The corri values obtained for epoxy coating, epoxy-CaCO3 coating, epoxy-3 wt% SLS/CaCO3 coating and epoxy-5wt%SLS/CaCO3 coating were 1.12 × 10−6, 3.73 × 10−7, 1.90 × 10−7, and 1.72 × 10−7 A·cm2 after 15 days of immersion, respectively. Surprisingly, the icorr value of modified coating was one orders of magnitude higher than that blank epoxy coating. The Ecorr value for the epoxy-5wt%SLS/CaCO3 coating remained stable (from −0.371 to −0.378 V), while the Ecorr value of the neat epoxy coating decreased in the range from −0.430 to −0.556 V. The IE% values of epoxy coating containing 3wt% SLS/CaCO3 and 5wt%SLS/CaCO3 were 83.02 and 84.64, respectively. Therefore, it suggests that SLS releases from CaCO3 micro-containers.As shown in 3 particles were blended into the epoxy coating. After the addition of CaCO3 particles, the fracture surface of the modified coatings manifested rough surface. CaCO3 micro-container exhibited the better compatibility with the blank epoxy coating. The EDX elemental mapping of the epoxy-CaCO3, epoxy-3 wt% SLS/CaCO3, and epoxy-5 wt% SLS/CaCO3 specimens is depicted in 3 particles were distributed evenly in the epoxy matrix.3 coating, epoxy-3 wt% SLS/CaCO3 coating, and epoxy-5 wt% SLS/CaCO3 coating were 0.592%, 0.405%, 0.312% and 0.233%, respectively. The modified coating possesses lower water absorption value and can be ascribed to the calcium carbonate filler reducing the porosity of the coating.Permeation of water and anti-corrosive behavior of the coating can be measured by the water absorption test. The water absorptions of all the coatings are displayed in 3, 3 wt% SLS/CaCO3, and 5 wt% SLS/CaCO3 were 100.2°, 100.5°, 106.1°, respectively, symbolizing the improved hydrophobicity of the modified coating specimens.The contact angle analysis is a frequently used method to confirm the wettability of the coating, which is related to the barrier protection effect of the coating .The wate3 coating, epoxy-3 wt% SLS/CaCO3 coating, and epoxy-5 wt% SLS/CaCO3 coating were 1, 0, 0, and 0, respectively. These observations illustrate that the modification by SLS/CaCO3 micro-containers can effectively enhance the adhesion strength of the pure epoxy resin. It can be inferred that the epoxy-SLS/CaCO3 coating possesses better adhesive property because the moderate amount of SLS/CaCO3 micro-container was added in neat epoxy coating. Furthermore, CaCO3 particles as filler will generate physical or chemical bond with the metal substrate, so as to ensure that the coating can adhere to the metal surface well [The results of the coating system after adhesion measurements are showed in ace well . 3 micro-containers, as shown in The coating hardness of the pure epoxy coatings was similar to those coatings modified with the CaCO2 and 2CaO·SiO2 are the main components of Portland concrete cement. A large amount of calcium hydroxide is generated after hydration reaction, which will lead to the concrete pore solution with high alkalinity [3) precipitates on the carbon surface and hinders the invasion of corrosive medium. Under severe environment, corrosive media such as aggressive chloride ion and carbon dioxide will cause the decline in the pH value of the concrete and the destruction of protective film on the metallic substrate [3 micro-containers in response to the change of pH. Increasing pH of the SCP solution due to continuous release of SLS results in the formation of passive film on carbon surface. Furthermore, SLS contains hydrophobic skeleton and hydrophilic groups (hydroxyl groups and sulfonic groups). The interaction between the SLS with sulfonic groups and positively charged metal surfaces is responsible for chemisorption. Moreover, the SLS is absorbed on the carbon surface due to the formation of coordinative bonds between the metal ions and the O element in phenolic hydroxyl group of SLS. The optimum addition of 5 wt% SLS/CaCO3 not only improves the self-healing capability of the pure epoxy coating but also avoids the consumption of corrosion inhibitor when the steel bar is in the passivated state.Importantly, 3CaO·SiOkalinity . In the ubstrate . At this3 micro-containers was investigated by UV-vis. The corrosion inhibition effect of incorporating various amount (3 wt% and 5 wt%) of SLS/CaCO3 microparticles with the epoxy coating in the SCP solutions was discussed by electrochemical measurements and a series of physical properties tests. The following observations were made:3 microparticles modified with sodium lignosulfonate had the same oval-shaped morphology as the unmodified one.The CaCO3 particles with or without SLS contain both the vaterite and calcite phases. Presence of sodium lignosulfonate in the CaCO3 particles leads to more calcite phase.XRD results showed that the CaCO3 micro-containers can be released under different pH conditions, with the extent of sodium lignosulfonate release in the acidic media being higher.The SLS loaded into the CaCO3 microparticles on the inhibition performances of the epoxy coating, especially the epoxy-5 wt% SLS/CaCO3 coating. These microparticles show better compatibility with the original epoxy coating.Electrochemical measurements demonstrated the beneficial role of the SLS/CaCO3 coating showed high hydrophobicity and good mechanical strength and exhibited better adhesion on the substrate.The epoxy-SLS/CaCOIn this study, calcium carbonate was used as pH-sensitive micro-containers for the loading of SLS as corrosion inhibitor, which were confirmed with FTIR, XRD, and SEM. Under the presence of different pH solutions, the response of SLS-loaded CaCO


Further, Gene Set Enrichment Analysis (GSEA) revealed that Wnt pathway-related genes significantly enriched in SCC. IHC staining showed gradually increased immunoreactivity score of Wnt3a and CBP and notable translocation of β-catenin from the membrane to the cytoplasm and nucleus during the lesion progression. The intensity and proportion of P16, Ki67 and CK17 staining also increased with the progression of cervical lesions, whereas minimal to negative Involucrin expression was observed in CIN2/3 and SCC. Therefore, canonical Wnt signaling may contribute to the progression of CIN to SCC and may be a potential therapeutic target.Piwil2 reprograms HPV-infected reserve cells in the cervix into tumor-initiated cells (TICs) and upregulates Wnt3a expression sequentially, which leads to cervical intraepithelial neoplasia (CIN) and ultimately squamous cell carcinoma (SCC). However, little is known regarding Wnt signaling in the maintenance of TIC stemness during the progression of cervical lesions. We herein investigated the expression of canonical Wnt3a signaling and related genes by microarray data set analysis and immunohistochemical (IHC) staining of samples obtained by biopsy of normal cervix, low- and high-grade CIN, and invasive SCC tissue. Array data analyzed by GEO2R showed higher expression levels of Wnt signaling and their target genes In 2015β-catenin with members of the T-cell transcription factor (TCF)/lymphoid enhancer-binding factor (LEF) family of transcription factors [β-catenin-dependent Wnt signaling pathway plays crucial roles in embryonic development and maintenance of homeostasis in mature tissues [Wnts are secreted cysteine-rich glycoproteins that act as short-range ligands to locally activate receptor-mediated signaling pathways , 6. A ke factors . To gene factors , 10. As  tissues , 12. Wnt tissues –15, and  tissues , 17.Min mouse has also been shown to exhibit an enhanced incidence (∼10%) of spontaneous mammary cancer and a greatly increased susceptibility (90%) to carcinogen-induced mammary cancer [Accumulating evidence suggests that the Wnt/β-catenin signaling pathway is often involved in oncogenesis and cancer development , 11, 18.y cancer , 22. Howy cancer . We alsoOur present findings provide novel pathological insights into Wnt3a signaling in the progression of CIN and aggressiveness of cervical cancer, thus defining a potential therapeutic target for cervical lesions.n = 20), CIN1 (n = 36), CIN2/3 (n = 32), and invasive SCC (n = 46) tissues.The project was approved by the Ethics Committee of China-Japan Friendship Hospital . One hundred thirty-four histological specimens, including hysterectomy tissues and loop/cone biopsied samples, were retrieved from the files of the Department of Pathology of China-Japan Friendship Hospital. The specimens consisted of normal cervix (2O2 for 10 min to inactivate endogenous peroxidase. The slices were incubated overnight at 4°C in a humidified chamber with the following primary antibodies: 1:500 diluted anti-Wnt3a (ab28472), 1:500 diluted anti-β-catenin (ab32572), 1:1000 diluted anti-CBP (ab50702), 1:100 diluted anti-cytokeratin 17 , 1:1000 diluted anti-involucrin (ab53112), 1:200 diluted anti-CDKN2A/p16INK4α , and 1:200 diluted anti-Ki67 (ab16667). All of the primary antibodies were purchased from Abcam, United States. The binding of the primary antibodies was visualized using the ChemMate detection kit . The slices were lightly counterstained with Mayer’s hematoxylin for 30 s.Four-micrometer-thick formalin-fixed, paraffin-embedded tissue sections were deparaffinized with xylene and rehydrated through a graded series of ethanol. Antigen retrieval was performed with 0.01 M citrate buffer (pH 6.0) in a microwave oven for 10 min. After cooling to room temperature, the slides were incubated with 3% HImmunoreactivity was semiquantitatively evaluated. The degree of positive staining for all antibodies was evaluated by score on a scale of 0–3 for strength of intensity of staining and on a scale of 0–4 for percentage of positive cells. The final total score was generated by the intensity score × proportion score. The intensity score was defined as follows: 0, no staining of epithelial cells; 1, weak staining; 2, moderate staining; or 3, strong staining. The proportion score was defined as follows: 0, no staining of the cervical epithelial cells in any field; 1, <10% of the epithelium stain-positive; 2, 11–50% stain-positive; 3, 51–80% stain-positive; or 4, >80% stain-positive. The total score ranged from 0 to 12. Two different pathologists evaluated all the specimens in a blinded manner. Notably, the distribution of staining in the intraepithelial lesions (CIN1 and CIN2/3) was compared with that of the normal cervical squamous epithelium. CIN tissues were categorized as having the thickness of the samples being from two-thirds to fully stained.https://www.ncbi.nlm.nih.gov/geo/). The platforms for these data sets are all based on GPL10558 and an Illumina HumanHT-12 V4.0 Expression BeadChip. The downloaded files were processed using the R package and then calibrated, standardized, and converted to log2 data, or normalized with cubic spline method. The limma package was used to standardize the gene chip and analyze the differential expression. The quantile normalized values for canonical Wnt signaling pathway associated genes were found out via VLOOKUP from the gene expression description tables. Boxplot was used to show the expression difference among groups of normal cervix, CIN and SCC.To investigate the expression pattern of genes associated with the activation of Wnt/β-catenin signaling in the progression of cervical lesions, the data set GSE64217 , GSE51993 , GSE67522  were downloaded from the GEO database . The significance of gene expression between groups in microarray data sets were analyzed with one-way analysis of variance. The comparison of immunoreactivity score between normal cervix and cervical lesions were performed with two-tailed Chi-square test. Pearson test was used to analyze the correlation between the expression of the biomarkers and the cervical lesion degree. A Wnt3a, CTNNB1 (β-catenin), and CREBBP (CBP) were significantly upregulated in the samples of precancerous and cancer of the cervix compared with those of the normal cervix, thereby contributing to the distinct upregulation of their downstream transcription factors KLF4, MYC, and SOX2, except KLF4 slightly downregulated in CIN (KRT17 (CK17), TP63 and CDK2A (P16), markers of HPV target cells/cervical cancer stem cells (CSCs), were apparently upregulated in CIN and SCC. In contrast, markers of squamous cell differentiation, LORICRIN, INVOLUCRIN and KRT4 (CK4), were significantly downregulated in CIN and SCC (KRT14 (CK14) and KLF4, showed in these GSE gene sets, were moderately downregulated in CIN tissues compared with normal cervix, nevertheless they were all significantly upregulated in SCC tissues , the data set GSE64217, GSE51993 and GSE67522 were analyzed by the GEO2R method on the GEO website. The boxplot of the distribution of the normalized expression values for selected samples demonstrates that the data were suitable for the analysis and “limma” , which wd in CIN . As prev and SCC . It was  tissues .In normal cervical squamous epithelium, Wnt3a staining was confined to the basal cells, whereas in CIN1 diffuse immunoreactivity was limited to the lower one-third of the epithelial layer, and in CIN2/3, the staining extended through the full thickness of the epithelial layer . Strong β-catenin score and its positive translocation from the membrane to the cytoplasm and finally to the nucleus was noted in cervical lesions at higher stages  of the CIN1 samples were membrane positive for β-catenin. Approximately 71.9% (23/32) of the CIN2/3 and 84.8% (39/46) of the SCC samples had cytoplasmic and even nuclear positive staining. The pattern of CBP expression in each stage of cervical lesion was similar to that of β-catenin with only nuclear immunoreactivity .A gradual increase in the r stages . Only 5%activity . Overallp < 0.01).Markers useful in the assessment of lesion grade were further observed. P16, which is valuable in the diagnosis of high-grade CIN, was positively stained in the upper two-thirds and full thickness of the epithelium in most cases of CIN2/3 and in all of cervical cancer tissues, while minimal or negative P16 was observed in cervical squamous epithelium of the normal cervix or CIN1 samples . As the β-catenin and coactivator CBP expression implied that the canonical Wnt signaling pathway was activated in high-grade lesions. Consistent with previous studies, CBP-Wnt activity was associated with tumorigenesis and stem cell maintenance because it modulates the levels of intrinsic pluripotency factors, such as Oct4, Nanog, and Sox2 [In this work, the characterization of and Sox2 . Here, Cand Sox2 , 24, wasβ-catenin, CBP, P16, Ki67, and CK17 were positively correlated with the cervical lesion grade , while the expression of Involucrin was negatively correlated with it   . Taken tβ-catenin in the cytoplasm and nucleus; nevertheless, β-catenin is mainly located at cellular junctions in the absence of Wnt ligands [In this report, we described the identification of canonical Wnt signaling activation in precancerous lesions and cervical cancer. Wnt proteins comprise a large family of secreted growth factors that play essential roles in regulating cell proliferation, survival, differentiation, tissue architecture, and organogenesis during embryonic development –9. Accum ligands –30.Wnt3a, β-catenin, and CBP were significantly upregulated in the CIN lesion and cervical cancer samples compared with those in the normal cervix sample. These bioinformatics data were further verified by immunohistochemical staining. In particular, a gradual increase in the β-catenin immunoreactivity score and its positive translocation from the membrane to the cytoplasm and finally to the nucleus were observed in CIN2/3 and SCC, which indicates the activation of Wnt signaling [β-catenin translocated to the nucleus and activated the downstream genes expression, including c-Myc, Sox2, Nanog, Oct4, Survivin and Cyclin D1, thus contributing to oncogenesis and cancer development [In this study, the gene expression profile data showed that within the top 250 differentially expressed genes, the canonical Wnt signaling genes ignaling , 8, 12. elopment , 27, 31.β-catenin and CBP/p300, which are cotranscriptional factors, influences Wnt/β-catenin signaling [CBP/β-catenin and its target genes of KLF4, MYC, and SOX2 were upregulated, generally synchronicity with the progression of cervical lesion grade. Consistent with the activation of CBP/β-catenin, markers of TICs CK17, CK14, TP63 and P16 were upregulated, while markers of squamous cell differentiation LORICRIN, INVOLUCRIN and CK4 were downregulated significantly in the CIN and SCC samples compared with normal cervix samples. However, the expression of KLF4 and CK14 were slightly decreased in CIN as compared with normal cervix. We speculated that the lesion grade (CIN1 or CIN2/3) and the status of high-risk HPV infection in CIN samples, to some extent, may interfere the results. The immunoreactivity scores analyzed with Spearman test showed that the expression of Ki67, P16, CK17 were positively correlated with the cervical lesion degree while Involucrin was just the opposite. Thus, both array and IHC data further verified that canonical Wnt signaling activation promotes cervical lesion progression, which indicates the potential role of Wnt3a in maintaining stemness of TICs reprogrammed by Piwil2 in our previous report and sequentially triggering the progression of CIN2/3 and SCC [The physical association between ignaling , 10, 32.ignaling , 33, wheignaling . The actignaling , 36. Forignaling –39. The  and SCC –42. TherIn conclusion, our observations demonstrate that the canonical Wnt signaling pathway was activated during the oncogenesis of cervical lesions. The CBP/β-catenin interaction played an essential role in the progression of CIN and SCC by maintaining the stemness of TICs . Therefo


Parkinson’s disease (PD) is a major neurodegenerative disease (ND), presenting a progressive degeneration of the nervous system characterized by a loss of dopamine in the substantia nigra pars compacta. Recent findings have shown that oxidative stress and inflammation play key roles in the development of PD. However, therapies remain uncertain and research for new treatment is of the utmost importance. This review focuses on the potential effects of using cannabidiol (CBD) as a potential therapeutic strategy for the treatment of PD and on some of the presumed mechanisms by which CBD provides its beneficial properties. CBD medication downregulates GSK-3β, the main inhibitor of the WNT/β-catenin pathway. Activation of the WNT/β-catenin could be associated with the control of oxidative stress and inflammation. Future prospective clinical trials should focus on CBD and its multiple interactions in the treatment of PD. Parkinson’s disease (PD) is a main neurodegenerative diseases (ND), in that degeneration of the nervous system is marked by a loss of dopamine in the substantia nigra pars compacta (SNPC). This disease is localized in the brain and/or in the spinal cord, patients remain without any symptoms for a considerable duration , 2. PD cAging is a main risk marker of neurodegeneration, as it may dysregulate the different signaling that modulate homeostasis processes in cells. Cells with neurodegeneration are the sites of numerous molecular and cellular dysregulation . NumerouCurrently, drug therapies the main efficient and widely utilized treatments in PD are the use of levodopa, DA agonists, amantadine, monoamine oxidase B (MAO-B) inhibitors , catechoCannabis sativa plant. The plant possesses many therapeutic properties for a range of neurodegenerative diseases [. CBD decreases the stimulation of GSK3-β, an negative modulator of the WNT/β-catenin pathway [Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid derived from the diseases –15 and, diseases –18. CBD  pathway , and has pathway , 21 and  pathway . The preSeveral findings have documented the stimulation of OS in PD . MitochoBody can produce free radicals of oxygen for oxidative metabolism. In the aerobic respiration, molecular oxygen (O2) is diminished to water molecules in mitochondria. Through this phenomenon, O2, H2O2 and OH are generated by a leakage of oxygen . Phagocy2O2, but during PD development, H2O2 is changed into hydroxyl radicals (OH) through iron by the Fenton reaction. Then, H2O2 and OH enhance OS [2O2 and OH oxidized GSH [Many enzymes, such as monoamine oxidase (MAO), L-amino acid oxidase and tyrosine hydroxylase, are implicated in metabolism of dopamine and in ROS production . ROS prohance OS . In the ized GSH , involviized GSH . DAergicized GSH . OS invoized GSH .Recent PD studies have presented that inflammation has a main action  by activWingless drosophila melanogaster” and its mouse homolog “Int”. The WNT/β-catenin pathway is implicated in many signals and molecular processes, including cell proliferation, embryogenesis, cell migration and cell polarity, apoptosis, and organogenesis [The WNT name is comes from “ogenesis . Neverthogenesis .AXIN, tumor suppressor adenomatous polyposis coli (APC), and glycogen synthase kinase-3 (GSK-3β)”. In WNT ligands absence, this complex enhances to phosphorylate cytoplasmic β-catenin and involves its proteasomal degradation. In presence of the WNT ligands, β-catenin binds to Frizzled (FZL) and LDL receptor-related protein 5/6 (LRP 5/6), thereby stopping the complex and preventing β-catenin proteasomal degradation. β-catenin translocates to the nucleus to bind with TCF/LEF. This in turn activates WNT target genes [The WNT pathway belongs to the family of secreted lipid-modified glycoproteins . WNT liget genes –67.GSK-3β is a major negative modulator of the WNT/β-catenin pathway –73. As aDysregulation in the WNT/β-catenin pathway is intricately involved in the development of PD , 8. WNT/FoxO (Forkhead box class O) transcription factors are important intracellular modulators of numerous molecular pathways, such as glucose production and cell response to OS . ROS proThe activation of the WNT/β-catenin pathway decreases inflammation and enhances neuroprotection through several interplays between microglia/macrophages and astrocytes , 97.Numerous findings have observed an interplay between the WNT/β-catenin and NF-κB pathways, major markers of inflammatory process . The NF-Cannabis sativa plant. The Cannabis sativa plant generates more than sixty-six components such as delta9-tetrahydrocannabinol (THC), causing psychological effects, and CBD, the main non-psychotomimetic component in the Cannabis sativa plant [Cannabis sativa for centuries. CBD, a major components of Cannabis sativa, has recently presented considerable interest for its potential role with respect to many neuropsychiatric disorders [Cannabinoids provide from a heterogeneous group of components characterized by 3 major components: endogenous, synthetic and phytocannabinoids , 109. CBva plant . CBD preva plant . CBD attva plant . Furtherisorders . CBD preisorders , includiisorders . In schiisorders , 115.OS contributes to neurodegeneration. Thus, neuron cells present a functional or sensory loss in neurodegeneration. For life, even if oxygen is needed, an unbalanced metabolic process and an increased production of reactive oxygen species ends up in several NDs, including AD and PD. Free radicals lead to damages in protein and DNA, stimulate inflammation processes and apoptosis . Some fiIn the CNS, CBD has been shown to possess anti-inflammatory actions, thus being useful for neuro-inflammatory diseases , and theRecent clinical investigations have presented the interest of using CBD for its antiparkinsonian properties –131. CBDA study have observed that mutant OCD mouse models presented stimulated GSK-3β activity, suggesting that GSK-3β downregulation can provide a therapy for perseverative behavior .Dysfunction of GSK-3β is implicated in the pathogenesis of several disorders, such as neuropsychiatric disorders . GSK3β iCBD downregulates expression of GSK-3β through the promotion of PI3K/Akt signaling , 142. PICBD can reduce the activity of the pro-inflammatory factors COX-2 and NF-κB. These effects are stopped by the combination of CBD and PPARγ receptors. The neuroprotective effects of CBD are generated by anti-inflammatory actions modulated by both CB1 and PPARγ . By inteThe energy production and metabolism of glucose implicated in OS are regulated by the FOXO transcription factors . This reCBD can reduce the redox balance through the modification of both the rate and activity of oxidants and antioxidants . CBD stoThe antioxidant activity of CBD is characterized by the stimulation of redox-sensitive transcription factor associated with the Nrf2 (Nuclear factor-erythroid 2 related factor 2) , which cCannabinoids present anti-inflammatory action by endogenous receptors, including CB1 and CB2 . N-OleoyTo date, few studies have studied CBD as a potential alternative therapeutic solution in treating PD. However, CBD is attracting increasing interest in this context because of its possible inhibitory effect on OS and inflammation and the fact that, at low doses there are few side effects. WNT/β-catenin pathway activity is diminished in the development of PD. By stimulating the WNT/β-catenin pathway, through the decrease of GSK-3β, CBD may be an integral part of an innovative therapeutic treatment of the condition. Future investigated studies should therefore focus on CBD and its many relationships with the development and treatment of PD.


Lysophosphatidic acid (LPA) is a pleiotropic lipid messenger that addresses at least six specific G-protein coupled receptors. Accumulating evidence indicates a significant involvement of LPA in immune cell regulation as well as Schwann cell physiology, with potential relevance for the pathophysiology of peripheral neuroinflammation. However, the role of LPA signaling in inflammatory neuropathies has remained completely undefined. Given the broad expression of LPA receptors on both Schwann cells and cells of the innate and adaptive immune system, we hypothesized that inhibition of LPA signaling may ameliorate the course of disease in experimental autoimmune neuritis (EAN).55–78) in female Lewis rats. Animals received the orally available LPA receptor antagonist AM095, specifically targeting the LPA1 receptor subtype. AM095 was administered daily via oral gavage in a therapeutic regimen from 10 until 28 days post-immunization (dpi). Analyses were based on clinical testing, hemogram profiles, immunohistochemistry and morphometric assessment of myelination.We induced active EAN by inoculation of myelin protein 2 peptide (P2Lewis rats treated with AM095 displayed a significant improvement in clinical scores, most notably during the remission phase. Cellular infiltration of sciatic nerve was only discretely affected by AM095. Hemogram profiles indicated no impact on circulating leukocytes. However, sciatic nerve immunohistochemistry revealed a reduction in the number of Schwann cells expressing the dedifferentiation marker Sox2 paralleled by a corresponding increase in differentiating Sox10-positive Schwann cells. In line with this, morphometric analysis of sciatic nerve semi-thin sections identified a significant increase in large-caliber myelinated axons at 28 dpi. Myelin thickness was unaffected by AM095.1 signaling may present a novel therapeutic target for the treatment of inflammatory neuropathies, potentially affecting regenerative responses in the peripheral nerve by modulating Schwann cell differentiation.Thus, LPA Immune-mediated neuropathies represent a heterogeneous group of rare peripheral nerve disorders comprising both acute and chronic forms. The prototype of an acute monophasic inflammatory polyneuropathy is the Guillain–Barré syndrome (GBS), with a reported incidence rate of 1–4 cases per 100,000 worldwide .While GBS and CIDP are distinct disorders, they share clinical and pathogenetic aspects: immunomodulatory therapies such as plasma exchange or intravenous immunoglobulins (IVIg) represent effective treatment options in both conditions. Moreover, the presence of cellular infiltrates consisting mainly of T-lymphocytes and macrophages in nerve biopsies of GBS and CIDP patients as well as in the respective animal models, further supports the idea of an immune-mediated pathogenesis for either condition , 4.Given the substantial body of evidence indicating autoimmune-driven damage to neurons and glial cells as major pathological hallmark of inflammatory neuropathies, cellular and humoral immune responses against the peripheral nervous system (PNS) have mostly been considered in isolation, largely ignoring the acute and potentially long-lasting impact on physiological homeostasis in the peripheral nerve. While the detrimental effects of innate and adaptive immune responses have been well-defined in autoimmune neuropathies , demyeliSchwann cells, as the myelin-forming glial cell population of the PNS, have long been considered as passive bystanders in peripheral neuroinflammation. While Schwann cells have clearly been shown to fulfill important functions beyond building myelin sheaths , such asA recent study took the effort to analyze Schwann cell differentiation in NOD/B7-2-knockout mice, a spontaneous inflammatory demyelinating neuropathy model that shares several pathological features with CIDP. It was demonstrated that the onset of neuroinflammation resulted in an upregulation of dedifferentiation-associated genes. Most notably, the downregulation of Krox20, the master regulator of peripheral myelination, was paralleled by the induction of c-Jun expression, a dedifferentiation marker . These f1receptor expressed on Schwann cells, contributing to the inverse regulation of the positive and negative differentiation factors Sox10 and Sox2  anti-CD3 ; mouse monoclonal [1F4] anti-CD3 ; mouse monoclonal [OX42] anti-CD11b/c ; rabbit polyclonal anti-Sox2 IgG ; rabbit monoclonal [EPR4007] anti-Sox10 .For the preparation of semi-thin sections, sciatic nerves were fixed in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde for 24 h at 4 °C. Further processing and staining procedures were performed as described previously . BrieflyMorphometric analysis was performed by a blinded investigator using ImageJ . For the evaluation of g-ratios and axonal diameters, the circumference of axons and their respective myelin sheaths was measured within randomly selected fields. A minimum of 200 axons per nerve was evaluated. For the calculation of g-ratios, axonal circumference was divided by the circumference of the respective myelin sheath.t-test; multiple comparisons were performed by multiple t-tests corrected by the Holm–Sidak method. Non-parametric data were analyzed by Mann–Whitney test. Statistical significance is indicated by asterisks with P ≤ 0.05*, P ≤ 0.01**, and P ≤ 0.001***.Analysis of images was performed using ImageJ . For the quantification of cell populations, three longitudinal sections per animal were photographed with a high magnification (Leica DMi8). Cells were counted manually by a blinded investigator using ImageJ and either adjusted to the analyzed area or normalized to total cell count of DAPI-positive cells, respectively. Data analysis and compilation of graphs was performed using Microsoft  Excel and GraphPad  Prism 7. Statistical analysis was conducted using Student’s 1 antagonist AM095 at 10 mg/kg via oral gavage once daily in a therapeutic regime, starting with the onset of first clinical signs of EAN, approximately 10 dpi. No effect of AM095 on disease severity was noted up to the peak of the disease. However, a significant improvement and markedly accelerated recovery was observed in the AM095-treated versus vehicle group shortly after the beginning of the remission phase from 20 dpi onwards up to the end of the experiment at 28 dpi . The animals were treated with the specific LPA+ T-lymphocytes as well as CD11b/c+ myeloid cells at the peak of disease (14 dpi) and during remission phase (21 dpi) on sciatic nerve sections by immunohistochemistry. At the peak of disease (14 dpi), neither the number of T-lymphocytes nor myeloid cells were changed by AM095  and in areas with greater distance to infiltrates (termed “para-infiltrative”) Fig. . Infiltr1 using AM095 reduced the expression of the negative regulator Sox2 while increasing the expression of Sox10 in this murine model of nerve injury. Moreover, this effect was associated with reduced demyelination in response to LPA treatment in murine DRG cultures [In the past decade, enormous progress has been made in understanding the regulation of Schwann cell differentiation dynamics, both in developmental and pathophysiological contexts. Several positive and negative regulators of Schwann cell differentiation have been identified. On the transcriptional level, the most prominent positive differentiation factors include Oct6 and Sox10, concertedly inducing expression of the master regulator of peripheral myelination, Krox20 –26. Negacultures . Consistcultures . Acetylacultures .1 antagonism might predominantly affect regenerative responses in the peripheral nerve rather than modulating inflammatory processes. Therefore, administration of AM095 might be of particular interest in a therapeutic rather than a preventative context. To assess the impact of AM095 on nerve regeneration, semi-thin sections of sciatic nerves were examined at 28 dpi to determine morphometric parameters. While there was no overall impact of AM095 on myelin thickness and axonal size distribution, we detected a significant increase in large-caliber myelinated axons under AM095 treatment, further strengthening the argumentation for regenerative effects of the compound.Interestingly, in the study presented herein, amelioration of clinical scores following AM095 treatment was statistically significant only from 20 dpi onwards. This finding suggests that LPA2 receptor signaling, but not LPA1 [+ T-lymphocytes or CD11b/c+ myeloid cells at the peak of disease. Limited evidence pointed to a slightly accelerated immune cell clearance at the beginning of the remission phase following AM095 treatment. However, there was no impact on the number of circulating immune cells. Based on our clinical, morphometric and immunohistochemical findings, these data strongly indicate that LPA1 antagonism might predominantly modulate nerve regeneration during the remission phase.T-lymphocyte chemotaxis and matrix metalloproteinase secretion have been shown to be stimulated by LPAnot LPA1 . As suchTo better understand the mechanism by which AM095 accelerated recovery, markers for Schwann cell differentiation were explored using immunohistochemical detection of the aforementioned negative and positive differentiation factors Sox2 and Sox10 on longitudinal sciatic nerve sections obtained from rats over the course of the remission phase at 21 and 28 dpi. At both time points, the number of Sox2-expressing cells was significantly decreased under AM095 treatment, and this effect was even more pronounced at 28 dpi. This reduction in Sox2-positive cells was paralleled by a significant increase of Sox10 expressing cells at 28 dpi, pointing to a greater number of redifferentiating Schwann cells at this stage. Notably, a greater proportion of Schwann cells expressed Sox2 in closer proximity to cellular infiltrates with CD3 positivity. This finding suggests that immune cells might foster Schwann cell dedifferentiation via LPA in a paracrine manner. The function of LPA as a pro-inflammatory paracrine messenger has already been extensively illustrated in inflammatory conditions such as cancer-related or airway inflammation and rheumatoid arthritis . In this1 antagonism may provide a potential therapeutic target to block inflammatory glial cell activation and propagate remyelination in autoimmune neuropathies.Collectively, these findings indicate that a promotion of Schwann cell redifferentiation might be a relevant therapeutic strategy in inflammatory neuropathies both by clinical and morphometric measures and that modulating LPA signaling using selective LPA


ABSTRACT IMPACT: Our data demonstrate that VC2 oncolytic virotherapy has significant clinical potential. OBJECTIVES/GOALS: Use our novel oncolytic herpes simplex virus type I (HSV-1), VC2, to understand how oncolytic virotherapy affects the immunosuppressive tumor microenvironment as a mechanism of efficacy. METHODS/STUDY POPULATION: We tested the efficacy of VC2 as an oncolytic virotherapy (OVT) in a syngeneic B16F10-derived mouse model of melanoma. We modified the B16F10 to express nectin-1 (B16F10n-1), the major receptor for HSV-1. Engrafted B16F10n-1 tumors were intratumorally treated with either phosphate-buffered saline (PBS) or 1x10^6 pfu VC2. At indicated time points, treated tumors were excised and processed for immunohistochemistry or flow cytometry analysis. For our experimental metastasis studies, mice were intravenously challenged with B16F10n-1 cells. For our depletion studies, CD4+ and CD8+ T cells were depleted in mice by treatment with mouse anti-CD4 and anti-CD8 monoclonal antibodies respectively, while the control mice were given Rat IgG2b isotype. RESULTS/ANTICIPATED RESULTS: We found that VC2 slowed tumor growth rates and significantly enhanced survival times over control treated mice. VC2-treated mice that survived initial tumor engraftment were able to reject a second tumor challenge and were also resistant to lung colonization  of tumor cells. Furthermore, VC2 treatment promoted increased intratumoral T cell infiltration and induced a strong antitumor effect that decreased growth rates of distant, untreated tumors. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our data demonstrate that VC2 OVT has significant clinical potential. Furthermore, due to the increased survival rates and CD8+ T cells dependence, our model will enable study of the immunological correlates of protection for VC2 OVT and OVT in general, as well as to inform the rational design of future OVs with improved therapeutic potentials.


Notch and Wnt are two essential signalling pathways that help to shape animals during development and to sustain adult tissue homeostasis. Although they are often active at the same time within a tissue, they typically have opposing effects on cell fate decisions. In fact, crosstalk between the two pathways is important in generating the great diversity of cell types that we find in metazoans. Several different mechanisms have been proposed that allow Notch to limit Wnt signalling, driving a Notch-ON/Wnt-OFF state. Here we explore these different mechanisms in human cells and demonstrate two distinct mechanisms by which Notch itself, can limit the transcriptional activity of β-catenin. At the membrane, independently of DSL ligands, Notch1 can antagonise β-catenin activity through an endocytic mechanism that requires its interaction with Deltex and sequesters β-catenin into the membrane fraction. Within the nucleus, the intracellular domain of Notch1 can also limit β-catenin induced transcription through the formation of a complex that requires its interaction with RBPjκ. We believe these mechanisms contribute to the robustness of cell-fate decisions by sharpening the distinction between opposing Notch/Wnt responses. This clearly raises the question of how so few signalling pathways can generate such diversity. The answer lies, in part, in crosstalk between the signalling pathways that allows one pathway to regulate or alter the output of another. The Notch and Wnt signalling pathways are a good example of this2. These pathways are often active at the same time to regulate the development and maintenance of a particular tissue but frequently have opposing effects on cell fate specification3. Therefore, it is not surprising that signalling through the two pathways is under strict control to prevent a conflict between them and to resolve into either Wnt-ON/Notch-OFF or Notch-ON/Wnt-OFF. This is particularly important to allow the progression of a cell along a particular lineage where subsequent steps are controlled by Wnt and Notch signalling. For example, Notch signalling promotes the differentiation of stem cells in both the skin and the mammary gland by inhibiting the Wnt signal that supports the self-renewal of these stem cells4. Similarly, resolving competition between the two pathways is required for the successful differentiation of enterocytes and secretory cells within the intestinal epithelium8.Animal development and adult tissue renewal are controlled by a handful of different signalling pathways that define a bewildering array of different cell types12. In the absence of Wnt ligand, the free cytosolic β-catenin interacts with a destruction complex, comprising CK1, APC, Axin and GSK3β, which phosphorylates β-catenin to target it for degradation via the proteosome. The binding of Wnt ligand to the cell surface receptors Frizzled and LRP recruits the cytoplasmic adaptor protein Dishevelled and the destruction complex to the membrane. Consequently, β-catenin is no longer phosphorylated and accumulates in the cytosol and nucleus. Within the nucleus, β-catenin induces the expression of downstream target genes by interacting with members of the TCF/LEF family of DNA binding proteins. Notch signalling is triggered by the interaction of Notch receptors and DSL  ligands on adjacent cells13. This leads to the proteolytic cleavage of Notch to release the intracellular domain (NICD), which translocates to the nucleus to induce target gene transcription in a complex with the DNA binding protein RBPj and the transcriptional co-activator Mastermind-like (MAML).It is well established that the key parameter of the Wnt signalling pathway is the stability and localisation of the soluble pool of β-catenin16 by associating with Armadillo/β-catenin present at the membrane and promoting its degradation through endosomal trafficking. In contrast, several other studies in Xenopus, mice and cell lines have suggested that the inhibition of Wnt signalling is mediated by the nuclear form of Notch, NICD, and may require the expression of downstream targets19. However, it is difficult to know from these studies whether one or both proposed mechanisms are required for Notch to affect the crosstalk on β-catenin, as many of the experiments have been completed with forms of the Notch protein that can localise to the membrane upon synthesis and the nucleus after γ-secretase mediated cleavage.Over recent years, several mechanisms have been proposed to explain the crosstalk between the Notch and Wnt pathways that allow signalling through the two pathways to be resolved into Notch-ON/Wnt-OFF. Studies in Drosophila and mammalian stem and cancer cells have suggested that Notch present within the plasma membrane can modulate the amount and transcriptional activity of Armadillo/β-cateninHere, we demonstrate that both a signalling inactive form of Notch that is restricted to the membrane fraction and a nuclear form of Notch, NICD, can inhibit Wnt/β-catenin signalling, although there is a mark difference in their potency. The membrane-restricted form of Notch sequesters β-catenin to the membrane fraction preventing its interaction with TCF/LEF and requires endocytosis to inhibit Wnt signalling. The nuclear form of Notch can also disrupt Wnt/β-catenin signalling by forming a complex with β-catenin, but does not require the transcription of downstream Notch target genes. Interestingly, we find that the interaction between β-catenin and nuclear Notch is stabilised by RBPj, although RBPj cannot form a complex with β-catenin itself. This may explain the marked difference in potency between the two different forms of Notch, as RBPj is nuclear restricted. Together, our results indicate that the interaction between Notch and β-catenin can limit Wnt signalling to establish a Notch-ON/Wnt-OFF state to allow robust cell-fate decisions during embryonic development and tissue homeostasis.20) or LEF1-VP16 . ΔEGF_N1 and NICD were both able to inhibit S45Fβ-catenin but neither could reduce LEF1-VP16 induced transcription  and a full length β-catenin protein fused with the C-terminal half of Venus GFP, (V2β catenin). Venus GFP will only refold to form a functional fluorescent protein if the TCF4 and β-catenin proteins interact28. We found that in the presence of NICD and ΔEGF_N1, TCFV1 and V2β-catenin still associated to form a functional Venus GFP ) independent; Su(H) is the Drosophila homologue of RBPj. This repression is then alleviated by a Wnt signal to initiate sense organ precursor and muscle progenitor cell development in a group of cells. Then there is a second ligand and Su(H) dependent Notch signal that resolves this cluster of cells to one or two cells that will complete their development into a sense organ precursor or muscle progenitor cell. Given our results, we would suggest that initial repression is mediated by a membrane bound Notch protein, whilst the second is mediated by a nuclear form of Notch. This also fits with the potency of inhibition we see. The repression mediated by the membrane bound form of Notch is modest and therefore easily alleviated by a Wnt signal to initiate development. The nuclear repression is much stronger and will overcome the Wnt signal to suppress sense organ precursor and muscle progenitor development in all but one or two cells. Interestingly, very similar observations have been made in mice during cardiac precursor cell development15, suggesting that this is a conserved mechanism across metazoa. These studies also show how it is important to regulate the transition from a Notch-ON/Wnt-OFF to Notch-OFF/Wnt-ON and back again during cell differentiation33.It is interesting that we have identified two distinct mechanisms by which Notch can inhibit Wnt signalling. Previous work in Drosophila on sense organ precursor and muscle progenitor cell development has suggested that Notch can suppress their development in two ways2 in a humidified incubator.HCT116 cells were purchased from ATCC. Human embryonic kidney cells that stably express the large T antigen of simian virus 40 (HEK293T) were obtained from Dr Anthony Brown  and from Dr Valerie Kouskoff . HCT116 and HEK293T cells were cultured in DMEM medium  supplemented with 10% FBS  and 50 μg/ml penicillin and 50 μg/ml streptomycin . Cells were maintained at 37 °C and 5% CO5 cells/well of a 24 well plate. After 24 h cells were transfected in triplicate with a 250 ng DNA cocktail containing the desired DNA plasmids, 50 ng of the required reporter plasmid , and 20 ng of pRL-CMV plasmid as an internal control. To ensure the transfections contained a constant amount of DNA, an appropriate amount of the plasmid pcDNA3.1(+) was added. The cell culture medium was changed 3 h after transfection when Lipofectamine and PLUS reagent  were used. Cells were lysed 48 h post-transfection using 1 × Passive Lysis buffer . Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter assay system , according to manufacturer´s instructions, with a MicroLumatPlus plate reader . Data are presented as mean fold change (± SEM) in relative luciferase units (RLU), compared to β-catenin, NICD or VP16-RBPj. Statistical analysis was perform using one-way ANOVA and Tukey’s post-hoc tests using Prism software  for more than two samples or with a Student T test for data with two samples.Cells were transfected using Lipofectamine and PLUS reagent  or X-Treme transgene 9 transfection reagent , according to manufacturers’ instructions. HEK293T cells were plated at a density of 2 × 10pLNCX + mWnt1 plasmid was obtained from Dr Anthony Brown . mβ-catenin cDNA was obtained from Geneservice  . The pcDNA3 + mN1 plasmid was obtained from Dr Jeff Nye . The pCMX + VP16-RBPj plasmid was obtained from Dr Tasuko Honjo 34. The cDNA encoding mHes5 was obtained from Dr Ryoichiro Kageyama . The pCS2 + LEF-VP16 plasmid was obtained from Dr. Rolf Kemler . The pcDNA3.1 Zeo + TCF4v1 and pcDNA3.1 Zeo + v2β-catenin plasmids were obtained from Dr Claudia Wellbrook . The Xβ-catenin plasmid was a gift from Dr Louise Howe . The plasmid pcDNA3.1(+), used as an empty vector in transfections, was obtained from . The p10xRbpj-luc Notch reporter plasmid was obtained from Dr Grahame MacKenzie . The pHes1-luc reporter plasmid was obtained from Dr Ryoichiro Kageyama . The pTOPflash Wnt reporter was obtained from Dr Louise Howe . The pRL-CMV reporter plasmid was obtained from . The pmRFP-C1 and pEGFP-Rab7 plasmid were obtained from Dr Andrew Gilmore .The following plasmids were kind gifts: The 35: pcDNA3.1 myc/HisA + mβ-catenin.The following plasmid has already been describedThe following plasmids were generated in our laboratory.pSecTagNC + ΔEGF_mN1: the cDNA encoding mouse Notch 1 molecule that lacks all 36 EGF-like repeats was generated by cloning a PCR fragment (using mN1 4409F and mN1 4901R primers) digested HindIII/SacI along with a SacI/HindIII fragment of the mN1 cDNA into pSecTagNC + ΔEGF + LNR_mN1 (HindIII).pcDNA3 + mNICD: the cDNA encoding myc tagged mNICD was generated by cloning the KpnI/BspEI fragment from pEGFP-N1 + mNICD into pcDNA3 + mN1 (KpnI/BspEI).pcDNA3.1(+) + VP16/Rbpj: was generated by digesting pcDNA3.1(+) KpnI/EcoRI (blunted) and ligated to remove the restriction sites from HindIII to EcoRI in the pcDNA3.1(+) multiple cloning site. The VP16-Tag was cloned from pCMX-N + VP16/Rbpj (HindIII). The Rbpj cDNA was cloned from pCMX-N + VP16/Rbpj (EcoRI).pcDNA3.1(+) + myc-mHes5: was generated by digesting pcDNA3.1(+) with PmeI and inserting a fragment containing the following sequences: Kozak, myc-tag epitope, EcoRI, HindIII, and BamHI, and then cloning mHes5 cDNA as an EcoRI/BamHI-digested PCR fragment generated using mHes5 73F and mHes5 663R primers.pcDNA3.1(+) + myc-hHey1: was generated by digesting pcDNA3.1 (+) with PmeI and inserting a fragment containing the following sequences: Kozak, myc-tag epitope, EcoRI, HindIII, and BamHI and then cloning hHey1 cDNA (I.M.A.G.E. BC001873) as an EcoRI/BamHI digested PCR fragment generated using hHey1 98F and hHey1 11077R primers.pmRFP + hHes5 & pmRFP + DNhHes5: these cDNAs were generated by digesting pcDNA3.1 + myc-hHes5 and pcDNA3.1 + myc-DNhHes5 with EcoRI/BamHI and inserting the fragment into pmRFP-C1 (EcoRI/BamHI).pcDNA6 V5/HisA + S45Fmβ-catenin: was generated by excising S45Fmβ-catenin from pcDNA3.1(+)/myc-HisA + S45Fmβ-catenin  as a KpnI/XhoI fragment and inserting it into pcDNA6/V5-HisA vector (KpnI/XhoI).pGL3basic + TCFAdTATA: this Wnt reporter plasmid was generated in two steps. Initially, p10xCBF1-luc was digested with XhoI (blunted)/BglII (blunted) and re-ligated to form pGL3basic + AdTATA. pGL3basic + AdTATA was then digested with XhoI and the SalI fragment containing the 4 TCF sites from pTOPFlash was introduced.The following plasmids were generated by mutagenesis.pSecTagNC + ΔEGF_mN1 W1758A and pSecTagNC + ΔEGF_mN1 R1994A: these plasmids were generated by PCR-based mutagenesis using the QuikChange Lightning Multi Site-Directed mutagenesis kit  using pSecTagNC + ΔEGF_mN1 as a template and primers: W1758A F and R1994AmutF, respectively.pcDNA3 + mNICD W1758A: this plasmid was generated by PCR-based mutagenesis using the QuikChange Site-Directed mutagenesis kit  using pcDNA3 + mNICD as a template and the primers W1758A F and W1758A R.pcDNA3 + mNICD R1994A: this plasmid was generated by PCR-based mutagenesis using the QuikChange Lightning Multi Site-Directed mutagenesis kit  using pcDNA3 + mNICD as a template and the primer R1994AmutF.pcDNA3.1(+) + DNhHey1, pcDNA3.1(+) + DNmHes5: these plasmids were generated by mutating the conserved DNA binding domain Glu-Lys-X-X-Arg (EK**R) to Alanines. This generates a non-functional protein that when transfected can dimerise with endogenous proteins disrupting their function. These DN proteins were generated by PCR-based mutagenesis using the QuikChange Lightning Multi Site-Directed mutagenesis kit  using pcDNA3.1(+) + myc-hHey1, pcDNA3.1(+) + myc-mHes5 as templates and primers: hHey1 E58AK59AR62A, and mHes5 E25AK26AR29A respectively.For list of the PCR and sequencing primers used see supplementary table 2 Table .For list of the mutagenesis primers used see supplementary table 3 Table .2, placed on ice and lysed in 1 ml of lysis buffer ) + 10 μl of 100 mM PMSF. Subsequently, cells were poured into a cold 2 ml dounce homogenizer and sheared with 30 strokes. Cell lysates were centrifuged at 1500×g for 5 min at 4 °C. The supernatant was centrifuged for 90 min at 100,000×g in a TLA 110 rotor in a Beckman Coulter Optima TLX-120 Ultracentrifuge at 4 °C. After the spin, 100 μl of the supernatant, containing the cytosolic fraction, were mixed with 100 μl of 2 × Laemmli buffer , boiled for 3 min and stored at − 20 °C.Cells were washed twice in 3 ml of 1 × TBS (10 mM Tris–HCl pH 7.4 + 140 mM NaCl) + 2 mM CaCl36. For list of the primary antibodies used see supplementary table 1  for Notch constructs, diluted 1:100 in blocking solution , 0.1% Triton-X100, and 0.05% NaN3 in TBS) in a humidified chamber for 1 h. Subsequently, cells were washed and coverslips were incubated with fluorescence goat α-rabbit Alexa 594 secondary antibody in blocking solution. Coverslips were mounted with VECTASHIELD mounting medium for fluorescence with DAPI H-1200 (Vector Laboratories). Images were captured with a Zeiss LSM 700, AxioObserver flexible confocal microscope , using the Zeiss ZEN 2011 software . The confocal software was used to determine the optimal number of Z sections when acquiring 3D optical stacks. Either maximum intensity projections of these 3D stacks or single z-section images are shown in the results. Or coverslips were mounted with Dako fluorescent mounting medium . Images were collected on a Leica TCS SP5 AOBS inverted confocal using a (63 × HCX PL Apo) objective. The confocal settings were as follows, pinhole 1 airy unit, scan speed 1000 Hz unidirectional, format 512 × 512. Single z-section images where taken.HEK293T cells were seeded on nitric acid treated coverslips at 4 × 10Total RNA was extracted from cells using peqGOLD TriFast solution  according to manufacturer’s instructions. A total of 2 μg of RNA were reverse transcribed to cDNA in a 20 μl reaction using the High Capacity RNA-to-cDNA Master Mix kit , following manufacturer’s instructions. The resulting cDNA was used as template for the quantitative PCR. Quantitative PCR was performed in triplicate, in a total volume of 20 μl, using the primers listed on supplementary table 4 Table , and FasSupplementary Information