The Wnt/β-catenin signaling pathway plays a major role in cell survival and proliferation, as well as in angiogenesis, migration, invasion, metastasis, and stem cell renewal in various cancer types. However, the modulation (either up- or downregulation) of this pathway can inhibit cell proliferation and apoptosis both through β-catenin-dependent and independent mechanisms, and by crosstalk with other signaling pathways in a wide range of malignant tumors. Existing studies have reported conflicting results, indicating that the Wnt signaling can have both oncogenic and tumor-suppressing roles, depending on the cellular context. This review summarizes the available information on the role of the Wnt/β-catenin pathway and its crosstalk with other signaling pathways in apoptosis induction in cancer cells and presents a modified dual-signal model for the function of β-catenin. Understanding the proapoptotic mechanisms induced by the Wnt/β-catenin pathway could open new therapeutic opportunities. The Wnt/β-catenin pathway is severely dysregulated in various cancer cell types ,2,3; itsThis dichotomic role of β-catenin can be explained considering that certain proteins show both oncogenic and suppressive actions on a subset of cancer cells ,12,13,14On a different note, it has been demonstrated that both overactivation and inhibition of the Wnt/β-catenin pathway induce antineoplastic effects even in the same cancer type ,17; thusARF (a Mdm2 inhibitor), promoting the nuclear accumulation of p53 . P. PBRAF-ama lines . These dF kinase .axin2, LEF1, and TNFRSF19 [axin1 siRNA significantly sensitized melanoma cells to rhTRAIL [It has also been reported that an activation of the Wnt/β-catenin signaling in WNT3-conditioned medium increased apoptotic cell death rates promoted by the recombinant human TNF receptor death-inducing ligand (rhTRAIL) in melanoma cells, by increasing the levels of the Bim and PUMA proapoptotic proteins, reducing the levels of the Mcl1 antiapoptotic protein, and upregulating mRNA expression for genes like TNFRSF19 . Further rhTRAIL . Convers rhTRAIL . It was  rhTRAIL ,204,205  rhTRAIL .In lung cancer lines, β-catenin induced the relocation and redistribution of death receptors like DR4 and DR5 on the cell membrane, inducing TRAIL-mediated extrinsic and intrinsic apoptosis by activating caspase-8 and -3 . A loss Arozarena et al. demonstrated that the expression of β-catenin and the melanocyte-specific transcription factor (MITF) are significantly reduced in areas of dermal invasion within primary tumors, and that an accumulation of cytoplasmic/nuclear β-catenin, which correlated with MITF expression levels in melanoma cells, inhibited invasion into 3D collagen systems . Inhibit675 β-catenin by a PKA-dependent pathway, while increasing MITF levels [Docosahexaenoic acid  was proved to reduce the proliferative and invasive potential, inhibiting the gelatinolytic activity of MMP-13, MT1-MMP, and MMP-2 in B16F10, WM115, and WM266-4 melanoma cells, by increasing the nuclear relocation of pSerF levels .675 reduced its proteasomal degradation and increased its nuclear expression [In fetal kidney HEK293 cells, β-catenin phosphorylation on Serpression . DHA alspression . These rpression .Medulloblastoma is one of the most frequent malignant brain tumors in the pediatric population. Age on diagnosis is usually 6–8 years, although it may occur in adult patients . Histologically, it is a cerebellar embryonic tumor. It is more common in male than in female patients, with a male–female ratio of 1.8: 1 . TranscrWNT medulloblastoma, which accounts for 10% of all medulloblastomas, shows a loss of chromosome 6 and activating mutations in the CTNNB1 gene, encoding for β-catenin . ClinicaWhile the canonical Wnt/β-catenin pathway is required for GNPs maturation, a constitutive activation of this pathway inhibits cell proliferation of GNPs . Poschl In addition, the activation of the Wnt/β-catenin pathway in Math1:SmoM2 Fi/+ ctnnb(ex3)Fi/+ mice (a well-known Shh-medulloblastoma model) significantly decreased medulloblastoma formation rates, resulting in a decreased cerebellar size . LiCl (aThe better outcome in WNT MB has been hypothesized to be due to the secretion of a soluble Wnt antagonist like Dickkopf 1 (DKK1) or the Wnt inhibitory factor 1 (WIF1) by tumor cells, which may reduce the Wnt/β-catenin pathway activity in neighboring endothelial cells, damaging the blood–brain barrier and making the tumor more susceptible to chemotherapy . In addiaxin2 mRNA and downregulating Sox2 and Bmil; L807mts, another GSK-3β inhibitor, can induce Wnt/β-catenin signaling in Group 3 and -4 medulloblastoma xenografts, decreasing tumor burden and increasing survival with respect to control xenografts [Orthotopic injections of WNT medulloblastoma to smaller, less invasive tumors increased mouse survival rates with respect to Group 3 xenograft mice . Meanwhinografts .TP53-mutant medulloblastoma [TP53-mutant medulloblastoma patients could be correlated to radiation resistance [TP53-mutant medulloblastoma cells, and it could represent an attractive therapy for high-risk medulloblastomas [Some authors suggested that the inhibition of stemness determinants could be a therapeutic strategy to transform malignant tumors into less aggressive cancers, like medulloblastoma ,227. Furblastoma . The poosistance . The conlastomas .p53-mutated) and D283MED (wild-type p53) cell lines; apoptosis and growth arrest in TP53-mutated medulloblastoma cells could be mediated by Wnt/β-catenin, bypassing the p53 pathway [In addition, Salaroli et al. showed that radiation triggers Wnt/β-catenin pathway activation with the ensuing cell cycle arrest and apoptotic cell death in medulloblastoma DAOY , which rapidly results in hepatomegaly and hepatocellular cancer ,241. TheWnt/β-catenin signaling suppressed the positive feedback loop between Notch and TAZ, by inducing the nuclear relocation of DP1 , which inhibits Notch signaling by promoting NICD degradation. In addition, the inhibition of Notch signaling in vivo significantly reduced the YAP/TAZ activity, along with liver tumorigenesis ,241. ThoLATS2 overexpression induces apoptotic cell death in SMMC-7721 and BeL-7404 liver cancer cell lines . The apoBax, Bcl-2, p2I, and p53, thus decreasing cell survival and favoring apoptosis in HepG2 cells [Qing-Qiang et al. demonstrated that trichostatin A (a deacetylase inhibitor) inhibits cell proliferation and induces cell cycle arrest and apoptosis on HepG2 hepatoma cells by decreasing the activity of histone deacetylases (HDAC1 and HDAC3) and activating β-catenin . When chG2 cells ,246.Overall, these results suggest that the Wnt/β-catenin pathway induces an antineoplastic effect by engaging in crosstalk in Hippo and Notch signaling, along with other pathways.Over the years, the role of the Wnt/β-catenin pathway in the embryologic development of hematopoietic stem cells (HSCs), as well as in the pathogenesis and progression of cancer, has been studied. Various gene mutations in Wnt signaling molecules have been proved to induce a cytoplasmic or nuclear accumulation of β-catenin. Such mechanism has been observed in several neoplasm types, including hematologic ones . Thus, dCtnnb-1 gene, which induced a higher expression of activated β-catenin, were found to downregulate Bcl2 and upregulate Casp3 with respect to controls [In the first place, Wnt/β-catenin activation was observed to induce apoptosis, as proved by β-catenin stabilization both in in vivo and in vitro models; mutant HSCs with deletion of exon 3 in the controls . In factcontrols .2/M phase, a lower number of cells in the S phase, and increased levels of p21WAF1, a key mediator in cell cycle arrest, were also observed. Meanwhile, an upregulation of β-catenin induced apoptosis [Histone acetylation and deacetylation play a major role in tumorigenesis. Acetylation is known to activate transcriptional activity, while deacetylation inhibits it . Shao N.poptosis .A dose-dependent loss of cell viability was observed with Wnt agonists in a monocytic leukemia model using the RAW 264.7 and J774.1 murine cell lines. The binding of Wnt agonists to their receptors led to dysfunctional activity of GSK-3β, along with β-catenin stabilization and nuclear accumulation .In a novel approach, RNA interference has been used to silence genes in oncology . Particu2 phase, and decreased the expression of cyclin D1 [On the other hand, higher apoptosis rates were observed in multiple myeloma cells with a silenced β-catenin pathway, increasing the expression of phosphorylated p53, active caspase-3, and Bax, while the expression of Bcl-2 was decreased, suggesting a mitochondrial involvement . Interveyclin D1 . LiCl inyclin D1 . A deepeyclin D1 .Mst1 overexpression causes apoptosis by increasing ROS generation, the levels of the Bax proapoptotic protein, and the activity of caspase-9, while decreasing the antioxidant activity of SOD, GSH, and GPX, as well as the levels of the antiapoptotic protein Bcl-2 in the squamous cell carcinoma of the head and neck (SCCHN) Cal27 cell line and in Tu686 cells; the mechanism is thought to involve the regulation of mitochondrial fission via the β-catenin/DRP1 pathways . An inhi127 phosphorylation of YAP, as well as inducing a decrease in amphiregulin, the connective tissue growth factor, and Survivin mRNA [Mst1 overexpression has also been reported to decrease cell viability and induce apoptotic cell death in the human non-small cell lung cancer (NSCLC) A549 line both in vitro and in vivo, by promoting the cytosolic relocation and Servin mRNA . Mst1 alvin mRNA . In addivin mRNA .On the other hand, a lower β-catenin expression correlated with a poorer prognosis , a more The tumor suppressor candidate 3 (TUSC3) inhibited cell growth and induced apoptosis in A549 cells. TUSC3 also promoted increased levels of LC3-II and Beclin-1, favoring autophagosome formation, while lowering the phosphorylation (inactivation) of β-catenin and promoting its nuclear translocation . The losOther studies have described a nuclear induction of β-catenin in human pancreatic cancer cells by SAHA, which resulted in an inhibition of cell proliferation and in apoptosis induction . The uprInterestingly, Ursolic acid exerts its antineoplastic effect on SK-OV-3 ovarian cancer cells by inducing β-catenin stabilization and promoting the phosphorylation (inactivation) of GSK-3β . TreatinMYCN mRNA expression, regulating the p53 and Wnt/β-catenin pathways [MYCN oncogene is amplified in 25% of neuroblastomas, and it has been linked to therapeutic resistance [MYCN overexpression in neural crest progenitor cells leads to neuroblastoma formation [MYC-amplified neuroblastoma. Treatment with ICG-001  and the Wnt agonist 1  significantly impaired cell viability in IMR32 neuroblastoma cells [MYC mRNA levels and decreased cell viability by inducing apoptosis, while ICG-001 increased MYCN transcripts and reduced cell viability and proliferation.It was reported that LY20314 (a GSK-3β inhibitor) inhibits cell proliferation in human neuroblastoma cell lines while increasing β-catenin levels . Duffy epathways . The MYCsistance . MYCN ovormation . It has ma cells . Wnt agoThese results suggest that altering β-catenin thresholds in a cancer cell, either below or above normal levels, will be detrimental to cell survival. MYCN has also been suggested to block the function of Wnt/β-catenin signaling by promoting the binding of MYCN to β-catenin, thereby stopping its transcriptional activity . On the It has been proposed that TGF-β induces apoptosis in prostate cancer cells through Smad 7, which interacts with TAK1 and MKK3, leading to the activation of p38 and the ensuing activation of AKT, the inactivation of GSK 3β, and the stabilization of β-catenin ,273. FurThe works herein reviewed suggest that, under certain circumstances, it is possible to induce apoptosis in cancer cells by activating the Wnt/β-catenin signaling pathway and/or receptors with tyrosine kinase activity that leads to β-catenin stabilization, either by deactivating the degradation complex, α-catenin, or the Notch signaling pathway, or by activating the Hippo pathway, which in turn inactivates the YAP/TAZ transcriptional factors.All these events result in a release and accumulation of β-catenin, causing its nuclear translocation and its transactivating capacity. Among other mechanisms, the Wnt/β-catenin pathway can induce apoptosis by downregulating antiapoptotic proteins and inducing the transcription of p53 and c-myc, which in turn can induce the transcription of proapoptotic proteins .However, the effect of the Wnt/β-catenin signaling pathway in cancer cells is extremely complex and context-dependent. It can be involved in the regulation of cell growth, proliferation, and death. For years, a dysregulation of the Wnt/β-catenin pathway was regarded as contributing to cancer invasion and metastasis, modulating the tumor microenvironment, and promoting resistance to treatment. Nevertheless, further studies indicated that this initial picture was an oversimplification. Instead, the Wnt pathway, either acting by a β-catenin-dependent or an independent mechanism, can inhibit or promote cancer progression in a stage- and cancer-type-specific manner.On the other hand, it has been proposed that the Wnt/β-catenin signaling and its crosstalk with other signaling pathways, including Hippo, DRP1, Notch, EGFR/RAS/RAF/MEK/ERK, LKB/AMPK, Rb, c-myc, p53, and FOXO, plays a key role in the regulation of cell growth, proliferation, and death. The study of the crosstalk between Wnt/β-catenin signaling and other well-characterized signaling pathways will deepen our understanding of the cell signaling network. In time, it may help us to discover valuable therapeutic targets to treat cancer.


This work demonstrates that the application of even moderate pressures during cure can result in a remarkable enhancement of the thermal conductivity of composites of epoxy and boron nitride (BN). Two systems have been used: epoxy-thiol and epoxy–diamine composites, filled with BN particles of different sizes and types: 2, 30 and 180 μm platelets and 120 μm agglomerates. Using measurements of density and thermal conductivity, samples cured under pressures of 175 kPa and 2 MPa are compared with the same compositions cured at ambient pressure. The thermal conductivity increases for all samples cured under pressure, but the mechanism responsible depends on the composite system: For epoxy–diamine composites, the increase results principally from a reduction in the void content; for the epoxy–thiol system with BN platelets, the increase results from an improved matrix-particle interface; for the epoxy–thiol system with BN agglomerates, which has a thermal conductivity greater than 10 W/mK at 44.7 vol.% filler content, the agglomerates are deformed to give a significantly increased area of contact. These results indicate that curing under pressure is an effective means of achieving high conductivity in epoxy-BN composites. Epoxy composites filled with boron nitride (BN) are widely used for heat management in Insulated Metal Substrates (IMS). Such materials must satisfy a number of requirements, including adhesion, processability, electrical insulation, and high thermal conductivity. The last of these has attracted considerable attention, and the state of the art in polymer-based composites in general ,2, and iIt is widely recognized that the thermal conductivity of epoxy-BN systems increases with increasing BN content ,4, at leThe effect of BN filler size highlights the importance of the matrix–particle interface, and attempts have been made to improve this interface by the surface treatment of the particles or with the use of coupling agents. The results, however, are not entirely conclusive. While many authors report significant increases in the thermal conductivity ,12,13,142O3 rather than BN, despite the intrinsically lower thermal conductivity of the former. Processability is also a factor that must be borne in mind; although significantly higher thermal conductivities have been achieved in epoxy composites by the alignment of the filler particles by various means , these In the light of the above discussion, it would be interesting to find a more generic way in which the thermal conductivity of epoxy composites might be increased. There is, indeed, just such a possibility, though it has received little attention to date; this is the effect of the application of pressure during cure. In the first place, it might intuitively be expected that applying pressure would consolidate the material, improving the matrix–filler interface, and hence enhancing the thermal conductivity. Additionally, though, there is also the possibility of densifying the material, by cooling the epoxy matrix through its glass transition region under pressure ,22. In vSeveral authors have reported the use of pressure during fabrication of their epoxy-BN composites, but there are very few systematic studies of this effect on the thermal conductivity. Furthermore, there are several different ways in which the pressure can be applied. Some workers apply pressure to the composite mixture before curing at ambient pressure ,24,25. Tper se on the thermal conductivity cannot be identified.On the other hand, several workers simply use pressure, without specifying the magnitude, in a process often referred to as “hot pressing”, as part of the fabrication procedure of their epoxy composites ,27,28,29In the majority of studies, though, the composite is either first partially cured under pressure ,31,32,33In the latter category, the large number of additional factors which play a role in determining the thermal conductivity of these composites  make itis a function of the pressure applied.In order to identify the effect of pressure on the thermal conductivity of these epoxy-BN composites, it would clearly be necessary to make a systematic study. However, of all the studies discussed above, only Zhu et al.  and Hu eFrom the foregoing discussion, it is evident that it is not possible to obtain any clear idea about the effect of pressure during cure on the thermal conductivity of epoxy-BN composites from the results presented in the literature. Consequently, in the present work we make a systematic investigation of the effect of pressure during cure on the thermal conductivity of a number of different epoxy-BN composite systems, and show that significant increases can be achieved. The mechanisms by which these increases are obtained depend on the composite system, and demonstrate that enhancement of the thermal conductivity can occur in ways other than by orientation.3 and viscosity of 7000 to 9000 mPa.s at 25 °C. The principal cross-linking agent used was a thiol, pentaerythritol tetrakis (3-mercaptopropionate) , with a molecular weight of 488.66 g/mol, density of 1.28 g/cm3, and viscosity of 500 mPa.s at 23 °C. In order to initiate the cross-linking reaction of the epoxy with the thiol, a latent initiator, encapsulated imidazole  in the form of powder, was used. In addition, a polyoxypropylene diamine, Jeffamine D-230 , and a dicyandiamide, N,N-dimethyl-N-phenyl urea , were also used as alternative cross-linking agent and initiator, respectively, for comparative purposes.The epoxy resin used was diglycidyl ether of bisphenol-A, DGEBA , with a nominal molecular weight per epoxy equivalent (eq) of 182 g/eq, density of 1.17 g/cm3, 10 m2/g;PCTP2: 2 μm, 10 μm, 0.2 g/cm3, 1 m2/g;PCTP30: 30 μm, 100 μm, 0.6 g/cm3, 1 m2/g;PCTP30D: 180 μm, 1600 μm, 0.6 g/cm3, 3.5 m2/g.CTS7M: 120 μm, 180 μm, 0.5 g/cmDifferent grades of hexagonal BN filler  were used, shown in Various important aspects of these filler particles can be appreciated from this Figure. The 2 μm and 30 μm particles are both in the form of platelets, but the specific surface area is 10 times higher for the smaller particles; this has the effect that it is difficult to fabricate composites with a large content of the 2 μm particles. The 180 μm particles are also in the form of platelets, but there is a very wide distribution of particle sizes; according to the manufacturer, these particles are engineered for high shear mixing processes. On the other hand, the 120 μm particles are in the form of agglomerates, denoted as spherical powder by the manufacturer. The filler particles were used as received, without any surface treatment.The epoxy-BN composites were fabricated in the proportions given in All the components were mixed by hand for 15 min to ensure homogeneity and were then degassed under vacuum (<26 hPa) at room temperature for about 10 min. The quality of the dispersion can be evidenced by examination by Scanning Electron Microscopy (SEM) of the fracture surfaces of cured composite samples. An illustration of one such SEM micrograph is shown in T as a function of time, t, following the heat pulse input. The thermal conductivity, λ, was obtained [T as a function of the inverse square root of time, 1/√t. The instrument was calibrated with five different standards covering the range from 0.2 to 10 W/mK. The samples prepared at ambient pressure were cast in silicone molds 10 mm × 40 mm × 4 mm, and cured isothermally in an air-circulating oven at 70 °C for 3 h. Four measurements were made of the thermal conductivity of each of the samples, and the average value was taken. Typically, the standard deviation of these four measurements was ±0.1 W/mK.The thermal conductivity was measured using the Transient Hot Bridge method , in which a heat pulse is applied to a sensor placed between two surfaces of the sample material and the instrument measures the temperature change Δobtained  from an In order to obtain compressed samples, the device illustrated in 3.The density of the cured samples was measured by Archimedes method. The samples were first weighed in air at room temperature, and then when immersed fully in ethanol, suspended by a fine thread. From repeated measurements on the same sample, the uncertainty in the density is estimated as ±0.02 g/cmFully cured samples, similar to those used for the thermal conductivity measurements and prepared using the same isothermal curing procedure, were fractured and then the fracture surface was examined in a Scanning Electron Microscope . An accelerating voltage of 10 kV was used to give magnifications from 100× to 5000×.x-30, -50, -60, and -70, with x = 2, 30, and 180, cured in open molds without the application of pressure, was measured experimentally in earlier work [The density of epoxy–thiol-BN platelet composites, samples ETLBN3 for the epoxy–thiol system without any filler. In fact, the composite density calculated from the densities and weight fractions of the three components  and allowing for a 4% shrinkage on cure gives the dashed line, which agrees very closely with the experimental values, implying that there is an insignificant void content for filler volume fractions less than 34%. For the highest filler content of almost 45 vol.%, however, there is a significant deviation from this linear relationship, which is attributed to the presence of voids in the sample; at such high filler contents the composite is a very stiff paste, and the mixing procedure introduces air bubbles which cannot be removed by degassing.Here it can be seen that, for BN contents less than 40 vol.%, the density of the composites is independent of the BN particle size, and increases linearly with increasing filler content from a value of 1.25 g/cm3) cured with triethylenetetramine (0.982 g/cm3) and filled with 3–8 μm BN platelets (2.29 g/cm3), which gives a calculated density of the composite  which falls on the same dashed line as given in 3 used in our calculations is applied instead, together with a 4% volume shrinkage on cure, the calculated density corresponds very closely with the data of Lewis et al., suggesting that their composites are essentially void-free, as are ours for volume fractions up to 34%. This is consistent with their thermal conductivity results, which compare well with ours and with others in the literature [Comparison can be made with literature values for other epoxy-BN composites, though this is not straightforward: the density depends on the nature of the epoxy and hardener system as well as on the BN content, and there are also rather few studies in which density measurements are reported. For epoxy-BN composites prepared by curing at ambient pressure, data are available from two references in addition to Zhu et al. . Lewis eThe second reference for comparison of the density values is Chung and Lin , who use3), and with very high BN contents, well beyond those used here. The reported densities, for BN contents between 74 wt.% and 95 wt.% do not appear to show any systematic variation with either BN content or consolidation pressure, and fall well below the “theoretical” density, which increases from about 1.8 g/cm3 to about 2.2 g/cm3 over this filler range. Indeed, the reported density of approximately 1.0 g/cm3 for composite samples fabricated without consolidation before cure is apparently less than that of the unfilled epoxy system (1.14 g/cm3). This would appear to be consistent with the reported values of thermal conductivity for these composites cured without prior consolidation, and hence without significant orientation, which, at less than 4.0 W/mK, are very low for such highly filled systems [It should be noted that both Lewis et al.  and ChunThe effect on the density of curing the samples under a pressure of 175 kPa is illustrated in The difference between the effects of curing under pressure for the two composite systems is remarkable. For the epoxy–thiol composites, the increase in density resulting from curing under this rather low pressure of 175 kPa is small, as can be seen in 2/g, for these platelets in comparison with that for the larger platelets, 1 m2/g, which made it very difficult to fabricate composites with a high filler content of 2 μm platelets. It can be seen, for example, that no data are included in Strangely, however, the density appears to decrease for the samples filled with 2 μm platelets, though the difference is small and close to experimental uncertainty. This is probably related to the much higher specific surface area, 10 mIn comparison with the very limited effect of the pressure of 175 kPa on the density of the epoxy–thiol composites illustrated in It is remarkable that the large density changes observed in the epoxy–diamine composites shown in 3 and 1.69 g/cm3, respectively, are greater than the calculated density for 70% epoxy–thiol-BN composites, 1.64 g/cm3  of 30 μm BN platelets gives values of 8.77 W/mK and 10.96 W/mK for amine and cationic cure, respectively. These composite samples are prepared by hot-pressing at 20 MPa, but the authors verify that there is no orientation induced by this procedure. Our values for the epoxy–thiol system at 2 MPa are therefore only slightly less than those of Islam et al. at 20 MPa, which represent the highest values reported for isotropic epoxy-BN composites. It is possible that the epoxy–thiol values could be increased further by pressures higher than 2 MPa.These very high values of thermal conductivity obtained by Zhu et al.  are a coFor comparison, we note the following studies in which thermal conductivities are reported with values which most closely approach our own. Xu and Chung  report a2/g, for these BN particles, which creates a much larger interfacial area between matrix and filler.The apparent correlation between thermal conductivity and density demonstrated by The effect of applying pressure during cure on the pathways for heat conduction is illustrated in In comparison with the fracture surfaces of the epoxy–diamine composites, shown above in It can be seen that for the samples cured under pressure there is much better contact between the BN particles and an improved matrix–filler interface. The improvement noted between On the other hand, the epoxy–thiol composites with 120 μm agglomerates display a quite different relationship between thermal conductivity and density, in which the thermal conductivity increases dramatically without a significant increase in the density. This is very clearly seen in The SEM micrographs for the epoxy–thiol composites filled with BN agglomerates are shown in In The application of pressure during the cure of several different epoxy-BN composite systems has been shown to increase both the density and the thermal conductivity of these composite materials. In most cases there is a correlation between the density and the thermal conductivity, but the mechanisms for enhancing the thermal conductivity depend on the particular epoxy-BN system. For the epoxy–diamine system, the principal effect of applying pressure is to reduce the void content, which leads to a large increase in the thermal conductivity. On the other hand, for the epoxy–thiol system in which the BN filler particles are in the form of platelets, the enhancement in thermal conductivity results from an improvement in the matrix–filler interface, and the closer proximity of the particles. In contrast, for the epoxy–thiol system filled with BN particles in the form of agglomerates, the effect of pressure is to deform the agglomerates, leading to a greatly increased area of contact between the particles and a correspondingly dramatic increase in the thermal conductivity. The use of pressure during cure, particularly for epoxy-BN composites filled with particles in the form of agglomerates, is therefore considered to be an excellent procedure for achieving high thermal conductivities in these systems.


Human papillomavirus type 18 (HPV18) has high carcinogenic power in invasive cervical cancer (ICC) development. However, the underlying mechanism remains elusive. The carcinogenic properties of HPV18 require the PDZ‐binding motif of its E6 oncoprotein (HPV18 E6) to degrade its target PSD95/Dlg/ZO‐1 (PDZ) proteins. In this study, we demonstrated that the PDZ protein membrane‐associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) inhibited the Wnt/β‐catenin pathway, and subsequently cervical cancer (CC) cell migration and invasion, via decreasing β‐catenin levels. By reducing MAGI3 protein levels, HPV18 E6 promoted CC cell migration and invasion through activation of Wnt/β‐catenin signaling. Furthermore, HPV18 rather than HPV16 was preferentially associated with the downregulation of MAGI3 and activation of the Wnt/β‐catenin pathway in CC. These findings shed light on the mechanism that gives HPV18 its high carcinogenic potential in CC progression. By reducing the level of MAGI3 protein, HPV18 E6 promotes cervical cancer cell migration and invasion through activation of Wnt/β‐catenin signaling. Furthermore, HPV18 rather than HPV16 is preferentially associated with downregulation of MAGI3 and activation of the Wnt/β‐catenin pathway in cervical cancer. These findings shed light on the mechanism that gives HPV18 its high carcinogenic potential in cervical cancer progression. CCcervical cancerCINcervical intraepithelial neoplasiaCISin situcarcinoma FDRfalse discovery rateGSEAgene set enrichment analysisHG‐CINhigh‐grade cervical intraepithelial neoplasiaHPV16human papillomavirus type 16HPV18human papillomavirus type 18HR‐HPVshigh‐risk types of human papillomavirusesICCinvasive cervical cancerIHCimmunohistochemistryMAGI3membrane-associated guanylate kinase, WW and PDZ domain containing 3NHERF1+/H+ exchanger regulatory factor 1NaPBMPDZ‐binding motifPDZPSD95/Dlg/ZO-1TCGAThe Cancer Genome AtlasCervical cancer (CC) is the fourth most prominent type of cancer in women . Infectiin situ (CIS) progression to ICC. Among HR‐HPVs, human papillomavirus type 16 (HPV16) and human papillomavirus type 18 (HPV18) are the two of the most common HPV types and cause more than 70% of ICC cases [Persistent infection of high‐risk HPVs (HR‐HPVs) has been convinced to cause early carcinoma CC cases . HPV16 wCC cases . When anCC cases . These sSo far, the viral oncoproteins E6 and E7 from HR‐HPVs have been identified as the main cancer‐causing factors of CC . E7 mainMembrane‐associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) belonging to the PDZ family of protein was more efficiently degraded by HPV18 E6 rather than HPV16 E6 , 23. We 2. CHIR‐99021, a Wnt/β‐catenin activator, was purchased from TargetMol ; IWR‐1‐endo, a Wnt/β‐catenin inhibitor, was purchased from Selleck .C‐33A and HeLa cell lines were obtained from the National Infrastructure of Cell Line Resource . C‐33A and HeLa cells were cultured in MEM  and DMEM , respectively, with 10% fetal bovine serum  at 37 °C in an atmosphere containing 5% COpcDNA3‐V5/His‐MAGI3 and GFP/GFP‐MAGI3 plasmids were kindly provided by R. Hall . Flag‐tagged β‐catenin vector was kindly provided by W. Wu . HA/Flag‐HPV18‐E6 (HA/Flag‐18E6) plasmid was kindly provided by Karl Munger .Cells were transfected with Lipofectamine 3000 . The stably transfected HeLa cells were obtained as previously described .The knockdown cell lines were transiently transfected with two different siRNA, which target MAGI3 and HPV18E6, and the sequences obtained as described previously , 24 wereThe western blotting assay was performed as previously described . The pri4) or C‐33A (5 × 105) cells were suspended in 100 μL medium with 0.5% FBS and plated into Transwell chamber  that was precoated with fibronectin  on the lower surface, with (migration assay) or without (invasion assay) Matrigel , and to the lower chamber was added 600 μL medium with 10% FBS. After culturing at 37 °C for 24 h, the cells were fixed and stained with methyl alcohol and 0.5% crystal violet, respectively. Cells were counted in 5 random fields of view per well by ImageJ, and the results were expressed as mean ± SD.HeLa  by the Ethics Committee of the Shanghai Outdo Biotech Company, and the member of the National Human Genetic Resources Sharing Service Platform . The present study was performed in accordance with the ethical standards of the Institutional and National Research Committee and with the Declaration of Helsinki. Written informed consent was acquired from all patients.The human tissue microarrays containing CC and adjacent normal tissues from patients  were analyzed by immunohistochemistry (IHC), which was performed as described previously . The dathttp://www.broad.mit.edu/gsea/). Predefined gene sets were obtained from the Molecular Signatures Database, MSigDB (http://software.broadinstitute.org/gsea/msigdb). False discovery rate (FDR) < 0.25 was considered statistically significant.RNA‐seq data of CC samples were downloaded from The Cancer Genome Atlas (TCGA). Gene set enrichment analysis (GSEA) was performed as described . Consequently, both HPV18 E6 and HPV16 E6 promoted cell migration and invasion of C‐33A cells , which attenuated α‐actinin‐4 expression [HPV18 has shown a higher carcinogenic potential than HPV16 in development of ICC , 36, 37.pression  since thpression . Here, wAltogether, our results provide a mechanism by which HPV18 E6 promotes malignant progression of CC through reducing MAGI3 protein level, leading to Wnt/β‐catenin signaling activation. It provides promising clinical strategies for treatment with HPV18‐positive patients of CIN and ICC through the application of Wnt/β‐catenin signaling pathway inhibitors, which has been applied to phase II clinical trial , 39, or The authors declare no conflict of interest.JH, HL, RS, and ZY designed the experiments and analyzed data. HL, ZY, and JH prepared the manuscript. ZY and WL performed the experiments. DF contributed to sample collection, data analysis, and review of the manuscript. HW, SG, XC, YC, and JL contributed to bioinformatics and statistical analysis and critically revised the manuscript. QQ and XY analyzed data, and reviewed and revised the manuscript. JH conceived and supervised the project.Fig. S1. No statistical difference of MAGI3 mRNAs levels between HPV18+ vs. normal or HPV16+ tissues. The Scatter plots of MAGI3 mRNA level in normal, HPV18+ and HPV16+ CC clinical specimens from TCGA Click here for additional data file.


Tg) and thermal decomposition temperature (Td) of epoxy resin cured by LPFA decreased slightly compared with that of MeTHPA-cured epoxy resin. Based on such excellent flame retardancy, low viscosity at room temperature and ease of use, LPFA showed potential as an appropriate curing agent in the field of electrical insulation materials.A novel liquid phosphorous-containing flame retardant anhydride (LPFA) with low viscosity was synthesized from 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) and methyl tetrahydrophthalic anhydride (MeTHPA) and further cured with bisphenol-A epoxy resin E-51 for the preparation of the flame retardant epoxy resins. Both Fourier transform infrared spectroscopy (FT-IR), mass spectrometry (MS) and nuclear magnetic resonance (NMR) measurements revealed the successful incorporation of DOPO on the molecular chains of MeTHPA through chemical reaction. The oxygen index analysis showed that the LPFA-cured epoxy resin exhibited excellent flame retardant performance, and the corresponding limiting oxygen index (LOI) value could reach 31.2%. The UL-94V-0 rating was achieved for the flame retardant epoxy resin with the phosphorus content of 2.7%. With the addition of LPFA, the impact strength of the cured epoxy resins remained almost unchanged, but the flexural strength gradually increased. Meanwhile, all the epoxy resins showed good thermal stability. The glass transition temperature ( Epoxy resins have been widely used in coatings, electronic materials, adhesives, and matrices for fiber-reinforced composites due to their fascinating properties such as good bonding strength, dielectric property, dimensional stability and high hardness ,2,3,4. HNowadays, the development of halogen-free, low toxicity, low smoke, efficient flame retardant epoxy resins has been demanded for environmental protection. Phosphorus-containing flame retardants have been widely investigated due to their good thermal stability, excellent flame retardant performance and low toxicity ,14,15. TOwing to the high reactivity of O=P-H bond, DOPO derivatives can be not only incorporated into epoxy matrix for the preparation of intrinsic flame retardants, but also participate as modified curing agents in the curing of epoxy resin ,25. ReceN, N-dimethylbenzylamine was purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China.DOPO and MeTHPA ) were purchased from Aladdin Biochemical Technology Co., Ltd., Shanghai, China. Bisphenol-A epoxy resin E-51  was supplied by Yuanbang Chemical Manufacturing Co., Ltd., Shanghai, China. An amount of 100 g (0.6 mol) of MeTHPA was added into a 500 mL four-necked flask equipped with stirrer, condenser and thermometer, and heated to 120 °C. Then, 50 g (0.2 mol) DOPO was added into the flask with constant stirring. The reactants were further heated to 180 °C and reacted for 6 h under constant stirring until a yellow transparent anhydride LPFA was obtained . The MeTN, N-dimethylbenzylamine (0.5% by mass of anhydride) as an accelerator according to the stoichiometric ratio under mechanical stirring at 60 °C for 30 min. The well mixed samples were initially degassed in vacuum at 80 °C for 20 min, and then poured into the metal mold for further degassing. The samples were precured in mold at 120 °C for 2 h, demolded and post cued at 130 °C for 9 h, respectively. By altering the mass ratios of LPFA:MeTHPA over the range of 0:100, 50:50, 70:30, 86:14 and 100:0, a series of cured epoxy resins with different phosphorus contents was obtained, and the phosphorus contents of the final cured products were 0%, 1.20%, 1.74%, 2.24% and 2.70%, respectively. A control sample with the phosphorus content of 2.70% was prepared at the same conditions by adding DOPO just as a flame retardant to epoxy resin E-51\MeTHPA curing system, in which the reaction of DOPO and the epoxy network considered when calculating the stoichiometric ratio of resin and hardener.Bisphenol-A epoxy resin E-51 was cured by LPFA and MeTHPA using A was calculated using the Equation (1).A—acid value, mg/g; N—concentration of NaOH standard solution, mol/L; V1—amount of NaOH solution consumed by sample, mL; 56.11—molecular weight of KOH; W1—mass of sample, g.Acid value was measured as follows: 1 g (accurate to 1 mg) of the sample was dissolved in acetone in a 250 mL triangular flask. After adding 2–3 drops of phenolphthalein indicator, the solution was titrated with 0.1 mol/L NaOH standard solution to the end point and the acid value B—anhydride content, %; A—acid value, mg/g; N—concentration of NaOH standard solution, mol/L; V2—amount of NaOH solution consumed by sample, mL; W2—mass of sample, g; 56.11—molecular weight of KOH; 72.03—molecular weight of acetic anhydride.For anhydride content, a 0.3 g sample (accurate to 1 mg) was dissolved in 5 mL acetone in 250 mL iodine flask and added to 20 mL anhydrous methanol. The flask was shaken and left to stand for 5 min. Next, 3–5 drops of phenolphthalein indicator solution were added and the mixture was titrated with 0.1 mol/L NaOH standard solution.The viscosity of anhydride was measured by DV-S digital viscometer  and the temperature was 25 °C.−1 to 500 cm−1.Fourier transform infrared (FT-IR) spectrophotometer  was recorded in an attenuated total reflection (ATR) mode. Each sample was scanned with an average of 32 scans ranging from 4000 cmMass spectrometry (MS) characterization of LPFA was measured with an electrospray ionization/time of flight mass spectrometry  with 50% acetonitrile and 50% water as solvents under the conditions of positive ion mode spray voltage of 1.5 kV, negative ion mode spray voltage of −3.5 kV. The temperature of CDL is 200 °C. The atomized gas is nitrogen (purity ≥ 99.5%) and flow rate is 1.5 L/min. LCMS-IT-TOF was equipped with liquid chromatography, so we can separate PFA from LPFA and get the Mass spectrometry of pure PFA.31P NMR spectra were tested at 298 ± 0.1 K under the conditions of a frequency of 202 MHz and scanning times of 256.AVANCE AV-III HD 500 MHz nuclear magnetic resonance (NMR) measurement  was carried out using deuterated chloroform as the solvent. Chemical shifts were calculated relative to tetramethyl silane (TMS) for NMR control. All LOI values of the epoxy resins were measured on a limiting oxygen index instrument  with a test specimen bar of 120 mm × 6.5 mm × 3.2 mm according to the standard test GB/T 2406.2-2009 . Each teVertical combustion test of the cured products was measured with the sample size of 125 mm × 13 mm × 3.2 mm using UL94 horizontal and vertical flammability tester  at burning angle of 45° according to ASTM D3801. Each test was repeated at least five times and the average value was reported.−1.Thermogravimetric analysis (TGA) of the samples was performed with thermogravimetric analyzer  under a nitrogen atmosphere from 25 °C to 800 °C at a heating rate of 10 °C min−1 and nitrogen gas flow rate of 20 mL min−1. The DSC curves were recorded the second heating cycle.Differential scanning calorimeter (DSC) measurements were carried out on an instrument  from 25 °C to 180 °C at heating rate of 20 K∙minThe impact strength of the cured products was tested with micro control impact testing machine  according to GB/T 2567-2008, and the dimensions of the samples were 120 mm × 15 mm × 10 mm. The flexural strength and flexural modulus were measured with CMT4304 type microcomputer controlled electronic universal testing machine  according to GB/T 2567-2008. The test speed was set to 10 mm/min, and the dimensions of the samples were 120 mm × 15 mm × 10 mm. For each specimen, the average value was obtained from five replicates.−1 and the characteristic peak of C=C bond at 1670 cm−1 for MeTHPA was found. Whereas the absence of P-H peak at 2386 cm−1 in LPFA spectrum confirmed that LPFA had been synthesized by the reaction between the P-H bond of DOPO and the C=C bond of MeTHPA. In addition, the peaks at 1860 cm−1 and 1770 cm−1 were ascribed to -C=O stretching vibration of anhydride groups in the spectrum of LPFA. The absorption bands at 1600 cm−1 and 1237 cm−1 were attributed to benzene ring and P=O stretching vibration, which originated from the structure of DOPO, respectively +. The m/z 387 was assigned to [M+Na-H2O]+, which indicated a loss of H2O [s of H2O . These M1H NMR profile of LPFA is shown in δ (ppm) = 6.97–7.99 for -CH=CH- in aromatic ring; 5.46–5.94 for-CH=CH- in cyclic structure; 3.24–3.45 for -CH-CO-O-; 2.53 for -CH-P-; 1.66–2.25 for -CH-C- and -CH2-C-; 1.11–1.32 for-CH3. 13C NMR spectra of LPFA. The peaks at 171–174 ppm and 120–140 ppm were assigned to -CO-O- and -CH=CH- in aromatic ring, respectively. The peak of -CH-P was located at 45.7 ppm. The chemical shifts of 38.9–42.5 ppm were assigned to -CH-CO-O-, and those of 16.5–30.0 ppm were due to -CH, -CH2- and -CH3.31P NMR spectra of DOPO (a) and LPFA (b) are shown in Bisphenol-A epoxy resin E-51 was cured with anhydrides according to the stoichiometric ratio. The phosphorus content of cured products was changed by mixing different mass ratios of LPFA and MeTHPA. The LOI measurements and UL-94 test of the epoxy resins as a function of different phosphorus contents are listed in Thermogravimetric (TG) curves of the LPFA and MeTHPA-cured epoxy resins are shown in Tg) of cured epoxy resin was determined as inflection point and shown in Tg of the MeTHPA-cured epoxy resin was 123 °C, while that of the epoxy resin cured with LPFA was 105 °C. The Tg decreased with the addition of LPFA into epoxy resin, which can be attributed to the reduced crosslinking density of LPFA. This is also consistent with the result shown in Tg is an important parameter of polymer for electrical insulation. The minimum Tg of insulating materials is 90 °C according to the standard of the International Electrotechnical Commission (IEC) [Tg of the LPFA-cured epoxy resin was higher than 100 °C, which met the demand of epoxy resins for electrical insulation.on (IEC) . Therefo−2, basically in the same range as that of the MeTHPA-cured resins, suggesting that the incorporation of LPFA had little effect on the mechanical properties of epoxy resins. However, the flexural strength increased from 105.8 MPa to 117.8 MPa as an increase in ratios of LPFA and MeTHPA because LPFA, as a curing agent, with bulky structure can cause longer intermolecular distance. The increase in distance between the macromolecular segments provided more space for the motion of molecular chains during curing process. The flexural modulus increased from 3.398 GPa to 3.856 GPa as an increase in ratios of LPFA and MeTHPA because LPFA contained more rigid phosphaphenanthrene ring structure compared with MeTHPA. Moreover, the control is a blank experiment with DOPO just added as a flame retardant to epoxy resin E-51\MeTHPA curing system, in which the phosphorus content was as same as the LPFA-cured epoxy resin (LPFA:MeTHPA = 100:0). The impact strength and flexural strength of the cured product decreased with the simple addition of DOPO to epoxy resin\MeTHPA curing system, suggesting that LPFA as a modified curing agent minimized the negative effect of additives on the mechanical properties in the curing of matrix resin.The impact strength, flexural strength and flexural modulus of different anhydride-cured epoxy resins are shown in Liquid anhydride (LPFA) with excellent flame retardancy was synthesized with DOPO and MeTHPA. Then, a series of flame retardant cured epoxy resins with different LPFA and MeTHPA contents were successfully prepared. The flame retardancy of the epoxy resins have been remarkably enhanced with the addition of LPFA. The LPFA-cured epoxy resin (LPFA:MeTHPA = 100:0) passed UL-94V-0 rating. The LOI value was 31.2% when the content of phosphorus was only 2.7%. The glass transition temperature and thermal decomposition temperature of epoxy resin cured by LPFA decreased slightly compared with that of MeTHPA-cured epoxy resin. The mechanical properties of epoxy resins cured with LPFA were increased compared with the simple addition of DOPO to the epoxy resin\MeTHPA curing system at the same phosphorus content. Liquid anhydride is convenient to use at room temperature and is more popular as a curing agent for epoxy resins. This work provided a novel method to synthesize an effective phosphorous-containing liquid anhydride for applications in flame retardant epoxy resins.


Moreover, regulating the canonical Wnt pathway (Licl and IWP2) in vitro also affected hair cell polarity and indicated that Axin2 may be important in this process. In conclusion, our results not only confirm that the regulation of canonical Wnt signaling affects the number of hair cells in the utricle but also provide evidence for its role in polarity development.As part of the inner ear, the vestibular system is responsible for sense of balance, which consists of three semicircular canals, the utricle, and the saccule. Increasing evidence has indicated that the noncanonical Wnt/PCP signaling pathway plays a significant role in the development of the polarity of the inner ear. However, the role of canonical Wnt signaling in the polarity of the vestibule is still not completely clear. In this study, we found that canonical Wnt pathway-related genes are expressed in the early stage of development of the utricle and change dynamically. We conditionally knocked out  The cochlea and vestibule are important for hearing and balance, respectively. They detect sound and position signals through stereociliary bundle on the surface of hair cells (HCs) –4. The vβ-catenin-degrading complex composed of GSK-3, APC, and Axin. β-Catenin accumulates in the cytoplasm and is then transferred to the nucleus, where it controls embryogenesis and cell fate through T-cell factor/lymphoid enhancer-binding factor (TCF/LEF). The canonical Wnt signaling pathway and its downstream target genes play an important regulatory role in cell proliferation ). The Petri dishes were incubated at 37°C, with a concentration of 5% COThe reaction protocol for PCR was as follows: predenaturation at 94°C for 5 min, 32 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, elongation at 72°C for 1.5 min, and a final elongation at 72°C for 10 min.RNA was extracted from the utricle using the RNeasy Micro Kit , and cDNA was synthesized using the GeneAmp® PCR System 9700  by reverse transcription: 42°C for 15 min and 85°C for 5 s. The qPCR reaction protocol was as follows: 94°C for 30 s, followed by 45 cycles at 94°C for 5 s and 60°C for 30 s. Each sample was run in triplicate for analysis purposes. At the end of the PCR cycles, melting curve analysis was performed to validate the generation of the expected PCR product. The primer sequences were designed in the laboratory and synthesized by TsingKe Biotech  based on the mRNA sequences obtained from the NCBI database, as follows:GADPH , Apc , Wif1 , Camk2d , Fzd6 , Axin2 , Vangl2 , Prickle2 .Total RNA was extracted from the utricle  and then submitted to Otogenetics Corporation  for RNA-Seq assays. We assessed RNA integrity and purity using an Agilent Bioanalyzer . cDNA was generated from total RNA using the Clontech SMARTer PCR cDNA Synthesis Kit . Bioruptor  was used to fragment cDNA, an Agilent Bioanalyzer was used for profiling, and SPRIworks HT  was used to prepare the Illumina library. The quality, quantity, and size distribution of the Illumina library were determined using TapeStation (Agilent Technologies).https://david.ncifcrf.gov/) is a commonly used database for gene analyses. KEGG pathway enrichment analyses were performed using DAVID, and the TXT files of the results were downloaded for subsequent analysis. For the Bonferroni test, P values < 0.05 were set considered significant, and Log2 (fold_change) values between -1 and 1 were excluded from the analysis. STRING (https://string-db.org/) was used to analyse the interactions between proteins. Many signaling pathways play important roles in the development of the utricle. To better understand the role of the Wnt signaling pathway, we only included the Wnt pathway-related genes in this analysis.The DAVID database  was used to dry them. An E-1045 sputter coater  was used to coat specimens with 100 Å Au. A NOV A NanoSEM 230 scanning electron microscope  was used to scan the samples.Utricles were fixed with 2.5% glutaraldehyde at 4°C overnight and then processed using 2% tannin and 1% osmium acid. Graded ethanol series were used to dehydrate the samples, and liquid COβ-spectrin  primary antibody and Alexa Fluor 568 Donkey Anti-Mouse IgG  secondary antibody. FITC-conjugated phalloidin  was used to visualize actin in the stereocilia of sensory HCs.Mouse embryos were harvested at E18.5. Otocysts were dissected in cold PBS and fixed with 4% paraformaldehyde  for 1 h at 4°C. After washing with PBS, utricles were dissected and blocked with 10% donkey serum, and 0.1% Triton-X in PBS for 30 min at room temperature, followed by incubating with primary antibodies diluted in PBS containing 5% donkey serum and 0.1% Triton-X overnight at 4°C. The next day, after washing extensively with PBS, tissues were incubated with secondary antibodies at 1 : 1,000 dilution in 0.1% Triton-X100 (in PBS) for 2 h at room temperature. After extensive PBS washing, samples were mounted in antifade Fluorescence Mounting Medium (Agilent Technologies) on coverslips. We used mouse anti-μm2 regions in the middle of the utricle, defined as field 1, field 2, and field 3  at 40× magnification, and the whole utricle image was obtained by flattening the overlapping parts of original images using Photoshop CS4 (Adobe Systems). The overall outline of a utricle was drawn on the stitched confocal image, and the area of the sensory epithelia was measured using the Zen image processing system (Zeiss). The number of HCs on the composite image was counted manually using Photoshop CS4 (Adobe Systems). Cell number was scored in two regions divided by LPR, defined as lateral and medial region. Hair bundle orientation was scored in three 40 × 80  field 3 . The haiOur previous studies showed that E13.5 is a critical period for the formation of polarity in the utricle . Two cluβ-catenin, and Frizzled are important genes in the canonical/noncanonical Wnt signaling pathway. We detected these genes in the utricle by RT-PCR. Our research shows that these genes were expressed in the utricle of E13.5 mice  and 33,824.00 ± 10,515.93 μm2 (n = 5), respectively, P < 0.05 (n = 3), the total number of HCs was 995.67 ± 14.61, with 585.33 ± 33.40 and 410.33 ± 15.41 in the medial and lateral regions, respectively, whereas in the experimental group (n = 5), the total number of HCs was 156.80 ± 40.09 with 109.00 ± 30.25 and 47.80 ± 15.46 in the medial and lateral regions, respectively, P < 0.05  and a Wnt/β-catenin activator (LiCl) [μM/mL for LiCl and IWP2, respectively.To determine the role of the Wnt signaling pathway in the polarity of HCs in the utricle, we regulated Wnt signaling pathway activity r (LiCl) . AccordiUsing a scanning electron microscope, we found that the polarity of the utricle was affected by treatment with the inhibitor or activator. Compared to the control group Figures , the posAccording to RNA-seq results and common Wnt pathway genes, RT-qPCR was performed to detect canonical Wnt signaling pathway-related genes  and PCP core genes (Prickle2 and Vangl2) in cultured utricle in vitro to explore the possible mechanism of polarity change . We founin vitro or by knocking out β-catenin in vivo. The results showed that the canonical Wnt signaling pathway played a role in polarity development of the utricle.The utricle is the organ that senses balance. Proper utricle function depends on the polarization of hair bundles located on the HC surface. This cellular polarization has also been found in many epithelial cell types, including in the respiratory airway and lateral ventricles of the brain. The planar polarity of the utricle was described at three levels: subcellular, cellular, and tissue-wide . In the in vitro, the proliferation domain of Sox2-positive cells in the cochlea (E12.5) was significantly expanded, indicating that the canonical Wnt pathway regulates the proliferation and differentiation of HCs during early development of the cochlea [β-catenin initiated proliferation of sensory precursors cells within the cochlear sensory epithelium [in vivo when we conditionally knocked out β-catenin that the number of utricle HCs was affected in the mic. In addition, we found changes in the polarity of hair cells.Wnt signaling is a marker of embryonic development and has multiple functions at a number of developmental time points. The Wnt signaling pathway includes both canonical and noncanonical forms. The Wnt signaling pathway has been studied in the early development of the cochlea. The Wnt signaling receptors  can be detected in the cochlea of mice at E14.5 –36. The ithelium . Therefoβ-catenin. Dislocation of the hair bundle is a common phenotype of Wnt/PCP pathway mutants in the inner ear. The cochlea of the Wnt5a mutant is wider and shorter, with stereociliary bundle misorientation in accordance with a subcellular polarity [Indeed, the Wnt pathway also plays an important role in inner ear polarity regulation. The polarity of HCs in the cochlea is mainly reflected in their convergence and extension, an identical ciliary direction among all HCs, and a V-shaped stereociliary bundle with a uniform direction . In vestpolarity . After kpolarity , 46. In polarity , 47, 48.β-catenin pathway, such as Axin2. Thus, we suspect that the canonical Wnt pathway also plays an important role in regulating polarity. The canonical Wnt pathway has been implicated in the regulation of polarity in other organs and animals. Local activation of the canonical Wnt pathway regulates neuronal polarity and axonal outgrowth [β-catenin signaling pathway [β-catenin is the core protein in the canonical Wnt pathway that controls the expression of downstream target genes. A mouse with conditional knockout of β-catenin in the dorsal neural folds will exhibit spina bifida aperta, caudal axis bending, and tail truncation [β-catenin [β-catenin signaling pathway [β-catenin pathway is important in the ground hypothesis, which was suggested to explain the formation of LPR in the vestibule [β-catenin in Sox2-positive cells and found that the utricle polarity was subsequently affected: the directions of some HCs were disordered near the LPR in the lateral and striolar regions. The changed polarity was also found after activating and inhibiting canonical Wnt pathways in vitro. The changed polarity was also found after activating and inhibiting canonical Wnt pathways in vitro, and PCP core genes (Vangl2 and Prickle2) were no statistical difference with IWP2 treatment or Licl treatment at the RNA level, compared with the control group. However, Axin2 unexpectedly decreased in the two treatment groups. Axin2, a scaffolding protein of glycogen synthase kinase 3, is a negative regulator of Wnt signaling. In addition, Axin2 feedback loop is important with many studies in Wnt pathway, which could help to limit Wnt-initiated signal [Our results showed that the Wnt genes expressed on different days in the utricle were closely related to canonical Wnt/utgrowth . In addi pathway . β-catenuncation . Similar-catenin . However pathway . Some reestibule ; howeverestibule . We knocd signal . Axin2 id signal , 58. In d signal . These fβ-catenin in vivo, our data showed the main effect is on hair cell number and may reveal an impact on stereociliary bundle orientation. Combined with the in vitro results, we believe that the canonical Wnt pathway is important for controlling HC polarity during mammalian utricle development. Our findings may stimulate future studies on vestibule polarity development.In this study, we investigated expression changes of Wnt pathway-related genes during early development of the mouse utricle. In conditional knockout of


In this work, epoxy acrylate resin (EA) based on the industrial-grade bisphenol A-based epoxy resin (Ep6) and acrylic acid (AA) has been synthesized in order to develop hybrid resin comprising both epoxide group and reactive, terminal unsaturation. Obtained epoxy acrylate prepolymer was employed to formulate photocurable coating compositions containing, besides the EA binder, also cationic or radical photoinitiators. Hence, when cationic photoinitiators were applied, polyether-type polymer chains with pending acrylate groups were formed. In the case of free radical polymerization, epoxy acrylates certainly formed a polyacrylate backbone with pending epoxy groups. Owing to the presence of both epoxy and double carbon–carbon pendant groups, the reaction product exhibits photocrosslinking via two distinct mechanisms: (i) cationic ring-opening polymerization and (ii) free radical polymerization. Therefore, photopolymerization behavior of synthetized hybrid resin with various photoinitiators was determined via photo-differential scanning calorimetry (photo-DSC) and real-time infrared spectroscopy (RT-IR) methods, and properties of cured coatings were investigated. The performance of the following type of photoinitiators was tested in the cationic photopolymerization: diaryliodonium cations or triarylsulfonium cations, and the following type of photoinitiators were used to induce free radical photopolymerization: α-hydroxyketones, acylphosphine oxides, and their mixtures. Lastly, the basic physicomechanical properties of cured coatings, such as tack-free time, hardness, adhesion, gloss, and yellowness index, were evaluated. Some structural factors and parameters of cationic and radical photoinitiators and photopolymerization mechanisms affecting the epoxy acrylate hybrid coatings performance are discussed. The polymerization of multifunctional monomers or oligomers by UV curing has become a widely used technology that has found many industrial applications due to its unique advantages. The coating industry is the most important application of UV curing technology and used to protect the surface of materials with quick-drying varnishes or printing inks. Other uses include manufacturing solder mask, stereolithography, and microelectronic photoresists. In addition, quick-setting adhesives, coatings, sealants, and the production of composite materials are just some of the many applications of photopolymerization ,2,3. So In the photopolymerization process, the type of monomer and photoinitiator are of great importance, because the compositions can be polymerized using a free radical or cationic photopolymerization mechanism ,8. CurreTo overcome the limitations of acrylates and epoxides, a product can be synthesized based on acrylate and epoxy monomers. In this case, the monomers can polymerize by various mechanisms, and the resulting products combine the properties of both acrylates and epoxides, making them suitable for various applications . Epoxy aIn this contribution, the modification of epoxy resin with methacrylic acid, allowing the obtainment of a hybrid polymer containing both epoxy and acrylic groups, has been reported. Subsequently, photoreactive coating compositions were prepared from the obtained epoxy acrylate and cured using UV radiation. The samples were tested for the kinetics of the process and the properties of the cured coatings. Both the cationic and free radical photopolymerization of synthetized EA have been investigated by the photo-DSC and RT-IR method, and the suitability of various photoinitiators for photopolymerization of the hybrid systems using a UV lamp is evaluated.® (Ep6), with epoxide number of 0.51–0.54 mol/100 g and viscosity ranging from 10,000 to 15,000 mPa·s at 25 °C, and acrylic acid (AA), stabilized, with the purity of 99.5%, supplied by Acros Organics, Geel, Belgium. Triphenylphosphine (PPh3), Apollo Scientific, Bredbury, UK, was used as a catalyst in the reaction between Ep6 and AA, while hydroquinone  was used as a polymerization inhibitor. All chemicals were employed as received.The industrial-grade bisphenol A-based epoxy resins were purchased from “Organika-Sarzyna” S.A., Nowa Sarzyna, Poland. The following epoxy resins were used: Epidian 6The following titration reagents and indicators were used: glacial acetic acid, toluene, potassium hydroxide standard solution 0.1 M in ethanol (KOH), and crystal violet purchased from Chempur , chloroform form P.P.H. Stanlab , ethyl alcohol from Avantor , tetraethylammonium bromide provided by Acros Organics , perchloric acid standard solution 0.1 M in glacial acetic acid supplied by Fischer Chemicals , and Phenolophtalein 1% in ethyl alcohol solution from Eurochem BGD . All chemicals were analytical grade and were used as received.The diaryliodonium and triarylsulfonium cation-based photoinitiators were selected from the many cationic photoinitiators available. The following photoinitiators based on the diaryliodonium cation were used to induce cationic photopolymerization of hybrid resin: (4-methylphenyl)[4-(2-methylpropyl)phenyl]iodonium hexafluorophosphate (Irgacure 250) from BASF , bis(dodecylphenyl)iodoniumhexaflouroantimonate in propylene carbonate (Deuteron 1240) and bis-((C10-C13)alkylphenyl)-iodoniumhexafluoroantimonate (Deuteron 1250) from Deuteron , (7-methoxy-4-methylcoumarin-3-yl)phenyliodonium hexafluoroantimonate (Sylanto 7M-S) and (7-methoxy-4-methylcoumarin-3-yl)phenyliodonium hexafluorophosphate (Sylanto 7M-P) from Synthos S.A. . The following photoinitiators were selected from the cationic photoinitiators based on the triarylsulfonium cation: mixture triarylsulfonium hexafluoroantimonate salts (Cyracure UV6976) and mixture triarylsulfonium hexafluorophosphate salts (Cyracure UV6992) from Dow . Structures of the cationic photoinitiators are shown in As free radical photoinitiators for hybrid monomers, photoinitiators were used, which were divided into α-hydroxyketones, acylphosphine oxides, and their mixtures. The following compounds were selected from the photoinitiators with the structure of α-hydroxyketones: 1-hydroxycyclohexyl phenyl ketone (Omnirad 184), 2-hydroxy-1-{4-[4-(2-hydroxy-2-methyl-propionyl)-benzyl]-phenyl}-2-methylpropan-1-one (Omnirad 127) and 1-[4-(2-hydroxyethoxyl)-phenyl]-2-hydroxy-methylpropanone (Omnirad 2959) from IGM Resigns (The Netherlands). The structures of the free-radical photoinitiators α-hydroxyketones are collected in It has long been known that the effectiveness of a photoinitiator depends on matching the absorption spectrum with the emission spectrum of the light source. It is important that the maximum of the absorption peak is in the range of the lapa emission. Epoxy acrylates (EAs) were obtained by the addition of acrylic acid to epoxy resin. The synthesis was carried out in a 250 mL three-neck glass reactor , into which bisphenol A type epoxy resin (Epidian 6) was introduced. Then, hydroquinone  was transferred into the reactor as a radical scavenger at room temperature. Lastly, acrylic acid  and catalyst–triphenylphosphine in the amount of 0.8 wt.% (relative to the mass of AA) were added. The reaction mixture was heated to 70 °C with vigorous stirring (120 RPM) using an oil bath. Once the homogeneous mixture was obtained, the temperature was raised to 90 °C, and the reaction was carried out for 4 h in a nitrogen atmosphere, with stirring. At room temperature, the as-prepared EA appears as colorless, transparent, viscous liquids. The reaction product is presented in −1 at room temperature. The non-volatile-matter content (NV) was evaluated thermogravimetrically, using moisture analyzer MAX 60/NP , according to the ISO 3251:2019 standard. Partial acid values (PAVs) were determined by colorimetric titration according to EN ISO 2114:2000 standard. Epoxy equivalent (EE) was determined by means of colorimetric titration, according to the EN ISO 3001:1999 standard. The viscosity tests were carried out using a cone-plate viscometer LAMY RM-100 plus CP 2000. The test methods were described in detail in our previous article on epoxy acrylate prepolymers [The infrared spectra acquired with a Thermo Nicolet 380 FT-IR spectrometer. Sixteen scans were averaged for each sample in the range of 4000–400 cmpolymers . The mol2 to dryness.The coating compositions have been formulated using epoxy resin or synthesized epoxy acrylate and 3 wt.% of photoinitiator . The components were stirred together under dark conditions until a homogeneous mixture was obtained. Subsequently, the curing solution was applied to the glass substrates by means of a gap applicator (120 µm). The polymeric film was cured under a light source  at room temperature and irradiated under UV light with an intensity of 200 mW/cm2. A polymerization solution was composed of epoxy resin (Ep) or epoxy acrylate (EA) prepolymer and 3 wt.% of photoinitiator.The UV-curing process of epoxy or epoxy acrylate varnishes was isothermally monitored (25 °C) for 15 min by means of photo-DSC apparatus  equipped with UV light emitter Omnicure S2000 . The spectrum range of the lamp is 320–500 nm. The OmniCure S2000 is equipped with a 200-W UV lamp. This allows the generation of UV light with a power of 200 mW/cm−1 and the scanning range is 400–4000 cm−1. The recording interval of the spectrum was 10 s. Series real-time IR (RT-IR) was used to determine the conversion of epoxide groups or acrylic double bonds. More importantly, this spectroscopic technique permits in situ monitoring of the chemical processes via mimicking the disappearance of the characteristic bands of the reactive monomer subjected to UV exposure [2) and an IR analyzing light beam. The absorbance change of the epoxide group (C–O) and acrylate double bond (C=C) peak area was correlated to the extent of polymerization. The degree of conversion (DC) can be expressed by the following relations: DC (%) = (A0 − At)·100/A0, where A0 is the initial peak area before irradiation and At is the peak area at time t. The photopolymerization rate (Rp) was calculated by the following relations: Rp = dDC/dt, where t is the time of irradiation [Fourier transform infrared spectra (FT-IR) were obtained on a Nicolet iS5 instrument. The resolution is 4 cmexposure . The mixadiation .The following tests were performed in order to evaluate the mechanical properties of cured coatings: tack-free time, pendulum hardness test, adhesion, gloss, and yellowness index. Tack-free time was measured as a surface cure time according to ISO 9117. It is the time at which the coating is deemed to be properly adhered to and achieves the final technical parameters. The hardness of coatings was tested using Persoz pendulum hardness on the glass substrate  according to ISO 1522 standard. According to the PN-EN ISO 2409 standard, the adhesion to the glass substrate was determined . Gloss was measured by spectrometer GLS  according to ASTM D523. The yellowness index is a number calculated from spectrophotometric data that describes the change in color of test samples. This parameter was measured according to ASTM E313 using precision colorimeter NH-145 .−1), C–O (914 cm−1), and C–O–C (825 cm−1) stretching vibrations of oxirane groups [−1 originating from the carbonyl stretching mode in AA [−1, thereby suggesting that new bonds occur due to the reaction of carboxyl groups of AA and epoxide groups of Ep6, i.e., ester bonds. This agrees with the previous studies reporting the shift of the C=O band after the esterification reaction [−1, due to –O–H groups’ evolution [The addition reaction of AA to the epoxy resin was carried out in order to obtain a hybrid resin containing both epoxy and acrylic groups in the molecule, which will be used in the further part of the research to monitor the course of cationic and radical photopolymerization with the use of various photoinitiators. Herein, the process parameters were developed based on a literature survey ,28,29,30e groups ,32,33 dede in AA ,35 becomreaction . Lastly,volution ,33,37 reThe product of reaction was also characterized for the non-volatile-matter content (NV), partial acid value (PAVs), and epoxy equivalent (EE). Each of these can be regarded as a measure of reaction progress, which was referred to as the % of reacted epoxide groups as well as % of reacted AA and expressed as epoxy group conversion (EGC) and acrylic acid conversion (AAC). The synthesized EA prepolymer was 98.4% solids (NV). The viscosity of the resin increased and is 70 Pas. The acid number of the product (PAVs) was 2.9 mgKOH/g, which indicates a conversion of acrylic acid in the amount of 97.9%. In turn, the EE was 408, so the conversion degree of epoxy groups was 49.6%. The molecular weight of the obtained prepolymers was 977 g/mol.photo differential scanning calorimetry (photo-DSC) and Fourier transform infrared spectroscopy for real-time process monitoring (RT-IR) methods. In a first approach, the photoinitiated cationic or radical polymerization of the obtained EA prepolymer was monitored by means of the photo-DSC method. Photo-DSC offers a simple method of characterizing the UV-curing kinetics for the photopolymerization of UV-cured materials. Therefore, the profiles for the heat of reaction versus time provided by photo-DSC can be used to describe the photoinduced reaction kinetics and evaluate the polymerization rate. In turn, through the RT-IR method, the curves of the conversion of epoxy or acrylate groups and photopolymerization rate (Rp) of EAs compositions with various photoinitiators were investigated. As expected, the progressive disappearance of the various IR bands characteristic of the epoxy or acrylate double bonds was observed. The extent of the epoxy groups’ reactions was determined by the areas of the peak at 915 cm−1, which is due to C–O stretching in the epoxy ring. Simultaneously, the extent of double bond reactions was determined by the peak areas of the double bond peak at 1635 cm−1.The kinetics of photopolymerization is useful in understanding the rate and degree of curing. It is well known that acrylates and methacrylates do not polymerize by a cationic polymerization mechanism, while epoxy monomers do not polymerize by free-radical means. Hence, when cationic photoinitiators were applied, polyether-type polymer chains with pending (meth)acrylate groups were formed. In the case of free-radical polymerization, epoxy (meth)acrylates certainly formed a polymethacrylate backbone with pending epoxy groups . The pro2), it was shown that triaryliodonium salts polymerize the fastest , despite their UV-C and UV-B absorption characteristics (2). Additionally, the conversion of epoxy groups was also investigated. As shown in Owing to the presence of both epoxy and double carbon–carbon bonds in the prepolymer chain, the resulting EAs are expected to exhibit curing behavior via two distinct mechanisms: (i) cationic ring-opening polymerization or (ii) free radical polymerization. First, the kinetics of cationic photopolymerization of the mixture of unmodified epoxy resin (E) and various cationic photoinitiators (3 wt.%) were investigated. In the photo-DSC tests  cationic ring-opening polymerization and (ii) free radical polymerization. In the first approach, the photoinitiated cationic polymerization of the EA prepolymers was monitored by means of the photo-DSC method. The compositions used in the study included synthetized EA and a cationic photoinitiators (3 wt.%). The next part concerns photoinitiated free radical polymerization of acrylate groups of EA chains, which was also monitored by both photo-DSC  and RT-IIn order to determine the effect of the type  and the course of photopolymerization of systems with different photoinitiators, the basic properties of the cured films were investigated. The results are presented in Both coatings prepared from epoxy and epoxy acrylate resins obtained in the cationic process achieve the shortest tack-free time with use of certain photoinitiators in the form of diaryliodonium cation ((7-methoxy-4-methylcoumarin-3-yl)phenyliodonium hexafluoroantimonate (7M-S) and (7-methoxy-4-methylcoumarin-3-yl)phenyliodonium hexafluorophosphate (7M-P)). Although these photoinitiators lead to the lowest conversions of epoxy groups among all tested systems and run slower, their shortest tack-free time can probably be attributed to absorption in the longer wavelength range (350 nm), which is responsible for the surface drying of the coatings. In turn, the longest tack-free time has coatings with other diarylyodonium cations, whose absorption characteristics are at shorter wavelengths, i.e., in the range of UV-C radiation. It is well known that UV-C radiation penetrates deep into the coatings; therefore, it is difficult to obtain surface dryness. Epoxyacrylate coatings achieved a lower degree of conversion, and to obtain tack-free time coatings, they were cured for a longer time than epoxy coatings. Interestingly, however, EA coatings have a higher hardness compared to epoxy resin coatings. Despite the lower number of epoxy groups, epoxy acrylate resins should produce coatings of lower hardness than epoxy resins. Their higher hardness can be attributed to longer curing time, which is associated with cross-linking the polymer and greater post-curing of the coatings. It turns out that the choice of photoinitiator affects the adhesion. The coatings with triarylsulfonium cations showed the best adhesion. This may be related to the achievement of a high degree of conversion of the epoxy groups. All coatings obtained in the cationic process had a high gloss, and the highest yellowness was found in diarylyodonium cations, in particular 7MS and 7MP.In comparison to the cationic process, the radical process of curing epoxy acrylate formulations was characterized by a much shorter time-free time, and most coatings with radical photoinitiators achieve it within 3 s . This is probably related to the much higher rate of radical polymerization compared to the cationic process. However, despite obtaining surface dryness in a short time, a higher photopolymerization rate, and much higher conversion of unsaturated bonds from acrylate groups, the coatings obtained in the radical process have lower hardness compared to those obtained in the cationic process. This effect may be related to the formation of flexible networks in the form of polymethacrylate backbone with pending epoxy groups, as opposed to the more rigid networks obtained by the cationic process, resulting in polyether-type polymer chains with pending unreacted acrylate groups. The stiffness of polyether networks may also result from the possibility of forming hydrogen bonds, which additionally stiffen the structure. It has also been shown that the selection of a radical photoinitiator affects the photopolymerization rate, conversion, tack-free time, and the properties of cured coatings, in particular, adhesion, gloss, and yellowness.In this paper, an epoxy acrylate resin based on the industrial-grade bisphenol A-based epoxy resin and acrylic acid have been synthesized in order to develop hybrid resin comprising both epoxide group and reactive, terminal unsaturation. The significant advantage of this type of resin is the presence of two functional groups in one molecule, which can be used in the photopolymerization process and obtain coatings with a wide range of properties or used for subsequent modification. The obtained epoxy acrylate prepolymer was employed to formulate photocurable coating compositions containing, besides the EA binder, also cationic or radical photoinitiators. Owing to the presence of both epoxy and double carbon–carbon pendant groups, the reaction product exhibits photocrosslinking via two distinct mechanisms: (i) cationic ring-opening polymerization and (ii) free radical polymerization. The mechanism of photopolymerization, the type of the polymer network formed, and the selection of the photoinitiator have been shown to affect the kinetics of the process as well as the properties of the cured coatings. The cationic polymerization proceeds slower and leads to a lower degree of reaction compared to the radical process. As a result of this process, the surface dryness of the cured coatings is achieved in a longer time; however, the coatings have a higher hardness and gloss. It has also been shown that the kinetics of polymerization and the properties of cured coatings differ when using various types of photoinitiators. The properties of the coatings obtained by the cationic and radical processes may differ, among others, because they form different polymer networks. When cationic phtoinitiators were applied, polyether-type polymer chains with pending acrylate groups were formed. In the case of free radical polymerization, epoxy acrylates certainly formed a polyacrylate backbone with pending epoxy groups. The polyether-type polymers produce higher hardness coatings in relation to polyacrylates.