This research addresses the need for a multiscale model for the determination of the thermophysical properties of nanofiller-enhanced thermoset polymer composites. Specifically, we analyzed the thermophysical properties of an epoxy resin containing bisphenol-A diglyceryl ether (DGEBA) as an epoxy monomer and dicyandiamide (DICY) and diethylene triamine (DETA) as cross-linking agents. The cross-linking process occurs at the atomistic scale through the formation of bonds among the reactive particles within the epoxy and hardener molecules. To derive the interatomic coarse-grained potential for the mesoscopic model and match the density of the material studied through atomic simulations, we employed the iterative Boltzmann inversion method. The newly developed coarse-grained molecular dynamics model effectively reproduces various thermophysical properties of the DGEBA-DICY-DETA resin system. Furthermore, we simulated nanocomposites made of the considered epoxy additivated with graphene nanofillers at the mesoscopic level and verified them against continuum approaches. Our results demonstrate that a moderate amount of nanofillers (up to 2 wt.%) increases the elastic modulus and thermal conductivity of the epoxy resin while decreasing the Poisson’s ratio. For the first time, we present a coarse-grained model of DGEBA-DICY-DETA/graphene materials, which can facilitate the design and development of composites with tunable thermophysical properties for a potentially wide range of applications, e.g., automotive, aerospace, biomedical, or energy ones. In recent years, there has been a growing interest among researchers and industrialists in polymer nanocomposites, which are composed of polymers that incorporate nanofillers and have superior mechanical, electrical, and thermal properties compared to neat polymers. Various types of nanofillers and additives, such as carbon nanofibers, graphene, graphene oxide, iron, iron oxide, silica, and alumina nanoparticles, are utilized in conjunction with thermoset polymers, such as epoxies, as matrix materials to form nanocomposites ,2,3. CarEpoxy resins are produced through cross-linking between epoxy monomers with multiple epoxide groups and hardeners containing active groups such as hydroxyls and amines. The stable covalent bonds among the different chains in cured epoxy resins often result in superior thermophysical characteristics . TherefoThe thermophysical properties of nanocomposites play a critical role in designing high-performance smart applications and verifying the integrity of structures or devices. Baptista et al.  investigExperimental procedures are often costly, and therefore, physics-based computer modeling has been developed as a valuable complement to experimental and analytical methods. In recent years, molecular dynamics (MD) simulations have emerged as a promising tool for predicting material properties using well-parametrized and validated interatomic potentials. MD simulations have also been employed to investigate thermoset polymers. Fully atomistic models have been developed through MD simulations to evaluate various characteristics of epoxies ,21,22,23Gavrielides et al.  investigAlthough MD simulations have been widely used to predict and interpret various heat and mass transfer mechanisms at the nanoscale, full atomistic models are subject to significant time and scale constraints, making them unsuitable for accurately modeling the thermal and mechanical characterization of polymers and nanocomposites with elevated concentrations of fillers. Coarse-graining (CG) is a technique for upscaling an atomistic model by grouping multiple atoms into a single entity, resulting in reduced degrees of freedom and larger timesteps with respect to MD simulations owing to the softness of interatomic potentials. The CG approach comprises two primary steps: (i) grouping the model into larger elements rather than single atoms, and (ii) establishing interparticle potentials, which are highly dependent on the material and application. The literature describes a set of procedures that classify the iterative determination of effective pair potentials for particles by accurately matching the material’s structural properties, such as its radial distribution function (RDF), beginning with the potential of mean forces derived via Monte Carlo (MC) or Boltzmann inversion techniques ,28,29,30Regarding previous CG simulations of thermoset composites, Komarov et al.  employedHence, provided that appropriate material modeling tools are available, understanding and predicting the properties of graphene-reinforced epoxy nanocomposites across different lengths and time scales can potentially facilitate the development of innovative materials. As depicted in The epoxy system investigated in this study is made of DGEBA as the epoxy monomer and DICY and DETA as the curing agents. The DGEBA-based epoxy system is a preferred choice for high-performance applications due to its superior mechanical and thermal properties, excellent adhesion to various substrates, and chemical resistance ,37,38. IThe mesoscopic model is characterized by the atomic composition of the CG beads, their interconnections, as well as bonded and non-bonded potential interactions. The initial stage involves atoms-to-bead mapping, wherein atoms are categorized into respective beads. In the case of the DGEBA model, atoms comprising the epoxy group were assigned to E beads, oxyphenylene groups to G beads, and dimethyl groups to B beads. In the DETA model, primary and secondary amine groups were assigned to Ad and Ae beads, respectively. Finally, in the DICY model, amine groups were assigned to Ay beads, whereas non-amine groups were assigned to Y beads see .During the curing process, reactive beads of DGEBA, DETA, and DICY form bonds, where beads Ad, Ae, and Ay (based on amines) bond with E beads, leading to the formation of a cross-linking point. Beads Ad and Ay have the potential to react twice, resulting in the formation of Ad1, Ad2, Ay1, and Ay2 beads  method. The IBI method is a structure-based coarse-graining method that determines the effective pair potential of particles in thermal equilibrium. It efficiently calculates potentials for the coarse-grained model based on structural characteristics, such as the radial distribution function (RDF), which is derived from atomistic simulations . AccordiAfter generating the RDF for a specific degree of freedom  from the atomistic configuration, Boltzmann inversion is applied to obtain the corresponding interaction potential, that is:Gaussian . HoweverGaussian , namely:The iterative process involves refining the CG potentials obtained from direct inversion by applying a correction term. The correction is gradually reduced as the ith iteration of potential refinement, The iterative Boltzmann inversion method yields potentials that may not accurately reproduce the thermodynamic behavior of the system, such as the system pressure. To overcome this limitation, an additional correction term can be introduced as follows:After adjusting the pressure of the coarse-grained system, the target property (Graphene (Gr) is a material known for its exceptional mechanical strength and high surface-to-weight ratio, making it an excellent filler for nanocomposites . DespiteThe TersoffCG potential is directly derived from the full-atomistic Tersoff potentials  by adjus3) and CG simulations (113 × 113 × 113 Å3), as depicted in In the network structure of epoxy-based materials, the curing reaction between epoxy and hardener monomers is a crucial step that should be accurately reflected in molecular dynamics simulations, whether atomistic or coarse-grained. Cross-linking was performed separately for the atomistic and CG models to ensure consistency and reproducibility. To achieve this, a reactant mixture containing stoichiometric amounts of DGEBA, DICY, and DETA (100:5:5) was randomly seeded into a cubic box at low density using LAMMPS  . A multiFollowing the construction of the cross-linked epoxy resin system for both atomistic and coarse-grained models, the density was measured at different temperatures, ranging from 350 K to 430 K, considering the NPT ensemble. To ensure equilibrium, the system underwent a 5 ns equilibration process in each instance. The glass-transition temperature was then determined by analyzing the discontinuity of the density as a function of temperature.x, y, and z) to obtain an average value along with standard deviation for the computed properties. This deformation process follows Hooke’s law:Subsequently, the elastic (Young) modulus and Poisson’s ratio of the epoxy resin were computed using molecular dynamics simulations, where the system was subjected to uniaxial deformation and the mechanical response was recorded. The Young modulus and Poisson’s ratio were obtained from the uniaxial tensile deformation simulations conducted in all three directions  to obtain an average value along with standard deviation. The method involved creating two lower-temperature regions at opposite ends of the simulation box and imposing a certain amount of heat in the middle region . As a result, a temperature gradient was established in the model, and heat flow occurred throughout the simulation domain via velocity exchanges between the atoms of the “cold” and “hot” regions. Periodic boundary conditions were applied in all directions . Once the system reached a steady state, the amount of energy transferred per unit of time and cross-sectional area from the hot region to the cold region was calculated as:To investigate the thermal conductivity of the system, we employed a Müller–Plathe method based on non-equilibrium molecular dynamics (NEMD) simulations  in all tgiven as :(6)λ=limIn addition to thermal conductivity, this study also aimed to determine the specific heat capacity of the model at constant pressure. To achieve this, we recorded the average enthalpy and temperature at different temperatures ranging from 300 K to 350 K, considering the NPT ensemble. The resulting data were then plotted as an enthalpy-temperature plot. The slope of this plot provided the specific heat capacity The predicted thermophysical properties of epoxy/Gr nanocomposites obtained from the developed mesoscopic model were then compared with two continuum models, namely mean field (MF) and finite element method (FEM). The MF homogenization approach utilized first-order Mori and Tanaka  . To prepare these models, we first randomly distributed the constituent monomers of the epoxy resin in stoichiometric proportion  within a simulation box. The time integration was applied first in the NVT ensemble and later in the NPT ensemble at a temperature of 300 K and pressure of 1 atm. After this initial distribution and equilibration, we applied a cross-linking procedure to achieve the desired cross-linking degree of the epoxy resin. The model consisted of approximately 13,500 atoms. We used the density of the epoxy resin model as an indicative parameter to assess its validity. The densities of the epoxy models with 20%, 30%, 50%, and 70% cross-linking degrees were found to be 1.134 g/cmted in 3 ,57,58.To determine the glass-transition temperature of the epoxy resin model with a 70% cross-linking degree, we conducted equilibrium simulations considering the NPT ensemble. Specifically, we progressively increased the system temperature from 350 K to 430 K in steps of 10 K over a period of 2 ns while maintaining a pressure of 1 atm. From these simulations, we obtained densities at various temperatures and plotted the resulting density-temperature curve. By identifying the junction point of the two fitting lines in the density-temperature plot, we calculated the glass-transition temperature. As shown in The mechanical properties of the epoxy resin were determined from stress-strain curves. The elastic modulus of the 70% cross-linked epoxy resin was found to be 2.99 GPa using the linear theory of elasticity, which agrees well with the range of experimental values from 2.5–5.0 GPa ,60,61,62Additionally, atomistic simulation was performed to calculate the thermal conductivity of the 70% cross-linked epoxy model, resulting in an average thermal conductivity of 0.163 W/mK at 300 K, which is in close agreement with literature values ranging from 0.18–0.2 W/mK . The speTo develop the coarse-grained force field for the epoxy resin, an atomistic model was specifically designed with a degree of cross-linking of 50%, as illustrated in Together with the coarse-graining of potentials, we developed a specific cross-linking procedure based on LAMMPS scripts for the epoxy resin CG force field. Following cross-linking, the new angle types generated through the procedure were assigned, and the charges of beads were updated according to their types. This method enables more accurate and efficient simulation of cross-linked epoxy systems using the developed CG force field. To assess the stability of the CG force field, a long simulation was conducted on a cross-linked CG epoxy system using a time step of 1.0 femtoseconds . The simulation results, presented in 3 at 300 K and 1 atm, which is in good accordance with atomistic simulation and experimental values ranging from 1.07 to 1.20 g/cm3 [The accuracy of the CG model was assessed by comparing the density, glass-transition temperature, elastic modulus, Poisson’s ratio, thermal conductivity, and specific heat capacity against both atomistic simulation and experimental values from the literature. This investigation focused on the CG epoxy model with 70% cross-linking, as Fourier-transform infrared spectroscopy (FTIR) studies monitoring the curing process of epoxy resins have indicated that the cross-linking degree typically falls between 50% to 100%, depending on the temperature . After c20 g/cm3 ,57,58. TThe Young’s modulus and Poisson’s ratio were obtained from the stress-strain plot using the CG model developed here, as shown in A tabulated comparison between the atomistic and mesoscopic model predictions, as well as literature measures for the thermophysical properties of the considered epoxy, are reported in 2, with the armchair and zigzag directions represented by the x and y directions, respectively, and a thickness of 0.335 nm.The thermophysical properties of a monolayer graphene sheet were computed using the optimized parameters of the TersoffCG potential. To generate the CG graphene model, the atomistic bond distance (C-C) of the graphene model was changed to the CG bond distance (C-C) using Visual Molecular Dynamics (VMD) . The sizThe system was equilibrated considering an NVT ensemble at 300 K for 100 ps before a uniaxial tension deformation was applied along the armchair and zigzag directions. The stress-strain curve in The thermal conductivity of the monolayer CG graphene model was then investigated using NEMD simulation for different sample lengths ranging from 20 nm to 100 nm. The relationship between the inverse length  were introduced into the epoxy resin. DGEBA, DICY, DETA, and Gr molecules were randomly seeded into a cubic box using LAMMPS, followed by a multistep cross-linking process. Equilibration procedures were performed, including energy minimization, using NVT and NPT ensembles at a temperature of 300 K and pressure of 1 atm, and a time step of 1 fs. The system was equilibrated after 4–5 ns, and the equilibrated systems were then used to determine the thermophysical properties of the resulting nanocomposite. To facilitate comparison with previous results, a cross-linking degree of 70% was considered for the epoxy matrix. 3), elastic constant (1.419 GPa), Poisson’s ratio (0.3), specific heat capacity (550.60 J/kgK), and thermal conductivity (0.099 W/mK), as well as the graphene density (1.28 g/cm3), elastic constant (916 GPa), Poisson’s ratio (0.14), specific heat capacity (526.95 J /kgK), and thermal conductivity (13.95 W/mK for the simulated length). The RVE model was created by assigning the same mass fraction  to the CG model.The input parameters for the RVE continuum models to be compared were obtained from the CG simulation results, including the parameters for epoxy such as density (1.24 g/cmE) with respect to that of pristine epoxy (0E) as a function of the graphene weight fraction for CG, MF, and FEM models. The elastic modulus is enhanced with all models with an increase in the graphene weight fraction. In the CG MD simulations, the elastic modulus increases from 2.32% (0.5 wt.%) to 9.16% (2.0 wt.%), whereas in the FEM simulation, the elastic modulus increases from 1.19% (0.5 wt.%) to 7.04% (2.0 wt.%), compared to the neat epoxy. Both FEM and MF models exhibit a similar trend. The effect of graphene on the thermal conductivity of the epoxy/Gr nanocomposite was also investigated, and the results are presented in 3, coherently with previous investigations employing atomistic as well as CG MD models of epoxy resin [Clearly, the size of the simulation domain may significantly impact the measured properties. Employing a larger domain size allows for improved statistical sampling, leading to more precise and reliable outcomes. Tsige et al.  observedxy resin ,90,91,92Overall, at least for the considered nanocomposites, both mesoscopic and continuum models provide consistent enhancements in the thermophysical properties of neat epoxy when graphene fillers are included. A detailed list of all the results is available in This study employed multiscale computational techniques to investigate the impact of graphene on the thermophysical properties of epoxy matrix composite. By integrating atomistic, mesoscopic, and continuum models, we accurately predicted properties including density, glass-transition temperature, elastic modulus, Poisson’s ratio, and thermal conductivity. Initially, a full atomic model of the epoxy resin was constructed using a COMPASS force field, yielding precise predictions for various properties. However, the specific heat capacity was found to be overestimated compared to the experimental values.Subsequently, a mesoscopic (coarse-grained) force field was developed using the iterative Boltzmann inversion (IBI) method, based on the atomistic model. The developed mesoscopic model exhibited significant agreement with the atomistic and experimental values for density, glass-transition temperature, and Poisson ratio. However, the predicted values for Young’s modulus and thermal conductivity were 47% and 40% lower than the atomistic values, respectively. The specific heat capacity was also underestimated compared to the experimental values. For graphene, a coarse-grained force field based on the TersoffCG potential was employed, with optimized parameters for mechanical characteristics. This model demonstrated good agreement with published MD and experimental results for the mechanical properties than the thermal conductivity.Furthermore, the developed mesoscopic models were utilized to determine the thermophysical properties of epoxy–graphene nanocomposites and compared them to similar continuum models, showing consistent results. The findings indicated that the increasing weight percentage of graphene-enhanced elastic modulus and thermal conductivity while reducing the Poisson’s ratio. Despite the coarse-grained model having lower prediction accuracy compared to full atomistic models, its 10× simulation speed enables the modeling of material sizes beyond classical MD simulations. In the future, this approach could be extended to investigate the thermophysical behavior of epoxy resin systems with other carbon-based nanoparticles, such as graphene oxide, reduced graphene oxide, and carbon nanotubes.


Sacrococcygeal masses encompass a diverse range of pathologies. Prenatal ultrasound facilitates early detection of congenital sacrococcygeal masses. We present the case of a newborn of a 22-year-old woman who was identified to have a sacrococcygeal mass by prenatal ultrasound that was initially diagnosed as sacrococcygeal teratoma. On examination after delivery, a large midline mass in the sacrococcygeal region was observed, which was globular in shape and had smooth, thin skin with bluish discoloration. Magnetic resonance imaging revealed a cystic lesion that protruded through a caudal sacral defect, consistent with a sacrococcygeal meningocele. The patient underwent surgical repair of the meningocele without any intraoperative complications and had preserved motor function in the lower extremities after the procedure. This case underscores the challenge of distinguishing sacrococcygeal teratoma from meningocele based on clinical presentation and prenatal ultrasound findings. An accurate preoperative diagnosis is essential for effective surgical planning. Sacrococcygeal masses may arise from various etiologies such as congenital, traumatic, neoplastic, or inflammatory pathologies . CongeniWe describe the case of a female newborn delivered at 37 weeks gestation with a prenatal diagnosis of a sacrococcygeal mass. The mass was identified during the routine antenatal ultrasound examination in the first trimester and appeared as a complex mass with heterogeneous echogenicity in the sacrococcygeal region. The patient was delivered via cesarean section, and the mother had an unremarkable pregnancy history. Upon physical examination, a large midline mass in the sacrococcygeal region was observed. The mass had a globular shape, was covered by smooth and thin skin, and displayed bluish discoloration Figure . The carTo better diagnose and characterize the sacrococcygeal mass, magnetic resonance imaging (MRI) was performed. The results of the MRI showed an extradural cystic lesion without a solid component or enhancement. The lesion had a similar signal intensity to that of cerebrospinal fluid, with low signal intensity in T1-weighted images and high signal intensity in T2-weighted images. Additionally, it was protruding through a caudal sacral defect, and these findings were consistent with a sacrococcygeal meningocele (Figure After discussing the findings with the parents, it was decided that surgical repair of the meningocele would be performed. The surgical technique used in this case involved preoperative planning of the incision to ensure optimal cosmetic results, as well as preoperative marking of feeding vessels such as arteries and veins.During the surgery, the surgeon performed a delicate dissection of the meningeal sac, with careful attention to bleeding control, resulting in a total blood loss of 50 ml. Fortunately, no blood transfusion was needed. The herniated sac was large, had a thin vascular wall, was filled with cerebrospinal fluid, and did not contain any neural tissues. It was in direct contact with the rectum anteriorly and displaced the coccyx posteriorly. The perineum was divided and displaced laterally, and the sac was completely removed after securing both the urethra and rectum. The glucose level of the sac content was 55 mg/dl, which was comparable to that of cerebrospinal fluid. Primary closure of the perineum was then performed, followed by the closure of the incision edges and skin layers using absorbable stitching. The end result was a considerable cosmetic improvement of the buttocks, and remarkably, there was no need for a wound suction drain. The patient tolerated the procedure well, and no intraoperative complications were noted Figure .After the surgery, the patient exhibited preserved motor power in the lower extremities, and normal urination and bowel movement resumed. No evidence of sphincteric injury was observed. The patient was discharged after two days, in good general and neurological condition. It is worth noting that the baby was followed up for 1.5 years postoperatively, during which no neurological complications were observed.The case presented highlights the complexity and challenges in the diagnosis and management of neural tube defects. Neural tube defects are a heterogeneous group of abnormalities that involve the central nervous system, with varying degrees of severity . These dThe etiology of neural tube defects is multifactorial, with both genetic and environmental factors contributing to their development. Studies have shown an increased prevalence of neural tube defects among first-degree relatives of affected patients, indicating a genetic component to their pathogenesis . HoweverIn the present case, the initial clinical diagnosis of a sacrococcygeal teratoma highlights the challenges in differentiating between different types of sacrococcygeal masses. Sacrococcygeal teratoma is a rare tumor that arises from totipotent stem cells and is composed of two or three germ cell layers. It is considered the most common tumor in the neonatal period and can be diagnosed prenatally during an ultrasound examination . HoweverThe treatment options for sacrococcygeal meningocele depend on several factors, including the size, location, and presence of associated anomalies -2. ExcisIn conclusion, we presented a case of a sacrococcygeal meningocele in a newborn who was successfully treated with surgical intervention, resulting in a favorable prognosis. Our case underscores the challenge of distinguishing between sacrococcygeal teratoma and meningocele based on clinical presentation and antenatal ultrasound findings. Accurate preoperative diagnosis is essential for optimal surgical planning and management.


Abnormal inactivation of the Wnt/β-catenin signaling pathway is involved in skin diseases like androgenetic alopecia, vitiligo and canities, but small-molecule activators are rarely described. In this study, we investigated the stimulatory effects of escin on the canonical Wnt/β-catenin signaling pathway in cultured human dermal papilla cells (hDPCs). Escin stimulated Wnt/β-catenin signaling, resulting in increased β-catenin and lymphoid enhancer-binding factor 1 (LEF1), the accumulation of nuclear β-catenin and the enhanced expression of Wnt target genes in cultured hDPCs. Escin drastically reduced the protein level of glycogen synthase kinase (GSK)-3β, a key regulator of the Wnt/β-catenin signaling pathway, while the presence of the proteasome inhibitor MG-132 fully restored the GSK-3β protein level. The treatment of secreted frizzled-related proteins (sFRPs) 1 and 2 attenuated the activity of escin in Wnt reporter assays. Our data demonstrate that escin is a natural agonist of the canonical Wnt/β-catenin signaling pathway and downregulates GSK-3β protein expression by facilitating the proteasomal degradation of GSK-3β in cultured hDPCs. Our data suggest that escin likely stimulates Wnt signaling through direct interactions with frizzled receptors. This study underscores the therapeutic potential of escin for Wnt-related diseases such as androgenetic alopecia, vitiligo and canities. The Wnt/β-catenin signaling pathway plays a critical role in various cellular and physiological processes, such as cell proliferation, differentiation, survival, migration and polarity, during development and tissue homeostasis. Controlling the level of transcriptionally active β-catenin via the protein (β-catenin) destruction complex is central to the pathway since it determines the expression of Wnt target genes . The β-cAesculus hippocastanum, L).As an essential regulator of tissue homeostasis and regeneration, Wnt/β-catenin signaling is tightly controlled in spatiotemporal patterns. Thus, dysregulation of the Wnt/β-catenin pathway is closely associated with many health conditions, including cancer, hair loss, pathological wound healing and osteoporosis ,10,11,12In this study, we examined the impact of escin on the Wnt/β-catenin signaling pathway in Wnt reporters and cultured human dermal papilla cells (hDPCs), and elucidated the underlying mechanisms of regulation. We found that escin is a canonical agonist of the Wnt/β-catenin signaling pathway. Our data demonstrate that escin stimulates Wnt signaling by facilitating the proteasomal degradation of GSK-3, possibly disrupting the β-catenin destruction complex in cultured hDPCs, and that the Wnt-stimulating activity of escin is likely mediated through the direct interaction with frizzled receptors. Escin and MG132 were purchased from Sigma-Aldrich . Recombinant Wnt3a and secreted frizzled related proteins (sFRPs) were purchased from R&D systems .−1 penicillin G and 100 µg·ml−1 streptomycin . Human DPCs were purchased from Promocell . The cells were cultured in basal medium supplemented with a supplement mix that contained 4% FBS, 0.4% bovine pituitary extract, 1 ng·ml−1 basic FGF and 5 μg·ml−1 insulin. Cells were maintained in a humidified incubator at 37 °C with 5% CO2.The cell line of the Wnt reporter WRHEK 293A was purchased from AMSBIO . Cells were cultured in MEM  supplemented with 10% FBS , 100 U·ml5 cells per well) were seeded in black 96-well plates and cultured for 24 h. Cells were treated with various concentrations of escin and incubated for another 24 h. Cells were then lysed by adding 50 μL of 1× Passive Lysis Buffer  to each well and shaking for 10 min. GFP expression levels  were assessed by measuring the fluorescence at a 488/510 nm wavelength using VICTOR3 . Then, 50 μL of luciferase substrate solution  was added and luciferase activity was measured using VICTOR3. Luminescence (TCF/LEF activity) values were normalized to GFP (cell viability) values.WRHEK293A cells (3 × 106 cells per dish) were seeded in 100 mm culture dishes and cultured for 24 h. Cells were treated with escin at various concentrations for 24 h, and then, washed with ice-cold PBS and lysed on ice in M-PER buffer  supplemented with CompleteTM protease inhibitor cocktail and phosphatase inhibitor . A total of 40 μg of protein was analyzed via Western blotting with antibodies to evaluate the following proteins’ expression levels: β-catenin , GAPDH , LEF1, Axin2  and GSK-3β . Western blot was analyzed using a chemiluminescence detector .DPCs (1 × 106 cells per well) were seeded in 6-well plates and cultured for 24 h. Then, the cells were treated with escin at various concentrations for 24 h. Total RNA was isolated using an RNA isolation kit  according to the manufacturer’s guide. cDNA was synthesized via reverse transcription using an eCube cDNA synthesis kit  with a PCR thermocycler  according to the manufacturer’s protocol. cDNAs obtained from control cells and escin-treated cells were subjected to real-time PCR analysis. The TaqMan probes used in this study were as follows: GAPDH assay id 4352934E; CTNNB1 assay id Hs00355045_m1; LEF1 assay id Hs01547250_m1; Frizzled3 assay id Hs00907280_m1; Frizzled4 assay id Hs00201853_m1; Frizzled5 assay id Hs00258278_s1; Frizzled7 assay id Hs00275833_s1; CCND1 assay id Hs00765553_m1; Myc assay id Hs00153408_m1; and DKK-1 assay id Hs00183740_m1. TaqMan One Step RT-PCR Master Mix Reagent  was used. The PCR reactions were performed using an ABI7500 Real-Time PCR system following the manufacturer’s protocol. The resulting data were analyzed using ABI software .DPCs (1 × 104 cells per well) were seeded in 96-well plates and cultured overnight. After washing with PBS, DPCs were fixed with 4% paraformaldehyde at room temperature for 10 min. Cells were then permeabilized with PBS containing 0.1% triton × −100 and blocked with PBS containing 5% FBS and 1% BSA. After consecutive incubation with primary antibodies  at 4 °C for 12 h and Alexa 488 nm or Alexa 594 nm conjugated secondary antibodies  at room temperature for 1 h, nuclei were stained with DAPI  in the dark for 10 min. High-resolution fluorescence images were taken using an EVOSTM FL Auto2 Imaging System .DPCs  of at least five independent experiments, unless otherwise indicated. The statistical significance of the difference was determined using Student’s We found that escin, a natural compound, stimulated Wnt signaling in Wnt reporter HEK293 cells. As shown in To ensure the Wnt-stimulating activity of escin in normal cells, the effects of escin on the Wnt/β-catenin signaling in cultured hDPCs were investigated. The treatment of escin significantly increased the amount of cellular β-catenin protein, an indicator of Wnt signal activation, in a concentration-dependent manner a. The mRThe effects of escin on the mRNA expression of several Wnt target genes and receptors in hDPCs were investigated. As shown in To elucidate the mechanism by which escin stimulates Wnt/β-catenin signaling, we scrutinized the protein levels of GSK-3β and Axin2, components of the β-catenin destruction complex.Of note, escin drastically decreased the amount of GSK-3β protein in a concentration-dependent manner a. The prThere were no significant changes, however, in the mRNA levels of GSK-3 and Axin c. To detThe secreted frizzled related proteins (sFRPs) were reported to inhibit Wnt signaling by interfering with the binding of Wnt ligands with their receptors, the frizzled receptors. To elucidate whether escin stimulates Wnt signaling through direct interaction with frizzled receptors, the effect of escin on Wnt reporter activity was evaluated in the presence of sFRP1 and sFRP2 in WRHEK293A reporter cells. As shown in In this report, we provide evidence, for the first time, to our knowledge, that the natural compound escin is an activator of the canonical Wnt/β-catenin signaling pathway . Wnt-stiIn the present study, we found that escin remarkably stimulated Wnt/β-catenin signaling in stable TOPFlash reporter cells . In ordeThe β-catenin destruction complex, consisting of Axin, adenomatous polyposis coli (APC), casein kinase (CK)-1 and GSK-3β, is reported to play a crucial role in the pathway by tightly controlling the level of nuclear, transcriptionally active β-catenin ,35. In tAnother control point of the Wnt signaling pathway is the scaffold protein Axin, which is thought to be the concentration-limiting component of the β-catenin destruction complex ,39,40. TOur data demonstrate that the activation of Wnt signaling by escin resulted from the post-translational modifications of GSK-3 and AXIN since escin did not elicit any changes in mRNA levels c, which To investigate the possible interactions between escin and Wnt receptors, Wnt reporter cells were treated with secreted frizzled-related proteins (sFRPs) in the presence of escin. sFRPs are known to inhibit Wnt signaling by binding to Wnt ligands, thus depleting free, active Wnt ligands. Each sFRP , however, was reported to show a different mode of action when modulating Wnt/β-catenin signaling, depending on the concentration balance of the Wnt and sFRP proteins. We found that the treatment of sFRP1 and sFRP2 almost completely blocked escin-induced Wnt signal activation, implying a possible direct interaction between escin and frizzled receptors . The preOur findings demonstrate that the natural compound escin stimulates canonical Wnt/β-catenin signaling through the downregulation of GSK-3β protein expression by facilitating the proteasomal degradation of GSK-3β. Our data strongly demonstrate the possible therapeutic potential of escin for treating and/or preventing Wnt-related disorders, especially skin-associated ones such as hair loss , canities and vitiligo, without safety concerns, since escin has long been used for dermatologic and cosmetic purposes.


Recent theories describe perception as an inferential process based on internal predictive models adjusted by means of prediction violations (prediction error). To study and demonstrate predictive processing in the brain the use of unexpected stimulus omissions has been suggested as a promising approach as the evoked brain responses are uncontaminated by responses to stimuli. Here, we aimed to investigate the pupil’s response to unexpected stimulus omissions in order to better understand surprise and orienting of attention resulting from prediction violation. So far only few studies have used omission in pupillometry research and results have been inconsistent.This study adapted an EEG paradigm that has been shown to elicit omission responses in auditory and somatosensory modalities. Healthy adults pressed a button at their own pace, which resulted in the presentation of sounds or tactile stimuli in either 88%, 50% or 0% (motor-control) of cases. Pupil size was recorded continuously and averaged to analyze the pupil dilation response associated with each condition.Results revealed that omission responses were observed in both modalities in the 88%-condition compared to motor-control. Similar pupil omission responses were observed between modalities, suggesting modality-unspecific activation of the underlying brain circuits.In combination with previous omission studies using EEG, the findings demonstrate predictive models in brain processing and point to the involvement of subcortical structures in the omission response. Our pupillometry approach is especially suitable to study sensory prediction in vulnerable populations within the psychiatric field. For example, a novel sound in a series of standard sounds may require additional cognitive resources for processing, or an unfamiliar environmental sound may elicit encoding of new memories. These and other stimulus-related factors can impact pupil dilation , 22, butAn innovative approach to studying surprise is through the use of stimulus omissions, where expected standard stimuli are occasionally replaced by an unexpected stimulus absence to elicit surprise. The surprise response to omission can be explained in terms of predictive coding, a neurocognitive theory that posits that the brain uses prior knowledge to continually generate predictions about incoming sensory information. When these predictions are incorrect compared to actual sensory input, this results in a prediction error or surprise response that is used to update and refine the brain’s internal models, allowing for more accurate predictions in the future , 23, 24.While theoretically appealing, the use of stimulus omissions has produced somewhat inconsistent results in previous pupillometry research. Cooper et al.  observed3027Pupil dilation studies have predominantly focused on the visual and auditory modalities, with comparatively few examining the somatosensory modality . SimilarThe current study utilizes a tried-and-tested paradigm adapted from EEG studies to examine omission responses in the auditory and somatosensory modalities using pupillometry. This paradigm has been proven to consistently elicit EEG responses to omission in both modalities , 34. The2.2.1.A total of 40 participants took part in the experiment . All participants were right-handed, as measured by a German version of the Oldfield Scale . All par2.2.2.2.1.A total of 48 different common environmental sounds  rated as identifiable by an independent sample of participants  were us2.2.2.Presentation of tactile stimuli was performed using pulses of pressurized air (3 bar) that inflated a membrane, which was controlled using a somatosensory stimulus generator  that was placed outside the chamber. Tactile stimulus duration was approximately 30 ms. Two membranes were placed on the left middle and index fingers at the volar aspect of the distal phalanx . In the2.4.Pupil diameter measurements were converted to mm as suggested by Steinhauer et al. . Eye sac2.5.2.5.1.t-tests, either a one sample test (to compare with motor activity) or two-tailed paired test  were used where the null hypothesis corresponded to a standardized effect size δ = 0, while the alternative hypothesis was defined as a Cauchy prior distribution centered around 0 with a scaling factor of r = 0.707 . For Bayesian repeated-measures ANOVA [rANOVA; see  against the null model, which included subjects and random slopes. The BFinclusion factor across matched models was calculated for all variables to determine the evidence provided by the data for an effect if comparing all matched models including vs. excluding the effect. Bayes Factor (BF10) was calculated using 10.000 sample repetitions (the JASP default) and was interpreted following Lee & Wagenmakers (10 > 1). For frequentist t-tests and rANOVA effect size was reported using Cohens d and the generalized η2 [ηG2;  to confirm the habituation effects. Next, we replicated all analyses of the planned data analysis (see above) on the data observed in the first block per condition only.A second, post-hoc data analysis of omission results was decided upon 3.3.1.Participants were generally able to keep a stable pace between button presses throughout the experiment, where the aim was to keep inter-press interval around 3 s. Group average for motor-control condition was 2.77 s (SD = 0.41 s), for auditory 88%-condition 2.75 s (SD = 0.38 s), for auditory 50%-condition 2.85 s (SD = 0.44 s), for somatosensory 88%-condition 2.80 s (SD = 0.43 s) and for somatosensory 50%-condition 2.82 s (SD = 0.43 s).3.2.3.2.1.3.2.1.1.10 = 63, d = 0.607, t(39) = 3.839, p < 0.001). PDR amplitudes in response to omissions in the 88%-condition were similar between modalities. The data provided moderate evidence against an effect of modality  = 0.860, p = 0.395).In line with our hypothesis, we observed larger PDR amplitudes in response to omissions in the 88%-condition compared to the motor-control condition . The dat3.2.1.2.10 = 1.194, d = 0.330, t(39) = 2.086, p = 0.044). PDR amplitudes in response to omissions in the 50%-condition were similar between modalities. The data provided weak evidence against an effect of modality  = 1.382, p = 0.175).Although we hypothesized similar PDR amplitudes between omissions in the 50%-condition compared to the motor-control condition, results were not clear, showing slightly larger PDR amplitudes in response to omissions in the 50%-condition compared to the motor-control condition. The data provided inconclusive evidence for a condition effect  = 3.218, p = 0.081, ηG2 = 0.013), modality  = 1.790, p = 0.189, ηG2 = 0.009), or the probability condition by modality interaction  = 0.246, p = 0.623, ηG2 = 0.000).Contrary to our hypothesis, we observed similar PDR amplitudes in response to omissions in the 88%-condition compared to omissions in the 50%-condition. PDR amplitudes were similar between modalities. The probability condition (88%-condition vs. 50%-condition) by modality (auditory vs. somatosensory) rANOVA favored the null model. The frequentist rANOVA showed no effects for probability condition (3.2.2.3.2.2.1.st block vs. 88%-condition 2+ blocks) by modality (auditory vs. somatosensory) rANOVA favored the model including block, providing moderate evidence (BF10 = 5.452). The frequentist rANOVA showed a significant effect for block  = 9.091, p = 0.005, ηG2 = 0.032), no effect for modality  = 0.433, p = 0.514, ηG2 = 0.003), and no modality by block interaction effect  = 0.035, p = 0.853, ηG2 = 0.000).Larger PDR amplitudes in response to omissions were observed in the first block of the 88%-condition compared to later blocks in the 88%-condition . PDR amp3.2.2.2.10 = 1.744, d = 0.362, t(39) = 2.291, p = 0.027).Slightly larger PDR amplitudes were observed in the first block of the motor-control condition compared to the second block of the motor-control condition. The data provided weak evidence for a block effect  = 4.120, p < 0.001). PDR amplitudes in response to omissions in the first block of the 88%-condition were similar between modalities. The data provided moderate evidence against an effect of modality  = 0.469, p = 0.641).In line with earlier hypotheses, we still observed larger PDR amplitudes in response to omissions in the first block of the 88%-condition compared with the first block of the motor-control condition. The data provided decisive evidence for a condition effect  = 0.731, p = 0.469). For differences between modalities, see section 3.2.1: 50% omission versus motor control.In line with earlier hypotheses, we now observed similar PDR amplitudes in response to omissions in the 50%-condition compared to the motor-control condition. The data provided moderate evidence against an effect of condition (BF3.2.2.5.st block vs. 50%-condition) by modality (auditory vs. somatosensory) rANOVA favored the model including probability condition, providing strong evidence for a larger PDR in the first block of the 88%-condition compared to the 50%-condition (BF10 = 12.741). Inclusion Bayes Factor provided strong evidence in favor of including probability condition (BFinclusion = 12.684) but moderate evidence against including modality (BFinclusion = 0.326) and the modality by probability condition interaction (BFinclusion = 0.223). The frequentist rANOVA showed a significant effect for probability condition  = 13.534, p < 0.001, ηG2 = 0.049), no effect for modality  = 0.735, p = 0.397, ηG2 = 0.005), and no modality by probability condition interaction effect  = 0.006, p = 0.937, ηG2 = 0.000).In line with earlier hypotheses, we now observed larger PDR amplitudes in response to omissions in the first block of the 88%-condition compared to omissions in the 50%-condition. PDR amplitudes were similar between modalities. The probability condition , with the 88%-condition being the only one in which the omission was expected to be surprising. Omission responses were observed in the pupil for both modalities when comparing the 88%-condition with the motor-control condition, showing similar omission PDRs across modalities. In contrast, no omission effects were observed when comparing omissions in the 88%-condition with omissions in the 50%-condition. Further analysis revealed that the amount of exposure to a condition  can impact the amplitude of the omission response, hindering detection of the response in the average over all blocks. When the number of presented blocks was matched between conditions, a clear pupil dilation could be observed in the 88%-condition compared with the 50%-condition. In the following we will discuss these results one by one.4.1.Similar to EEG studies, the current study used a motor-control condition to determine the elicitation of an omission response. In the motor-control condition, none of the button presses result in a stimulus, making the omissions completely predictable. The comparison of omissions in the 88%-condition with the motor-control condition therefore entails a comparison of two identical events (a button press without a stimulus) where only the associated surprise is manipulated. Controlling for motor activity is important even in pupillometry, as actions are known to be accompanied by a dilation of the pupil , 61. AlsThis study is the first to directly compare pupillary omission responses between different modalities. Results show evidence against a difference in omission PDRs between the two modalities. This aligns with the EEG findings of Dercksen et al. , who obs164.2.To confirm that the amplified PDR in the 88%-condition was caused by surprise, a 50%-condition included occasional stimuli but, like the motor-control condition, no expectation of stimuli. The cross-condition comparison again involved two identical events (a button press without a stimulus), where now both conditions included stimuli. Contrary to our hypothesis, there was no evidence for a larger omission PDR in the 88%-condition compared to the 50%-condition . This coPrevious pupillometry studies typically compared unexpected omissions with standard stimulus responses to assess the presence of an omission response. This approach may be problematic because the pupil dilates in response to both surprise as well as stimuli. Interestingly, the present study demonstrates how this comparison can be influenced by stimulus-related activity and lead to incorrect conclusions regarding the omission response. In the tactile modality, the relatively small PDR to stimuli is similar to the motor-control and thus allows for the detection of the surprise response in the omission PDR . However664.3.This study offers a robust and unconfounded approach for investigating the neural mechanisms of surprise. Understanding how the brain responds to surprise has become increasingly relevant not only for a fundamental understanding of the brain, but also in the context of various clinical disorders. For example, several accounts of autism and schizophrenia put the prediction processes that give rise to surprise (or prediction error) at the core of these disorders . Given t5.The current study presents conclusive evidence of a pupil response to the unexpected omission of a stimulus, presumably reflecting surprise and the orienting of attention. This finding aligns with the interpretation of the oP3 component observed in EEG omission studies and indicates the involvement of subcortical structures such as the locus coeruleus and superior colliculus in the omission response. There was no indication of an amplitude difference between modalities, suggesting similar, modality-unspecific activation of the involved brain circuits. Additionally, this study was able to identify two important factors that might have contributed to unreliable omission results in past pupillometry studies. First, habituation over experimental blocks apparently plays a substantially larger role in pupillometry compared to EEG studies. Second, stimulus processing effects might mask omission responses when compared with the standard stimulus. These effects should be carefully considered in future study designs. Finally, omission studies using pupillometry may be well suited for use in clinical studies that investigate surprise processing.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics committee of the medical faculty of the OVGU Otto von Guericke University, Magdeburg, Germany. The participants provided their written informed consent to participate in this study.TD: conceptualization, methodology, software, validation, formal analysis, investigation, data curation, writing—original draft, writing—review and editing, visualization. AW: conceptualization, methodology, software, formal analysis, writing—review and editing. NW: conceptualization, methodology, resources, writing—review and editing, supervision, project administration, funding acquisition. All authors contributed to the article and approved the submitted version.This work was supported by the Center for Behavioral Brain Sciences Magdeburg financed by the European Regional Development Fund (ZS/2016/04/78120) and Leibniz Association (P58/2017). Funding sources did not have any involvement in study design, data collection/analysis/interpretation, writing of the report, or decision to submit for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The response rate to immune checkpoint inhibitor (ICI) monotherapy in hepatocellular carcinoma (HCC) is as low as 20%; therefore, it is important to identify the patient subgroups that respond to ICI. β-catenin activation is considered to be a key factor of ICI resistance. β-catenin activation is involved in immunologically cold tumor formation in the tumor microenvironment. Overall, controlling β-catenin activation may improve response rates of HCC tumors to ICI. β-catenin modulators and certain kinase inhibitors can suppress β-catenin activation; therefore, combining these drugs with ICIs is expected to further improve the therapeutic effect of ICIs in HCC.Recently, the therapeutic combination of atezolizumab and bevacizumab was widely used to treat advanced hepatocellular carcinoma (HCC). According to recent clinical trials, immune checkpoint inhibitors (ICIs) and molecular target agents are expected to be key therapeutic strategies in the future. Nonetheless, the mechanisms underlying molecular immune responses and immune evasion remain unclear. The tumor immune microenvironment plays a vital role in HCC progression. The infiltration of CD8-positive cells into tumors and the expression of immune checkpoint molecules are key factors in this immune microenvironment. Specifically, Wnt/β catenin pathway activation causes “immune exclusion”, associated with poor infiltration of CD8-positive cells. Some clinical studies suggested an association between ICI resistance and β-catenin activation in HCC. Additionally, several subclassifications of the tumor immune microenvironment were proposed. The HCC immune microenvironment can be broadly divided into inflamed class and non-inflamed class, with several subclasses. β-catenin mutations are important factors in immune subclasses; this may be useful when considering therapeutic strategies as β-catenin activation may serve as a biomarker for ICI. Various types of β-catenin modulators were developed. Several kinases may also be involved in the β-catenin pathway. Therefore, combinations of β-catenin modulators, kinase inhibitors, and ICIs may exert synergistic effects. Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related mortality worldwide ,2. HCC eCTNNB1 mutation is a typical driver mutation that is found in approximately 30% of HCC cases [It is clear that the effects of cancers on the immune microenvironment differ depending on the carcinogenic pathway ,15,16,17CC cases .In 2015, Spranger et al. reported that Wnt/β-catenin pathway activation inhibits cytotoxic T cell infiltration in the immune microenvironment of malignant melanoma, resulting in resistance to immune checkpoint inhibitors . FurtherIn 2019, Galarreta et al. reported the role of β-catenin pathway activation in HCC . AccordiBasic research in mice revealed that immune exclusion is associated with β-catenin activation. Associations between β-catenin activation and the immune microenvironment were also reported in human HCC.In 2020, we examined the association between oncogenic pathways in 154 resected human HCC specimens. We reported a relationship between PD-L1 expression in HCC and eight carcinogenic pathways  based on immunohistochemical and genetic analysis [p < 0.01 and p = 0.03, respectively). Therefore, it is postulated that the PD-1/PD-L1 antibody response is difficult to achieve.Among the HCC variants, those with genetic mutations that lead to β-catenin activation had significantly lower CD8-positive cell infiltration and PD-L1 expression (p < 0.0001).There were also reports on the therapeutic efficacy of ICI in human HCC. Harding et al. reported response rates in 27 patients with HCC treated with ICIs . Progressive disease (PD) was found across all 10 cases that possessed genetic mutations that activated the Wnt/β-catenin pathway . On the p < 0.0001) and OS (OS p = 0.0048).In 2021, we investigated the background factors and prognosis of 34 patients with unresectable HCC that were treated with anti-PD-1 antibody monotherapy by assessing pre-treatment tumor specimens . OverallAccording to the above results, β-catenin activation is considered to be involved in ICI resistance in HCC. Furthermore, the effects of ICI are thought to be low in tumor immune microenvironments that possess poor CD8-positive cell infiltration and PD-L1 expression. Nonetheless, these studies evaluated only a small number of cases; therefore, further research with a larger sample size is required.CTNNB1 mutation). The corresponding expression statuses of the immune-related gene groups were then compared [p = 0.0490). Previous studies reported that IFNγ is an important cytokine in the immune response against tumors, and that PD-L1 expression is induced by IFNγ [In order to further investigate the role of β-catenin activation, we performed genetic analysis in groups with and without a β-catenin activating mutation , and IDH1 mutations were high in Cluster4. Cluster5  exhibited a particularly low lymphocytic infiltration and a relatively high percentage of M2 macrophages. Cluster6 (TGF-β dominant) was characterized by relatively high transforming growth factor-β (TGF-β) signature, with homogeneous and abundant Th1 and Th2 infiltration. Cluster3 (Inflammatory) and Cluster4 (Lymphocyte depleted) were the predominant clusters observed in HCC tumors. Additionally, specific driver mutations were reported to be correlated with leukocyte levels. Driver mutations that correlated with low leukocyte levels in all carcinomas were CTNNB1, NRAS, and IDH1; driver mutations that correlated with high leukocyte levels were BRAF, TP53, and CASP8.In 2018, Thorsson et al. reported several immune subclasses across various cancers . They peCTNNB1 mutation and possesses poor immune cell infiltration.In 2017, Sia et al. described an “immune class” (herein referred to as the “inflamed class”) following gene expression analysis of 956 HCC cases. Approximately 25% of HCCs belong to the inflamed class. Specifically, the inflamed class is characterized by severe lymphocytic infiltration and elevated PD-1 and PD-L1 expression . ApproxiKurebayashi et al. histologically classified the immune microenvironment using immunostaining of 919 regions in 158 HCC cases . AccordiCTNNB1 and ARID2 in cancer cells.Fujita et al. evaluated immune-related genes in 234 HCCs cases and classified these HCC tumors based on local immune responses . Overallp = 2.35 × 10−6). Regarding non-inflamed class, the excluded subclass had the lowest tumor immune cell infiltration and a higher incidence of CTNNB1 mutations than the intermediate subclass .Montironi et al. analyzed 240 HCC cases and proposed an immune-like subclass within the inflamed class ,30. The ACVR2A mutations was higher in NASH-HCC than in non-NASH-HCC. However, with regard to the molecular class, NASH-HCC was significantly less likely to belong to the CTNNB1 subclass than non-NASH-HCC  [CTNNB1 mutation was determined to be a dominant event in the coding DNA sequence of NAFLD-HCC (33%). Immune exclusion correlated with CTNNB1 mutations in patients with NAFLD-HCC Specifically, CTNNB1 mutations lead to immune exclusion via the upregulation of TNFRSF19, which subsequently represses senescence-associated secretory phenotype (SASP)-like cytokines (including IL6 and CXCL8). Nonetheless, this phenomenon can be reversed by using the Wnt-modulator ICG001 [In 2021, Pfister et al. reported that ICIs had an inferior effect on NASH-HCC even when lymphocyte infiltration was observed. NASH-specific CD8-positive cells induce hepatocellular injury and fibrosis, which results in the promotion of hepatocarcinogenesis ,32. Addi = 0.02) ,34. In ar ICG001 .Basic research revealed that the Wnt/β-catenin pathway is complex and involves various molecules ,46,47,48Tumor-associated macrophages (TAMs) are major components of the tumor immune microenvironment and play an important role in the progression of HCC. Tumor cell–derived Wnt ligands stimulate M2-like polarization of TAMs through canonical Wnt/β-catenin signaling, resulting in tumor growth and immunosuppression. . At presX protein is a hepatitis B virus (HBV)-encoded oncogenic protein that promotes HCC progression. HBV X protein (HBx) interacts with non-muscle myosin heavy chain IIA (MYH9) and induces its expression by modulating GSK3β/β-catenin/c-Jun signaling. Alternatively, silencing MYH9 blocks HBx-induced GSK3β ubiquitination, thereby activating the β-catenin destruction complex . ElevateGSK3β phosphorylates Axin1 to facilitate the ubiquitination and proteasomal degradation of β-catenin . IFN indCasein kinase 1 (CK1) phosphorylates disheveled (Dvl) and promotes Dvl signaling . It was GSK3β phosphorylates APC to facilitate the ubiquitination and proteasomal degradation of β-catenin ,68,69. CGSK3β phosphorylates β-catenin, promoting its ubiquitination and proteasomal degradation ,68,69. CMesenchymal epithelial transition factor (MET) phosphorylates β-catenin on tyrosine residues, leading to the dissociation of β-catenin from MET and corresponding nuclear translocation of β-catenin . FibroblSia et al. reported that the CTNNB1 class of HCC tumors exhibits overexpression of protein tyrosine kinase 2 (PTK2) signaling . PTK2, aThe broad involvement of Wnt signaling in cancer progression drove extensive research efforts to target the Wnt pathway using small molecules .IWP is a specific small-molecule inhibitor of the Wnt pathway that blocks Porcupine, an enzyme that promotes the acylation of Wnt proteins. The IWP2 compound can inactivate Porcupine with a high degree of selectivity ,83,84. AAs previously discussed, β-catenin activation participates in the formation of an immunologically cold tumor with poor immune cell infiltration. Some kinase inhibitors may modulate β-catenin activation and have the potential to alter the tumor immune microenvironment from cold to hot .MET inhibitors may theoretically be effective in regulating the β-catenin pathway. MET inhibitors include cabozantinib, tepotinib, golvatinib, and capmatinib. Furthermore, cabozantinib is used for advanced hepatocellular carcinoma based on corresponding clinical trial results .EGFR, FAK, and Src inhibitors may theoretically affect the β-catenin pathway. EGFR inhibitors include erlotinib, gefitinib, lapatinib, cetuximab. Additionally, defactinib inhibits FAK and saracatinib inhibits Src.To date, vascular endothelial growth factor (VEGF) was not shown to be involved in β-catenin activation. Nonetheless, VEGF contributes to tumor angiogenesis and effects the tumor microenvironment by inducing the infiltration of various cells, including Tregs, TAMs, and myeloid-derived suppressor cells (MDSCs), and inducing the release of immunosuppressive cytokines. Therefore, VEGF inhibition may improve the tumor microenvironment. It was reported that VEGF inhibition reduces TAMs and Tregs in the tumor microenvironment, decreases TGF-β and IL-10 expression, decreases the expression of T-cell exhaustion markers, such as PD-1 and TIM-3, and increases the release of immunostimulatory cytokines .β-catenin activation and FGFR4 expression were also reported to be positive correlation in hepatocellular carcinoma . LenvatiAccording to prior basic research, the following mechanism was suggested for the β-catenin-dependent regulation of the tumor immune microenvironment. β-catenin activation reduces the production of chemokines such as CCL5, which inhibits the recruitment of CD103+ dendritic cells and prevents antigen presentation by dendritic cells. As a result, T-cell activation is inhibited and tumor infiltration of CD8-positive cells is prevented ,12,19.Considering the corresponding results in human HCC, one possible hypothesis regarding the relationships between β-catenin pathway activation, CD8-positive cells infiltration, PD-L1 expression, IFNγ, and ICI efficacy are as follows. In HCCs with β-catenin activation, dendritic cells cannot be recruited to the tumor, which ultimately prevents antigen presentation and T cell activation. Consequently, immune exclusion occurs, and IFNγ is not released because CD8-positive cells do not target the tumor without detecting antigen presentation by the dendritic cells ,10,20. FCTNNB1 mutations are found not only in immune-excluded subclasses but also in the inflamed immune-like subclass [Therefore, β-catenin activation may serve as a biomarker to predict the efficacy of ICI treatment. According to Montironi et al.’s classification, subclass . Alternasubclass . FurtherRegulating the Wnt/β-catenin pathway may alter the tumor microenvironment from immunologically cold to hot. It may be possible to strongly inhibit the Wnt/β-catenin pathway by combining kinase inhibitors and Wnt/β-catenin inhibitors. If this β-catenin pathway can be controlled, there is a possibility that the efficacy of ICIs improved; therefore, combining the use of these kinase and Wnt/β-catenin inhibitors with ICIs may result in a synergistic effect. In recent years, clinical trials also began in combination with Wnt/β-catenin inhibitors, kinase inhibitors, and ICIs. A phase 2 study is ongoing to assess the objective response rate of E7386 in combination with pembrolizumab or of E7386 in combination with pembrolizumab plus lenvatinib in HCC .According to prior studies, the tumor immune microenvironment can become “immunologically cold” following the activation of β-catenin; this immunologically cold tumor environment is important for the corresponding reduction in ICI response rate. The β-catenin pathway also plays an important role in immune subclassification. Specifically, it was reported that genetic mutations and various other factors, such as kinase activity, are involved in β-catenin activation. Regulating β-catenin pathway activation may improve the immune microenvironment; therefore, various β-catenin modulators are under development. Additionally, certain protein kinases are understood to indirectly act on the β-catenin pathway; these kinases are also expected to be utilized as novel targets for improving the immune microenvironment. The combined use of kinase inhibitors, ICIs, and β-catenin modulators may be an effective strategy for the treatment of HCC subtypes with β-catenin activation. Therefore, future reports regarding the development of these therapeutic strategies are expected.


The Wnt pathway has been recognized for its crucial role in human development and homeostasis, but its dysregulation has also been linked to several disorders, including cancer. Wnt signaling is crucial for the development and metastasis of several kinds of cancer. Moreover, members of the Wnt pathway have been proven to be effective biomarkers and promising cancer therapeutic targets. Abnormal stimulation of the Wnt signaling pathway has been linked to the initiation and advancement of cancer in both clinical research and in vitro investigations. A reduction in cancer incidence rate and an improvement in survival may result from targeting the Wnt/β-catenin pathway. As a result, blocking this pathway has been the focus of cancer research, and several candidates that can be targeted are currently being developed. Flavonoids derived from plants exhibit growth inhibitory, apoptotic, anti-angiogenic, and anti-migratory effects against various malignancies. Moreover, flavonoids influence different signaling pathways, including Wnt, to exert their anticancer effects. In this review, we comprehensively evaluate the influence of flavonoids on cancer development and metastasis by focusing on the Wnt/β-catenin signaling pathway, and we provide evidence of their impact on a number of molecular targets. Overall, this review will enhance our understanding of these natural products as Wnt pathway modulators. Despite major advancements in cancer treatment and remission rates, many challenges remain in cancer management. Resistance to therapy, which is linked to disease recurrence and metastasis, still poses serious issues that require attention. Cancer is one of the main ailments that places a burden on the world’s healthcare systems. There were approximately 19 million new cases of cancer in 2020, and approximately 10 million people died from cancer worldwide . AccordiThe early stages of cancer development are caused by an aberrant modulation of the transcription factor β-catenin, which is a crucial part of the Wnt/β-catenin signaling pathway ,20,21,22Moreover, Wnt/β-catenin signaling coordinates with a number of cell signaling pathways, including the PI3K/Akt pathway, nuclear factor kappa-B (NF-κB), Sonic Hedgehog, Notch, Hippo/YAP, and epidermal growth factor receptor (EGFR), all of which play a crucial role in the carcinogenesis process ,30,31. TSeveral malignancies, such as ovarian, colon, pancreatic, skin, and lung cancers, are primarily caused by mutations in the Wnt/β-catenin pathway ,39,40,41Even when Wnt ligands are absent, the Wnt/β-catenin pathway is still active in several cancerous cells . APC mutThe cancer stemness paradigm, which describes the capability of cancerous cells to self-renew, has been used to explain a variety of malignant characteristics . The cruThe epithelial-to-mesenchymal transition (EMT), another feature of cancer stem cells and metastasis, also seems to be significantly influenced by the Wnt signaling pathway ,57,58. TEMT, tumor neoangiogenesis, and tumor advancement are all aspects of metastasis, which develops from a primary tumor site and spreads to target tissues and organs in multiple steps. Malignant cells move across blood vessels to particular organs and tissues and invade them through infiltration, resulting in secondary tumor development ,64. EmbrEMT is used to describe the conversion of polarized epithelial cells into migratory and invading mesenchymal cells ,67. SNAIExosomes have recently been identified as a possible mechanism by which cancers establish their metastatic microenvironment . ExosomeCirculating tumor cells (CTCs) are another mechanism implicated in how distant metastasis spreads . For proThe Wnt/β-catenin signaling pathway is implicated in tumor immune exclusion, in addition to its direct contribution to tumorigenesis ,79. AlthPhytochemicals, particularly polyphenols, are among the most diverse and extensively researched classes of naturally occurring substances. This class includes subgroups of flavonoids and nonflavonoids. Flavonoids, also known as polyphenolic chemicals, are one of the most distinctive families of substances involved in plant metabolism and comprise an extensive group of natural compounds . The subDue to the existence of polyphenolic constituents in tea, several epidemiological research studies have demonstrated a potent reduction in the prevalence of carcinoma among those who regularly consume tea . Green tResearch on breast carcinoma has demonstrated that EGCG can block Wnt/β-catenin signaling without altering the expression levels of β-catenin by activating a transcriptional repressor called HBP-1. This has been demonstrated to be mediated by increased HBP1 mRNA stability ,99. ChenIt has been demonstrated that β-catenin protein (intracellular) stability is controlled by two APC-dependent mechanisms. The (APC/Axin/CK1/GSK-3β)-mediated pathway is the first pathway, followed by the (APC/Siah-1)-dependent pathway. Investigations further revealed a later mechanism that is not needed for EGCG-mediated β-catenin destruction . AccordiGenista tinctoria L. plant and is soluble in different polar solvents. As previously mentioned, foodstuffs containing a soy base are the principal source of genistein , can be found in food either in its free or esterified form. It has long been known that using soy products is associated with a lower chance of developing cancer. This is largely because soy products contain high genistein levels. This substance is found in enistein . Althougenistein ,112. Morenistein . Genisteenistein ,115.Studies have demonstrated that genistein inhibits Wnt signaling, which is linked to a decrease in pre-neoplastic lesions in the colon of male Sprague Dawley rats. Moreover, genistein administration suppresses the level of Wnt key elements, including Cyclin D1, c-Myc Wnt5a, Sfrp1, Sfrp2, and Sfrp5 ,117. SubResearch using RT-PCR analysis has shown that genistein has anti-colorectal cancer properties mediated by the Wnt signaling pathway . In SW11Quercetin, a plant flavonol derived from the polyphenol family, is a beneficial, readily available, and extremely potent natural chemical. It is abundant in fruits, vegetables, leaves, and other plants. Quercetin is employed to treat a wide range of ailments, such as malignancies, diabetes, and coronary heart diseases. Numerous studies have examined the antitumor effects of quercetin on cancer progression through signal transduction pathways, including PI3K/protein kinase B (AKT), Wnt/β-catenin, Janus kinase (JAK), signal transducer and transcription activator (STAT), NF-kB, and mitogen-activated protein kinase (MAPK) signaling cascades . NumerouIn a different study, Park et al. proposed that quercetin is a potent inhibitor of β-catenin/Tcf signaling in colon cancer SW480 cell lines and that decreased β-catenin/Tcf transcriptional ability is a result of reduced β-catenin (nuclear) and Tcf-4 proteins . In a reScutellaria baicalensis. It has received a great deal of interest owing to its potential to inhibit cellular growth and apoptotic induction. Moreover, baicalein inhibits tumor growth by altering several cell signaling pathways, including p-Akt, p-mTOR, p-IB, and NF-kB [Baicalein  is a member of active flavonoids that is mostly present in the dried roots of the medicinal herb nd NF-kB . In an ond NF-kB . Baicalend NF-kB . Osteocynd NF-kB ,133. Accnd NF-kB . It has nd NF-kB . Anothernd NF-kB .Scutellaria baicalensis, baicalin , also demonstrates antitumor properties by targeting the Wnt/β-catenin signaling pathway in a few studies. In human osteosarcoma cell lines, baicalin has been demonstrated to activate apoptosis and autophagy by inhibiting the β-catenin signaling pathways [Furthermore, another important flavonoid of pathways . Anotherpathways .Silibinin (flavonolignan) is a well-known natural dietary supplement extracted from milk thistle seed and has demonstrated biological activity against a range of malignancies via pleiotropic mechanisms . ExtensiApigenin  is a natural flavonoid abundant in many fruits and vegetables.The biological and pharmacological aspects of apigenin have been studied for many years. A growing body of studies have revealed that apigenin can modify the expression of important signaling pathways implicated in the carcinogenesis process, thereby inducing apoptosis . Recent According to Xu et al., apigenin reduced colorectal cancer cellular growth, migration, metastasis, and organoid development by impeding the Wnt/-catenin signaling pathway. Apigenin suppressed the stimulation of β-catenin/TCF/LEF signal by repressing the nuclear movement of β-catenin, which was increased by LiCl in a dose-responsive manner .A long noncoding RNA, H19, which is typically increased in HCC, is known to play a critical role in promoting carcinogenesis and cancer development. Apigenin was found to downregulate H19 in a mouse model of xenograft tumors, which resulted in the attenuation of canonical Wnt/β-catenin signaling and tumor development . FurtherLuteolin  is a flavonoid that is present in different plants, medicinal herbs, fruits, and vegetables. It functions as an antitumor agent against different forms of human malignancies, such as glioblastoma, pancreatic, prostate, breast, and colon cancers. Moreover, it prevents the growth of cancer cells both in vitro and in vivo by preventing the proliferation of tumor cells, shielding them from carcinogenic stimuli, activating cell cycle arrest, and causing cell death via various signaling pathways . In a 20Naringenin  is a prominent bioactive compound primarily found in citrus fruits, such grapefruits and other fruits, as well as in medicinal herbs. It belongs to the flavonoid class of polyphenols. As a herbal remedy, naringenin possesses significant pharmacological properties, including antioxidant, anti-inflammatory, neuroprotective, hepatoprotective, and anticancer activities, as per currently available reports. In vitro and in vivo investigations have demonstrated that carcinogens are rendered inactive after exposure to naringenin (pure), naringenin-loaded nanoparticles, or naringenin combined with chemotherapeutic drugs in a variety of malignancies. Naringenin suppresses the development of cancer through a variety of mechanisms, including pro-apoptosis, cell cycle arrest, inhibition of invasion, and modulation of several signaling pathways, including the Wnt/β-catenin, NF-kB, PI3K/Akt, and TGF-β pathways . In gastFisetin  has recently been identified as a Wnt/β-catenin signaling inhibitor . FisetinNatural bioactive substances obtained from plants have been used to treat various human health problems for a long time. Currently, they are a key source of drug discovery for the creation of contemporary medications. Despite the intriguing biological properties of many natural compounds, most of them cannot be used efficiently in therapeutic medications because of their intrinsic flaws, including low solubility, structural instability, short half-lives, poor bioavailability, and non-specific organ delivery. Furthermore, the use of these phytochemicals is further constrained by their poor pharmacokinetic profile, which includes rapid metabolism and short half-life in the body. To enhance their pharmacokinetic profile, derivatization of these phytochemicals is necessary. An ideal derivative is one that is comparable to or more effective than the original prototype in terms of solubility, bioavailability, tumor invasion, and dispersion. The development of new delivery methods, including nanotechnology, has led to several encouraging improvements in the pharmacokinetics of pharmaceuticals with limited solubility and low bioavailability and, in the case of phytochemicals, can dramatically overcome pharmacokinetic limitations. In conclusion, the Wnt/β-catenin signaling pathway is crucial for cancer growth and progression. Wnt communicates extensively with other signaling pathways, such as the NF-κB, JAK/STAT, and Notch pathways, which provide cancer cells with a survival advantage, thereby making it crucial to target this pathway. Moreover, Wnt/β-catenin activation is crucial for maintaining CSCs that are highly tumorigenic and treatment-resistant and contribute to the survival and recurrence of tumors. Many natural compounds have been discovered to be Wnt/β-catenin signaling modulators over the past 10 years, and the majority of research studies have focused on examining the effectiveness of these modulators as cancer prevention and/or treatment agents. Further research on the impact of flavonoids, either alone or in conjunction with chemotherapeutics, on the modulation of Wnt/β-catenin signaling will advance our knowledge of this signaling cascade and enhance tumor prevention and treatment.


The red palm weevil poses a significant threat to palm species, resulting in substantial economic losses. While multiple methods have been developed to control its infestations, there is an urgent need for eco-friendly insecticides that selectively target its critical systems or pathways. One such target is its digestive system, which is essential for its survival. This review highlights the potential of using the digestive system of the red palm weevil to manage its infestations. Proteomic and transcriptomic data analyses on the weevils have provided a better understanding of the protein and gene compositions in its digestive system. With technological advancements, a more comprehensive approach can be taken to explore the opportunities in manipulating the data on the digestive system of red palm weevil, leading to improved management methods.Rhynchophorus ferrugineus, commonly known as red palm weevil (RPW), is a high-risk insect pest that has become a threat to many important palm species. There are several dominant factors that lead to the successful infestation of RPW, including its stealthy lifestyle, highly chitinized mouthpart, and high fecundity rate. Due to that, millions of dollars of losses have been suffered by many countries invaded by RPW. Several methods have been designed to control its invasion, including the usage of insecticides, but many cause resistance and environmental pollution. Therefore, an environmentally friendly insecticide that targets specific systems or pathways in RPW is urgently needed. One of the potential targets is the digestive system of RPW, as it is the major interface between the insect and its plant host. The related knowledge of RPW’s digestive system, such as the anatomy, microflora, transcriptomic analysis, and proteomic analysis, is important to understand its effects on RPW’s survival. Several data from different omics regarding the digestive systems of RPW have been published in separate reports. Some of the potential targets have been reported to be inhibited by certain potential insecticides, while other targets have not yet been tested with any inhibitors. Hence, this review may lead to a better understanding on managing infestations of RPW using the system biology approach for its digestive system. Rhynchophorus ferrugineus (Coleoptera: Curculionidae), commonly known as the red palm weevil (RPW) (Olivier 1790), is a Coleopteran insect that has been classified as a significantly serious pest on the A2 list , according to the European and Mediterranean Plant Protection Organization (EPPO) . Th. Th84]. nhibitor .Anthonomus grandis) using RNAi recombinant transgenic cotton resulted in malformed first and third instar larvae. The report suggested that the knockdown of chitin synthase led to the obstruction of nutrient uptake in the gut [On the other hand, chitin synthase EC: 2.4.1.16) catalyzes the polymerization of the chitin polymer , a major.16 catal the gut .In another study, the expression profiling of RPW’s gut showed the identification of key enzymes in the digestion of plant cell walls , namely Myzus persicae, resulting in a high mortality rate. This study also highlighted that the insecticide tested was not harmful towards beneficial insect Chrysoperla carnea [Other than that, potentially targeted genes encoded for neuropeptide precursors and their receptors  were foua carnea .Hence, the gut transcriptome analysis of RPW shows that it can offer valuable insights into cellular processes that occur in the gut. The identification of those genes and neuropeptides is necessary for the development of potential insecticides to effectively manage and prevent the invasion of RPW in palm trees ,88,92.Having a comprehensive knowledge regarding the potential targets in the digestive system of RPW is helpful in developing and discovering new potential strategies in managing the RPW pest.Although certain potential targets have been found to be responsive to insecticides or inhibitors , severalAccording to Zulkifli et al. , among tCalotropis latex extract PIs were effective in inhibiting and reducing the activity of digestive protease enzymes in the gut of RPW by 39% and 18%, respectively, resulting in a decrease in the survival rate of the insect. This suggests that PIs obtained from plant extracts can serve as a valuable bioactive source for the development of biopesticides, essential for maintaining an ecological balance in the management of RPW insect pest infestations.The discoveries of selective inhibitors, such as specific inhibitors targeting the trypsin and peptidase enzymes, which are crucial components of insecticides, can result in the disruption of digestion in the target insect. Consequently, this can impede the insect’s growth and eventually lead to the mortality of the pest weevil. This corresponds with the findings by Mohamed et al. , who empAnother way of controlling RPW is by targeting the specific biosynthesis pathway involved in the digestive system of RPW. One of the potential target pathways for pest control in RPW is the chitin biosynthesis pathway . As mentStudying RPW’s digestive systems is crucial in agriculture, especially because of the detrimental impact of RPW as insect pests. With the advent of high-throughput analysis in metagenomics, proteomics and transcriptomics, the investigation of insects’ digestive systems has progressed to the systems’ biology level, encompassing the whole system rather than individual components. This approach offers insights into the dynamic nature of the digestive system and creates opportunities for further exploration .The ability of phytophagous insects to survive is largely dependent on their capacity to effectively consume their respective host plant. As for RPW, its ability to consume and digest the host plant was reported to be aided by its insect-associated microbial communities in the gut ,36. IdenAdditionally, gaining knowledge on the digestive enzymes that RPW produces when it infests different host plants can provide a way to learn how the insect picks a specific type of plant . This knAs part of RPW’s digestive system is lined with chitin, targeting the chitin biosynthesis pathway is also an opportunity for controlling and managing the pest . ChlorflThe review showed that there are many opportunities to explore and study the digestive system of RPW as a means for managing and controlling the infestation and spread of RPW as an insect pest. Metagenomic, proteomic and transcriptomic data were shown to be suitable approaches to further understanding the protein and gene constituents of RPW. However, with the advancement of technologies, the detection limit, the data generated and the tools for analysis no longer limit deeper exploration at the systemic level, opening the doors for gaining more knowledge on the digestive system of RPW. Hence, a more holistic approach could be used to produce a better way to manage RPW.