Caenorhabditis elegans development. By combining early studies with recent findings, the review is aimed to serve as an updated reference for the roles of Axin in C. elegans and other model systems.Scaffold proteins serve important roles in cellular signaling by integrating inputs from multiple signaling molecules to regulate downstream effectors that, in turn, carry out specific biological functions. One such protein, Axin, represents a major evolutionarily conserved scaffold protein in metazoans that participates in the WNT pathway and other pathways to regulate diverse cellular processes. This review summarizes the vast amount of literature on the regulation and functions of the Axin family of genes in eukaryotes, with a specific focus on  PreAxin1 mutations in many of the cell lines and a corresponding increase in the DNA-binding activities of TCF/LEF (T-cell factor/Lymphoid enhancer-binding factor 1) and β-catenin [Axin1 mutation was independent of WNT-β-catenin signaling [Cell proliferation is another process where Axin’s involvement has been investigated in considerable detail. The findings have shown that, as a negative regulator of the WNT-β-catenin signaling, Axin functions to inhibit cancerous growth and appears to act as a tumor suppressor . Altered-catenin . Interes-catenin . Moreoveignaling ,55. Furtignaling .Among its other roles, Axin appears to be necessary for maintaining cell survival, metabolic homeostasis, and thymic adipogenesis. Overexpression studies in transgenic mice and certain cultured cells demonstrated increased apoptosis, possibly due to activation of the cell-death pathway ,57. AxinDrosophila melanogaster Axin (D-Axin) homolog is necessary for the development of embryos and organs, such as the wings, eyes, heart, gut, and circulatory system [Tribolium castaneum Axin homolog (Tc-Axin) revealed its dynamic expression during embryogenesis. Tc-Axin was initially localized at the anterior pole, extending posteriorly during subsequent development and eventually becoming somewhat ubiquitous [C. elegans, two divergent Axin-like proteins, specifically PRY-1 (poly ray 1) and AXL-1 (Axin-like 1) have been identified. The roles of both these family members are discussed in a separate section.Studies in invertebrates have been instrumental in uncovering the developmental roles of Axin homologs in tissues and organs, in the context of intact animals. The y system ,60,61,62iquitous . This exIn summary, the roles of Axin family of proteins described above rely on its scaffolding properties that are mediated by conserved domains facilitating interactions with WNT-β-catenin pathway components . AdditioGiven that Axins play essential roles in metazoan development, it is not surprising that Axin family members form complexes with various cellular factors and components of signal transduction pathways A. While C. elegans have shown the presence of a divergent asymmetry pathway that regulates nuclear factor POP-1 in a WRM-1 (worm armadillo 1)/β-catenin-LIT-1 (loss of intestine 1)/NLK (Nemo-like kinase)-dependent manner . In theAxin family members also cooperate with an increasing number of proteins in other, non-WNT, processes. These interactions involve pathways such as JNK, TGF-β, p53, and AMPK. Tissue culture experiments involving kidney 293T cells and embryonic fibroblast cells showed that Axin binds to MEKK1 and MEKK4, two members of the MEKK (MAPK (Mitogen-activated protein kinase)/ERK  kinase kinase) family, through domains distinct from those involved in WNT signaling and activates the MKK4- and MKK7-  mediated JNK cascade ,68. ThisAxin’s function in TGF-β pathway involves regulation of transcription factor Smad3 activity to affect gene transcription. In human MSCs , Axin and GSK-3β physically interact to facilitate Smad3 phosphorylation by the active TβRI (TGF-β type-I receptor) kinase (reviewed in ). This iWhile the tumor-suppressor role of Axin has been traditionally investigated in the context of WNT-β-catenin signaling, some studies have demonstrated its interactions with p53, a DNA-binding protein that responds to genotoxic stress and controls cell proliferation and cancerous growth. During p53 signaling, Axin interacts with HIPK2 (homeodomain-interacting protein kinase-2) to facilitate p53 phosphorylation, which stimulates p53-dependent transcription of target genes . SubsequDrosophila, D-Axin was reported to physically interact with a component of the glucose-transport regulatory complex, DCAP (Drosophila catabolite activator protein), to increase glycogen utilization through insulin signaling and glucose transport [Drosophila Pygo2 (Pygopus) homolog to affect glucose metabolism [Research on Axin has also uncovered its role in controlling cellular energy, nutrient sensing, and metabolic processes. One of these processes involves glucose homeostasis. In ransport ,78. Addiransport . In an utabolism . Anothertabolism . Becausetabolism .Beyond their other functions, Axins also form complex with Dvl to facilitate cytoskeletal rearrangement during gastrulation (Reviewed in ) and oriC. elegans systems .In summary, Axin participates in both WNT-dependent and -independent signaling events A. By actThe crucial roles of Axin family members in metazoans depend on multiple mechanisms that operate at spatiotemporal and subcellular levels. Several reports have shown that Axin is regulated both at transcriptional and post-translational levels. Using an auto-feedback loop, Axin controls its own expression ,84,85,86The post-translational modifications of Axin include phosphorylation, ubiquitination, and SUMOylation. These alterations affect the subcellular localization, stability, or potential interactions of the protein with other factors . SimDrosophila system [Drosophila homolog, Iduna , acts as a key factor in the breakdown of ADP-ribosylated Axin [Axin is subjected to ubiquitin-mediated proteolysis through poly ADP-ribose modification. Experiments in cell culture systems showed that poly ADP-ribosylation of Axin by ADP-ribose polymerase enzymes, tankyrase 1 and tankyrase 2, targeted Axin for proteasomal degradation when the pathway was in the ‘off’ state ,102,103.a system ,105. Fena system  used humted Axin ,107. Othted Axin ,109.Axin is also known to be SUMOylated. The earliest role of SUMOylation in Axin regulation was revealed by the discovery of Axam (Axin associated molecule), an enzyme that possesses deSUMOylation activity ,111. AxaAdding to its complex mode of regulation, Axin is proposed to utilize an autoinhibitory mechanism. The N-terminus region of the protein was earlier suggested to play an inhibitory role in binding to its partner proteins ,114 and Drosophila system showed that Axin was negatively regulated by miR-315 via conserved 3′- UTR (untranslated region) miRNA consensus sequences [Axin2 transcript was targeted by let-7f at the 3′ UTR, by hsa-miR-34a at both the 5′ UTR and 3′ UTR, and by miR-205 at the 3′ UTR, which regulated expression of a WNT/β-catenin target gene and a β-catenin-activated reporter [Yet another mechanism by which Axin function is modulated involves miRNA (microRNA)-mediated gene silencing. Experiments in the equences . Likewisreporter ,118,119.The findings summarized above show that Axin is subjected to multiple modes of regulation. Although the full picture of its regulatory mechanism is far from complete, it is evident that alterations help modulate Axin’s function and its interactions with other cellular factors and signaling pathway components.C. elegans genome encodes two Axin family members, PRY-1 and AXL-1. Although both proteins act as scaffolds to recruit other factors, major domains (RGS and DIX) are not well conserved /β-catenin and pop-1 (posterior pharynx defect 1)/TCF/LEF and downstream of egl-20 (egg laying defective 20)/WNT and mig-5 /Dvl, thus establishing it as a core component of the canonical WNT signaling pathway in C. elegans , seam cells (V5 and V6), ventral hypodermal (P) cells (P7/8 to P11/12), body-wall muscle cells, and neurons in the head, tail, and ventral nerve cord. In addition, pry-1 continues to be expressed in all seam cells and QL/R cells through the late-L1 stage. At later stages, pry-1 expression persists in hypodermal cells and several neurons in the ventral cord, head, and tail ganglia [pry-1 expression is also observed in reproductive tissues, including vulval precursors and their progeny, as well as the male tail. A similar expression pattern of pry-1 ortholog, Cbr-pry-1, was also seen in Caenorhabditis briggsae, a sister species of C. elegans [C. elegans that are discussed below.The genetic epistasis experiments confirmed that, similar to mammalian Axin, iewed in ). Additiiewed in . Consistogenesis . At the  ganglia . Further elegans . In agreC. elegans Axin-like protein, AXL-1, also acts as a functional ortholog of Axin to regulate the canonical WNT signaling [The other ignaling . The proignaling   . In addi elegans .Below, we describe the major developmental events and tissues that depend on PRY-1 and AXL-1 function and their interactions with other cellular factors.C. elegans, pry-1 mutants exhibit defects in some of their neurons . These pry-1-mediated WNT signaling is essential for the migration of Q-lineage cells and guiding them along specific trajectories [mab-5 /Hox (homeobox) expression and to enable anterior migration of their descendants. Loss of PRY-1 function mimics constitutively active WNT pathways with high MAB-5 expression in the QR cell and their progeny, resulting in their migration in an opposite (posterior) direction [Molecular genetic studies have shown that ectories . PRY-1 airection  -hypersensitive strain [pry-1 RNAi caused defects in ventral cord neurons, such as branched commissures and abnormal midline crossing. In a separate study, Schneider et al. [pry-1 acts in a canonical WNT-β-catenin pathway to promote synapse formation of a specific motor neuron, based on results showing that pry-1 mutants enhanced movement defects in animals lacking unc-4 (uncoordinated 4)/Hox function.Other neuronal processes in which roles for e strain . It was r et al.  reportedpry-1 may also participate in neuroprotection in adults. This possibility is supported by our findings that pry-1 mutants show accelerated degeneration of dopaminergic neurons   [pry-1 mutant worms   C. Whetheiewed in ,130). On factor) , was sigblished) D. In theC. elegans is another process that depends on the pry-1-mediated, non-canonical WNT signaling pathway. This divergent pathway, known as the WNT-β-catenin-asymmetry pathway, has been shown to control the division of several different types of somatic cell, such as EMS blastomeres in the embryo and larval seam cells (reviewed in [Embryogenesis in iewed in ). Duringiewed in ,133 /Frizzled (Fz), DSH-2, and MIG-5 in the posterior cortex ,135, andSimilar to the EMS, the PRY-1-mediated WNT-asymmetry pathway is also essential for seam cell development (reviewed in ) FigureB. Seam cpry-1 mutation disrupts the nuclear localization of WRM-1, SYS-1, and POP-1 [wrm-1 and lit-1 suppressed the seam cell phenotype in pry-1 mutants whereas pop-1 RNAi exacerbated the defect.In support of its role in the asymmetric division of seam cells, nd POP-1 ,134,149,nd POP-1 ,66. Workpry-1 mutants, which revealed five DE  miRNAs of lin-4 (lineage defective 4) and let-7  families. Further experiments revealed that all DE miRNAs were repressed by PRY-1 in a POP-1-dependent manner [The temporal division pattern of seam cells relies on several heterochronic miRNAs and their target genes  generate rays whereas the V1–V4 lineages (with no mab-5 expression) give rise to alae [pry-1 function show defective alae and ectopic rays as the V cells can now differentiate to make more rays due to the inappropriate expression of mab-5 [mab-5 expression in pry-1 mutants inhibits the formation of the postdeirid, a sensory structure resulting from the differentiation of V5.pa descendants [In addition, stdeirid . Thus, i to alae ,153. Malof mab-5   and secondary (20 - P5.p and P7.p) cell fates. Mutations in pry-1 cause more than three VPCs to get induced and lead to the formation of ectopic pseudo-vulvae-like structures in adults, which is referred to as the multivulva (Muv) phenotype [pry-1 function leads to the constitutive activation of WNT signaling and the dysregulation of downstream targets. One such target is the homeobox family member, lin-39 (lineage defective 39), which is necessary for pry-1-mediated vulval development [Although ormation A. C. eleiewed in ). As a rhenotype   and showed that Cbr-pry-1 functions in vulva development in a Cbr-bar-1/β-catenin-, Cbr-pop-1/tcf/lef-, and Cbr-lin-39/hox-dependent manner [Pristionchus pacificus (Diplogastridae family), uncovered an Axin family member that is most closely related to C. elegans axl-1 [Ppa-axl-1 caused a Muv phenotype suggesting that the gene plays an important role in vulva development. Further experiments revealed that Ppa-axl-1 genetically interacts with WNT-β–catenin pathway components.Axin family members have also been genetically characterized in other nematodes. Our laboratory identified mutations in the t manner  /MAPK phosphatase [Experiments aimed at understanding the mechanism of n et al.  found thormation C. Subseqsphatase . How thepry-1-mediated signaling for proper differentiation of their progeny. These two cell lineages contribute to the formation of the ventral nervous system [pry-1-mutant animals, P11.p appears to adopt a P12.pa-like fate, based on the presence of two cells having the characteristics of P12.pa [bar-1 exhibit an opposite phenotype, i.e., two P11.p-like cells [P11 and P12 cells also depend on s system . Whereasf P12.pa   and 20 (P10.p) fates through the actions of the WNT, EGFR-Ras, and LIN-12 pathways. The P10.p and P11.p progeny differentiate to form a functional hook that includes sensory neurons, structural cell, and support cells. It was found that pry-1 mutants have ectopic hook-like structures due to inappropriate induction of some of the anterior Pn.p (n = 3–8) cells that normally fuse with the surrounding hypodermal syncytium [bar-1 and a downstream target (mab-5). Additional genetic experiments led the authors [0 fate. The neighboring P10.p cell receives a comparatively lower signal and thereby adopts a 20 fate.The role of g mating . Cell-mog mating . These tyncytium   serve as building blocks for lipids. Fatty acids are synthesized from thioesters or isoprene units via condensation reactions  and fats (fatty acid desaturases) participate in pry-1-mediated lipid metabolism. Consistent with these findings, pry-1 mutants exhibited defects in lipid content . We anand aging . Functio content A, egg laC. elegans, MUFAs and PUFAs  can be derived from saturated fatty acid precursors by the actions of fat desaturases  [pry-1 mutants had reduced transcription of fat desaturases and sbp-1. However, transcription of the nhr-49 and nhr-80 genes was unaltered. Thus, a working model is that pry-1 acts via sbp-1 to regulate the expression of fat genes that, in turn, regulate lipid synthesis [pry-1 mutants and supplementing the diet with oleic acid, a MUFA, rescued lipid defects in mutant animals [In d FAT-7) ,160. Thed FAT-7) ). More rd FAT-7) . We founynthesis . This mo animals .C. elegans system, Axin family members are reported to function in lipid biology. Axin expression in mice contributes to an age-related increase in adiposity in thymic stromal cells [The requirements of lipids in many biological processes and across multicellular eukaryotes is well documented. These macronutrients are vital for proper growth and reproduction; however, their excessive intake contributes to a variety of diseases in humans. Changes in lipid metabolism could affect a host of processes including signaling, cell structure, and aging . Outsideal cells . A recenal cells . TherefoAs described in the previous section, PRY-1 and AXL-1 show significant sequence divergence in GSK-3β and β-catenin binding domains. In spite of this, both of these proteins physically interact with BAR-1 and regulate WNT-β-catenin signaling. The functional conservation may be brought about by conserved structures of RGS and DIX domains, thereby making them bona fide Axin family members.C. elegans Axins participate in developmental processes such as cell proliferation, cell differentiation and cell migration. The examples include the formation of the vulva and male hook, P11/12 fate specification, and neuronal development. All of these involve interactions with WNT-β-catenin pathway components, suggesting that this might be the most ancestral mechanism of Axin function. Among other roles of PRY-1, its involvement in lipid metabolism is a recent discovery that may potentially fit into a broader role of Axin as an energy sensor and regulator in eukaryotes.Similar to other eukaryotes, C. elegans have also uncovered a unique Axin-mediated WNT signaling during embryogenesis and seam cell development. Typically, interactions between β-catenin and TCF lead to transcriptional regulation of the target genes. However, this divergent WNT signaling has evolved to utilize WRM-1/β-catenin to regulate nuclear localization of POP-1/TCF, leading to the specification of asymmetric cell fates. Thus, research in the C. elegans model has shed a new light on the novel mechanism of Axin function in eukaryotes.In addition to their conserved roles, studies in C. elegans, Axin’s role has been investigated during embryogenesis and larval development. Given the extensive conservation of genes and signaling mechanisms in C. elegans, future studies on PRY-1 and AXL-1 hold the potential to further advance our understanding of shared mechanisms of Axin function in animal development. By harnessing the power of C. elegans genetics, new and novel interacting partners of Axin could be identified. Additionally, the discovery of Axin-target genes could help unravel pathways and process-specific functions associated with this family of proteins.Axin is a well-characterized scaffold protein that is conserved in eukaryotes. While vertebrate genomes carry two Axin genes, a single ancestral family member is found in invertebrates. Axin’s function has been studied mainly in the context of WNT-β-catenin signaling; however, the protein also interacts with multiple factors belonging to WNT-independent signal transduction pathways. Over the years, studies have shown that Axin homologs are necessary for the development of various tissues and cell types. In


Its values ranged from 137 up to 108 °C after adding the phenolic-based components. A cone calorimeter was used to evaluate the burning behavior of the formulated thermosets. A significant reduction of the peak of heat release rate (pHRR) for combustion was detected. Indeed, with 10 wt.% of GA and EA, pHRR was reduced by 12 and 44%, respectively, compared to that for neat epoxy resin. GAD and EAD also induced the decrease of pHRR values by 65 and 33%, respectively. In addition, a barrier effect was observed for the resin containing GAD. These results show the important influence of the biobased phenolic compounds and their boron derivatives on the fire behavior of a partially biobased epoxy resin.The aim of this work was an investigation of the ability of gallic (GA) and ellagic (EA) acids, which are phenolic compounds encountered in various plants, to act as flame retardants (FRs) for epoxy resins. In order to improve their fireproofing properties, GA and EA were treated with boric acid  and ellagic acid derivatives (EAD)) to introduce borate ester moieties. Thermogravimetric analysis (TGA) highlighted the good charring ability of GA and EA, which was enhanced by boration. The grafting of borate groups was also shown to increase the thermal stability of GA and EA that goes up respectively from 269 to 528 °C and from 496 to 628 °C. The phenolic-based components were then incorporated into an epoxy resin formulated from diglycidyl ether of bisphenol A (DGEBA) and isophorone diamine (IPDA) . According to differential scanning calorimetry (DSC), the glass transition temperature (T Due to their intrinsic characteristics, such as mechanical and electrical insulating properties, chemical resistance under both acidic and basic conditions, and adhesive properties, epoxy resins are widely used for structural applications (in association with glass or carbon fibers), as well as in the coating, electronic, and adhesive industries . HoweverAmong others, halogenated FRs have been extensively used ,3. But tTannins are a group of phenolic biopolymers and macromolecules widely distributed in plant cells ,9. ThereIn the last ten years, a particular focus has been the development of condensed tannins-based FRs ,11,12. T−1) and an increase of the LOI values from 19.0 to 30.2% compared with that of the control cotton. However, more attention should be given to FRs based on hydrolysable tannins. Their aromatic rings and hydroxyl functions available for a chemical modification, as well as their abundance in plant biomass such as chestnut, tara, and oak trees ,26Ellagips of EA ,28. No sBoron derivatives of EA and GA (GAD and EAD). The boration of the phenolic compounds allowed improving their thermal stability. For both GAD and EAD a progressive weight loss was observed from 100 °C up to their main decomposition step. This phenomenon was associated with the dehydroxylation of boron derivatives [max1) for GAD and at 628 °C (Tmax1) for EAD, respectively. It was mainly ascribed to the pyrolysis of carboxylic and lactone groups. The main products related with this process, CO2 and CO, were detected in the off-gasses of the heating sample. No boron- and sodium-containing volatile compounds were detected. Therefore, it may be assumed that boron and sodium atoms mainly remain in the solid phase. In the case of GAD, it is noteworthy that the formation of a sodium carboxylate , total heat release (THR), and theoretical and experimental effective heat of combustion . All the results obtained by the PCFC analyses were in accordance with the TGA measurements  and their boron derivatives (GAD and EAD) were characterized by pyrolysis/combustion flow calorimetry (PCFC). The heat release rate (HRR) curves of the FR systems are shown in urements .−1 and the THR equal to 12.4 kJ g−1, respectively, while EA shows considerably lower pHRR and THR values for an organic molecule . As is shown in theo and EHCexp values—14.8 and 15.9 kJ g−1, respectively. Moreover, the ratio THR/EHCtheo is approximately 80%, indicating that the THR decrease is almost proportional to the char yield (circa 20%). As for EA, its EHCexp value is much lower than its EHCtheo—4.5 and 16.0 kJ g-1, respectively. The ratio THR/EHCtheo is 18%, meaning that the THR decrease is much higher that the char yield. However, thermochemistry calculations do not take into account the energy stored in the char. To correct this, the EHCtheo should be calculated again by considering the char composed only of carbon . It was observed that GA is the most exothermic compound with the pHRR equal to 293 W gz et al. . The EHCthe char .−1, respectively, while the THR values were equal to 0.3 kJ g−1 for GAD and 0.5 kJ g−1 for EAD, respectively. In addition, GAD and EAD showed a decrease of the EHCexp values compared to the EHCexp of GA and EA—0.6 and 1.0 kJ g−1, respectively. The EHCtheo values of GAD and EAD cannot be calculated because of their non-uniform chemical structure. That is why the EHCtheo and EHCexp values of GAD and EAD cannot be compared.The PCFC measurements also confirmed the improvement of GA and EA thermal characteristics by their functionalization. In fact, GAD and EAD exhibited the pHRR equal to 10 and 25 W gThe adhesion between the epoxy matrix and the FR additives as well as the influence of additives on epoxy crosslinking are key factors that may affect the thermal and mechanical properties of FR/epoxy systems. Therefore, dispersion within epoxy thermoset was studied through SEM observations, and differential scanning calorimetry (DSC) experiments were carried on.The SEM observations of the formulated epoxy thermosets are illustrated in Non-uniform dispersion of the pure phenolic components into the epoxy matrix was clearly observed. The agglomeration process of acicular particles of GA and spherical particles of EA into DGEBA/IPDA/GA and DGEBA/IPDA/EA thermosets, respectively, was noticed. This can be explained by (1) mixing parameters such as speed and time of dispersion, (2) some adhesion forces  holding the particles together , and (3)A chemical treatment is usually able to prevent such an agglomeration by changing interfacial bondings with the epoxy matrix . NeverthDSC analyses were performed on the non-cured epoxy resins to observe the influence of FR systems on epoxy crosslinking. The glass transition temperature was determined after the resins were cured according to the procedure described in −1, the neat epoxy resin showed an ideal exothermic peak (Tp) at 109 °C. Its total heat of reaction (ΔH) was equal to 431 J g−1 corresponding to 96 kJ ee−1. The cured DGEBA/IPDA thermoset exhibited a glass transition temperature (Tg) equal to 144 °C. These results are consistent with those previously reported [During a first heating at 10 °C minreported .p values of these resins were slightly reduced compared to the neat resin and ranged between 105 and 108 °C. The lowest and the highest values correspond to DGEBA/IPDA/GAD and DGEBA/IPDA/EAD thermosets, respectively. The ΔH values calculated by gram of material were logically reduced by about 10% with the incorporation of 10 wt.% of the additives compared to the neat thermoset. The enthalpies of reaction calculated by epoxy equivalent (ee) were quite close to that of the neat resin except for DGEBA/IPDA/EA. It allows concluding that GA, GAD, and EAD do not influence the cure kinetic of the epoxy resin. On the contrary, the use of EA as additive affects thermal properties of the epoxy thermoset. An exothermic peak at 99 °C with a shoulder between 120 and 150 °C was observed for the DGEBA/IPDA/EA thermoset  as already observed by the SEM analyses  and their derivatives (EAD and GAD) on the thermal stability and charring ability of DGEBA/IPDA resin. TGA curves under nitrogen are illustrated in max was reduced from 370 °C for DGEBA/IPDA, to 349 and 338 °C for DGEBA/IPDA/GA and DGEBA/IPDA/EA, respectively. Two phenomena could be responsible for this reduced thermal stability. The first one is the early stage of decomposition of the pure phenolic compounds. The effect is particularly noticeable for the GA-containing thermoset. The onset of weight loss at about 200 °C corresponds to the beginning of thermal decomposition of the pure phenolic additive  and experimental char yields at 900 °C of DGEBA/IPDA/GA thermoset were almost the same - 10.7 and 10.9 wt.%, respectively. Whereas the theoretical char900 value of DGEBA/IPDA/EA was lower than the experimental one—12.0 against 18.6 wt.%, respectively, it could be explained by some reactions between the particles of EA and the epoxy matrix . The formed products seem to promote the charring ability of the thermoset.The GA- and EA-containing epoxy thermosets showed a premature onset of decomposition compared to the unfilled epoxy resin. The Tadditive . A seconmax values of DGEBA/IPDA/GAD and DGEBA/IPDA/EAD were equal to 367 and 371 °C, respectively. It is assumed that the pro-degradant effect of hydroxyl groups from GA and EA was screened after functionalization. An increase of the char yield up to 18.0 wt.% was observed for both thermosets. The theoretical char yields at 900 °C of the epoxy resins were close to the experimental ones: 14.1 against 15.6 wt.% for DGEBA/IPDA/GAD and 14.1 against 15.8 wt.% for DGEBA/IPDA/EAD, respectively. These results allowed considering that GAD and EAD do not promote additional charring of the DGEBA/IPDA matrix.Concerning the GAD- and EAD-containing epoxy thermosets, they exhibited similar thermal stability to the neat epoxy resin. The T−1), CAr—O groups of phenol (1260 and 1176 cm−1), CAr=CAr (1603 and 1507 cm−1), CAr–H , and OH–phenolic groups of bisphenol A (3649 cm−1). The decomposition products as CO2 (2379 and 2311 cm−1), CO (2180 and 2107 cm−1), ammonia NH3 (965 and 931 cm−1), methane CH4 (3018 cm−1), and free H2O (3756 and 1595 cm−1) were also detected.The off-gases of the DGEBA/IPDA thermosets analyzed by FTIR correspond to those previously reported . The FTIThermogravimetric analysis can be completed by the study of the fire behavior of the formulated FR epoxy thermosets using PCFC and cone calorimeter measurements. −1 and 28.4 kJ g−1, respectively. Similar results were previously reported [−1. Regarding EA containing epoxy, this corresponds to a 10% reduction in good agreement with an additive mixing law. On the opposite, the DGEBA/IPDA/GA resin exhibits a 45% pHRR reduction, i.e., much higher than expected from the mixing law. It allows suggesting that this pure phenolic compound may influence the combustion pathway of the epoxy thermoset. The THR values of the GA- and EA-containing resins were reduced by about 10% compared to the uncharged epoxy thermoset and were equal to 25.8 and 25.9 kJ g−1, respectively. No significant variation was observed for the EHCexp values of the formulated thermosets. They were equal to 29.3 and 32.3 kJ g−1 for the GA- and EA-containing thermosets, respectively, compared to 31.8 kJ g−1 of the neat epoxy resin.The neat epoxy resin shows high pHRR and THR values during PCFC test—525 W greported . A decreexp values of DGEBA/IPDA/GAD and DGEBA/IPDA/EAD were decreased in proportions compliant with the mixing rule. Hence, no specific interactions with the polymer matrix were highlighted by this method.GAD and EAD led to the improvement of the fire behavior of the epoxy resin. The pHRR, THR, and EHCIt is well known that the PCFC test is not sensitive to physical effects while both chemical and physical actions are effective in cone calorimetry. Therefore, the fire behavior of the epoxy resins was also investigated using a cone calorimeter. It provides key information about flame retardant properties of materials such as time to ignition (TTI), HRR, pHRR, THR, and char content. The results of cone calorimeter test are summarized in −2 and 3.9 wt.%, respectively. The addition of the phenolic compounds and their derivatives in DGEBA/IPDA leads to the evolution of its burning behavior. The use of the pure phenolic additives induced a decrease of TTI values from 90 s for DGEBA/IPDA to 66 and 80 s for the GA- and EA-containing epoxy resins, respectively. These variations in TTI values are in good agreement with the lower thermal stability of these compositions as shown by TGA and PCFC. It should be noted that a decrease of TTI was also observed in the presence of the boron derivatives GAD or EAD despite their good thermal stability as observed in TGA. In a general way, the incorporation of 10 wt.% of the phenolic compounds or their derivative in DGEBA/IPDA allows reducing both the pHRR and THR. In the case of DGEBA/IPDA/GA, the pHRR value is reduced only by 12% while for DGEBA/IPDA/EA, the decrease is much more significant (by 44%). The most remarkable effect was obtained with GAD that induces a 65% reduction of pHRR while the results for EAD were a little bit disappointing with only 31% reduction of pHRR. In order to evaluate the influence of the additive dispersion in the epoxy matrix, the sample DGEBA/IPDA/EA was exposed to the incident heat flux by the side with the higher EA content referred to as DGEBA/IPDA/EA*. It was observed that TTI was decreased to 55 s. This result was associated with the early decomposition of this composition as observed by TGA. Nevertheless, the pHRR value was significantly reduced to 492 kW m−2 which corresponds to a decrease by 21 % compared to the DGEBA/IPDA/EA sample. It leads to the conclusion that the non-uniform dispersion of additives in the epoxy matrix may affect the fire behavior of material. A uniform dispersion of the additives in the epoxy matrix should be achieved to ensure a better efficiency as FRs. Apart from the decrease of pHRR, it is noteworthy that the use of phenolic compounds  induces a change in the HRR curve shape. Hence, for these compositions, the pHRR happens rapidly after ignition and then HRR tends to decrease. This behavior is typical of thick charring materials according to the classification of Schartel and Hull [−1, the HRC is equal to the pHRR. The method relies on the assumption that the barrier effect is only operative in the cone calorimeter but not in PCFC where samples are supposed to be at a uniform temperature. Consequently, the occurrence of a barrier effect can be evidenced by a deviation from the R1 = R2 line. As observed in According to the literature  and coneand Hull . It meanand Hull . This meThe barrier effect can also be grasped by observing the cone calorimeter test residues. −1 . The cycloaliphatic cross-linking agent isophorone diamine (IPDA) .Ellagic acid labeled EA (purity 97%) was purchased . Gallic acid labeled GA (purity 97.5%) was supplied . Boric acid labeled BA (≥99%) was provided . Sodium hydroxide abbreviated by SH (≥99%) was supplied. The conventional petroleum-sourced epoxy monomer used was diglycidyl ether of bisphenol A (DGEBA) a and wase (IPDA) b was supA two-step procedure was applied.The first step consisted in the preparation of BA and SH solution. Fifty milliliters of 0.2 M aqueous solution of BA and 21.4 mL of 0.2 M aqueous solution of SH were mixed in a 100 mL beaker. The mixture was stirred vigorously for 5 min at room temperature and then stored at 4 °C until use. The pH of the solution was 9.The second step corresponded to the functionalization. A 25 mL single-necked round-bottomed flask equipped with a magnetic stirring bar was charged with phenolic compound and aqueous solution of BA and SH. The pH of the solution was adjusted to 9 with 0.2 M aqueous solution of SH and then heated for 3 h at 50 °C under vigorous stirring. After that, the solution was freeze-dried over 48 h.The formation of boron complexes of the phenolic compounds was supposed under the conditions described above. The hydroxyl groups of phenolic compounds in the ortho-position help a complex formation with BA . HoweverDifferent chemicals are under liquid forms (DGEBA and IPDA) while other are under solid forms (FR systems). In order to obtain a homogeneous dispersion, GA and EA as well as GAD and EAD were firstly introduced in DGEBA by manual stirring. The prepared mixture was then heated for 15 min at 80 °C. The curing agent–IPDA–was then added in a 1:1 molar ratio of epoxy group to active H of amine. The system was stirred manually for 5 min. The mixture was cross-linked in a silicon mold to the following temperature program: 4 h at 80 °C and 1 h at 150 °C under inert atmosphere.−1; nebulize pressure, 3 bars; gas drying temperature, 200 °C; capillary voltage, 5000 V; fragmentor voltage, 1 V.Mass spectrometry (MS) analyses were performed on a Ion trap mass spectrometer with an electrospray ionization (ESI) interface  operating in negative ion mode. The samples of additives were dissolved in methanol and then filtered through a polymer membrane with a pore diameter of 0.45 µm. The injection of samples was made by syringe. The following conditions of the ESI interface were used: drying gas flow, 8.0 L min11B solid-state nuclear magnetic resonance (NMR) analyses were performed on an Avance III 600 MHz spectrometer  operating with 11B frequency of 192.4 MHz. A magic-angle spinning (MAS) rate of 20 kHz was used for all experiments. The samples were packed into a 4 mm outer diameter ZrO2 rotor using a double resonance probe.Boron and sodium concentrations were determined by inductively-coupled plasma–atomic emission spectrometer . Calibration curves were established by employing the certified samples of known boron and sodium concentrations. Peak intensity was converted into boron weight percentage using the calibration curves. All samples were analyzed three times. The calculations were based on a dry weight of samples.−1 under nitrogen atmosphere. The degradation temperatures (Tmax) and the char yields at 600 °C (Char600) and 900 °C (Char900) were determined for each sample. The theoretical the char content at 900 °C  measurements were obtained on a thermal analysis system Setsys Evolution (Setaram) . The initial weight of each tested sample was about 10 mg. The samples were heated from 30 to 900 °C at a heating rate of 10 °C min−1 in the spectral range of 4000–400 cm−1. The evaluation of the gas concentration during the test was measured using OMNIC© software (v. 9.2) from Thermo Fisher Scientific.A Fourier transform infrared spectrometer (FTIR) Antaris IGC  was connected to the previous thermal system. The temperatures of the transfer line and the gas cell were fixed at 210 °C. The FTIR spectrometer was settled to collect 8 interferograms at a resolution of 8 cm−1 between 30 °C and 250 °C to observe the exothermic temperature peak (Tp), the total heat of reaction (ΔH), and the enthalpy of reaction of the uncured samples and the glass transition temperature (Tg) of the cross-linked thermosets determined as the inflexion value in the heat capacity jump. The uncured samples were tested in duplicate, while the cures ones were tested in triplicate.Differential scanning calorimetry (DSC) measurements of samples were carried out under nitrogen atmosphere using a DSC Q200 . The thermal properties were analyzed at 10 °C min−1 under nitrogen flow. Then, decomposition gases were sent to a combustor where they were heated at 900 °C under air flow (N2/O2 = 80/20). In these conditions, combustion was considered to be complete. Heat release rate (HRR) value was calculated by oxygen depletion according to Huggett’s relation [exp) represents the released heat by mass loss and is calculated as a ratio between THR and mass loss measured using TGA. The theoretical effective heat of combustion (EHCtheo) of the pure phenolic compounds was also calculated [Pyrolysis combustion flow calorimetry (PCFC) analyses of materials were performed using a FAA Micro Calorimeter from Fire Testing Technology . About 2 mg of sample was placed in the pyrolyzer, undergoing an increase of temperature from 100 to 750 °C at 1 °C srelation . The totlculated . For thetheo of organic compounds can be calculated. All samples were analyzed in duplicate.Conforming to Huggett’s relation, the heat released by the combustion is 13.1 kJ per gram of consumed oxygen . Therefo3). The samples were exposed to an irradiance of 35 kW m−2 in the presence of a spark igniter to force the ignition. The peak of heat release rate (pHRR), the time to ignition (TTI), the total heat release (THR), and the residue content were measured. All blends were done in triplicate and results were averaged.Fire behavior of the epoxy resins was also studied using a cone calorimeter (Fire Testing Technology) according to the standard ISO 5660  was used to observe the dispersion of the FRs in the epoxy resin. Blended samples were prepared using cryo-fractured cross-sections and analyzed under high vacuum at a 12.5 V with a working distance of 10 mm.This paper reported the alternative using of bio-sourced phenolic compounds such as gallic and ellagic acids and their boron- and sodium-functionalized forms as FR agents in the epoxy resin. It was observed that EA may influence the cure kinetic of the epoxy thermoset, whereas the other additives act as invert additives. The incorporation of the FR agents caused the reduction of the glass transition temperature from 144 °C for the neat epoxy resin to 54 °C for the GAD-containing thermoset. This effect was mainly correlated to the low compatibility of the additives with the epoxy matrix as well as to their non-uniform dispersion and agglomeration within the epoxy resin. These phenomena were also confirmed by the SEM analysis. The TGA and PCFC measurements showed the decrease in the thermal stability of the epoxy resins containing the pure phenolic components, and it was mainly attributed to the early decomposition of the phenolic compounds. Nevertheless, this decrease in thermal stability was accompanied by an increase in char yield and reduction of flammability. The THR was reduced by 10% and the pHRR by 45% with 10 wt.% of gallic acid compared to that of the thermoset without FR. The char content of the epoxy resin was doubled with the incorporation of 10 wt.% of ellagic acid. It could be explained by the interaction of EA with the epoxy matrix as observed in the DSC analysis. The derivatives of gallic and ellagic acids also showed the improvement of thermal characteristics. The pHRR and the THR values were reduced by about 10% by using of 10 wt.% of the phenolic derivatives. The combination of both PCFC and cone calorimeter analyses has highlighted that the significant improvement of flame retardant properties was related to a physical barrier effect. These results prove the potential of the bio-sourced phenolic compounds such as gallic and ellagic acid, as well as their derivatives, as FRs for epoxy resin.The development of bio-based FRs is still the main purpose of the FR field. In view of the obtained results, the efficiency of the plant extracts  as FR agents for a thermoset matrix should be investigated. The efficiency of its functionalized forms is also proposed to be studied in following works. It is also suggested to investigate the influence of EA on the cure kinetic of the epoxy thermoset by the other methods. The uniform dispersion of the additives in the epoxy matrix should be achieved in order to better evaluate the influence of the phenolic additives on the fire behavior of the epoxy thermosets.


On multivariate analysis, female sex  and bidirectional endoscopy  were independently associated with post-endoscopic nausea. In conclusion, bidirectional endoscopy could increase hypoxia in elderly patients using pethidine and post-endoscopic nausea in female patients.Same-day bidirectional endoscopy has been reported to reduce recovery time, and procedure-related cost. The safety of bidirectional endoscopy vs colonoscopy only, while using midazolam and pethidine, has never been evaluated. We reviewed 1,202 consecutive patients who underwent bidirectional endoscopy or colonoscopy only with administration of midazolam and pethidine in Toyoshima Ensdoscopy Clinic. We compared the clinical characteristics and adverse events associated with method of endoscopy . Furthermore, multivariate logistic regression analyses were conducted to study the role of age, sex, use of sedative, polypectomy, and bidirectional endoscopy in adverse events. In the bidirectional endoscopy group, the doses of pethidine and midazolam, and the incidence rates of hypoxia and posto-endoscopic nausea were significantly higher. On multivariate analysis, age (odds ratio = 1.061,  However, propofol is not always available, because the manufacturers of propofol restrict its use solely to personnel trained in general anesthesia.,6 On the other hand, benzodiazepines such as midazolam are the most commonly used sedative, and these are generally given with an opioid analgesic such as pethidine or fentanyl for synergistic effect. The safety of bidirectional endoscopy vs colonoscopy only, while using midazolam and pethidine, has never been evaluated. This study evaluated the safety of same-day bidirectional endoscopy with administration of midazolam and pethidine.Lucendo ,8 All clinical investigations were conducted according to the ethical guidelines of the Declaration of Helsinki.This retrospective study was approved by the Ethical Review Committee of the Hattori Clinic on September 7, 2018. Written informed consent was obtained from the study participants.This study included subjects who agreed to participate in the study and underwent same-day bidirectional endoscopy or colonoscopy only at Toyoshima Endoscopy Clinic, an outpatient clinic specialized in endoscopy, between May 1, 2017 and August 31, 2017. Bidirectional endoscopy or colonoscopy only was performed to evaluate patients with gastrointestinal bleeding, including patients with positive results for the fecal occult blood test, iron-deficiency anemia, and abdominal pain and those who had undergone screening for cancer, polyps, atrophic gastritis, and physical check-up. The following demographic and clinical characteristics were collected from medical records: age, sex, doses of midazolam and pethidine, and adverse events. Adverse events included hypoxia during and after procedures and post-endoscopic nausea, abdominal pain, and gastrointestinal bleeding and perforation. Hypoxia was defined as reduction in oxygen saturation <90% for more than 20 s or implementation of oxygen inhalation based on the judgement of the on-site endoscopist. The diagnostic criteria for post-endoscopic nausea included nausea or vomiting within 24 h after endoscopy. Bidirectional endoscopy or colonoscopy only was performed by experienced endoscopists. The sequence for bidirectional endoscopy is EGD followed by colonoscopy. Before starting EGD, the pharynx of patients was topically anesthetized by gargling with 2% lidocaine hydrochloride viscous solution . Sedation with midazolam and/or pethidine was induced based on the patient’s willingness. Endoscopists were allowed to use clinical judgement in deciding the dose and type of sedative and analgesic medication; midazolam (0–10 mg) and pethidine (0–87.5 mg). For elderly patients (age ≥70 years), reduced doses of sedative were used. For insufflation, CO2 was administered in the absence of chronic respiratory failure. Colonoscope insertion in the cecum was accomplished using standard maneuvers. In the absence of contraindications, when the colonoscope reached the cecum, we administered 10 to 20 mg of scopolamine butylbromide. Lesions, diagnosed as adenomas or sessile serrated polyps, were removed by using hot or cold polypectomy with a snare or forceps or by endoscopic mucosal resection on the examination day. We did not resect the polyps with a diameter of 20 mm or more because they should be resected in the hospitalization facility.Patients, who participated in the study underwent colonic preparation using 2 L of polyethylene glycol solution administered orally 5 h before the procedure. Polyethylene glycol solution or magnesium citrate was added when the stool was not clear liquid.Patients were transferred to the recovery room after the procedure. All adverse events including hypoxia and nausea were evaluated by the recovery room nurse. Patients were requested to return for a follow up visit 10 to 14 days after the procedure for an explanation of the endoscopic findings, and they were also counselled for adverse events.t test or chi-square test. If a zero frequency arose in the chi-square test, 0.5 was added to all of the cells of a fourfold table to estimate odds ratio .We compared the clinical characteristics and adverse events between bidirectional endoscopy and colonoscopy only with the use of Welch’s p<0.1) were subsequently assessed using multiple logistic regression method to identify independent factors. Age, body weight, body mass index, and dose per kilogram of body weight of each drug were included as continuous variables in the univariate and multivariate logistic regression methods. A p value of less than 0.05 was considered statistically significant. The data were analyzed using the Stat Mate IV software .The clinical parameters were analyzed using univariate logistic regression analysis. The predictors found to be associated with hypoxia or post-endoscopic nausea on univariate analysis (p = 0.0078), and women were significantly less (p = 0.025). In the bidirectional endoscopy group, the dose of pethidine and midazolam and occurrence rates of hypoxia and nausea were significantly higher  than those in the colonoscopy only group , use of pethidine , with polypectomy , and bidirectional endoscopy  were significantly associated with hypoxia during and after the procedure. On multivariate analysis, age , bidirectional endoscopy , and use of pethidine  were independently associated with hypoxia during and after the procedure.Table p = 0.024) and bidirectional endoscopy  were significantly associated with post-endoscopic nausea. On multivariate analysis, female sex  and bidirectional endoscopy  were independently associated with post-endoscopic nausea.Table Same-day bidirectional endoscopy with administration of midazolam and pethidine has significantly increased hypoxia and post-endoscopic nausea. Furthermore, old age and use of pethidine are independent risk factors for hypoxia. Female sex is an independent risk factor for post-endoscopic nausea. To the best of our knowledge, this is the first report of increased adverse events in same-day bidirectional endoscopy.Peri-endoscopic sedatives and analgesics are often used to provide patient comfort and improve examination quality during endoscopic procedures. Benzodiazepines such as midazolam are the most commonly used sedative, and these are generally given to the patient along with an opioid analgesic for synergistic effect. However, respiratory depression is the major adverse effect of sedative use. Bidirectional endoscopy requires more sedation due to longer procedural time resulting in higher incidence of hypoxia. In the elderly patients, EGD and colonoscopy performed separately might be a better choice. The observed sex differences can be explained by the presence of a different socialization process for men and women that may influence the willingness to communicate distress. Other possible explanations include the interaction between sex hormones and opioid and the hormonal fluctuations associated with the menstrual cycle.,17 Although post-endoscopic nausea is a minor adverse effect, it may have an impact on the willingness to repeat endoscopy. Endoscopists should recognize that bidirectional endoscopy may increase post-endoscopic nausea in female patients using pethidine.Female sex and bidirectional endoscopy are independently associated with post-endoscopic nausea. In this study, all of the 11 patients who had post-endoscopic nausea used pethidine. Nausea and vomiting might be the result of stimulation of the medullary chemoreceptor trigger zone by pethidine.The limitation of this study is its retrospective design. A follow-up study should be performed prospectively to confirm and clarify the characteristics of adverse events after bidirectional endoscopy.In conclusion, bidirectional endoscopy could increase hypoxia in elderly patients using pethidine and post-endoscopic nausea in female patients.


However, the molecular mechanism of SMC differentiation from DPSCs has not been fully uncovered. The canonical Wnt signaling  pathway plays an essential role in stem cell fate decision. The aim of this study is to explore the regulation via GSK3β and associated downstream effectors for SMC differentiation from DPSCs. We characterized one of our DPSC clones with the best proliferation and differentiation abilities. This stem cell clone has shown the capacity to generate a smooth muscle layer-like phenotype after an extended differentiation duration using the SMC induction protocol we established before. We further found that Wnt-GSK3β/β-catenin signaling is involved in the process of SMC differentiation from DPSCs, as well as a serial of growth factors, including TGF-β1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor-homodimer polypeptide of B chain (BB) (PDGF-BB), and vascular endothelial growth factor (VEGF). Pharmacological inhibition on the canonical Wnt-GSK3β/β-catenin pathway significantly downregulated GSK3β phosphorylation and β-catenin activation, which in consequence reduced the augmented expression of the growth factors  as well as SMC markers  at a late stage of SMC differentiation. These results suggest that the canonical Wnt-GSK3β/β-catenin pathway contributes to DPSC differentiation into mature SMCs through the coordination of different growth factors.Smooth muscle cell- (SMC-) based tissue engineering provides a promising therapeutic strategy for SMC-related disorders. It has been demonstrated that human dental pulp stem cells (DPSCs) possess the potential to differentiate into mature bladder SMCs by induction with condition medium (CM) from bladder SMC culture, in combination with the transforming growth factor- A range of injuries or diseases including cancer, benign bladder contracture, and congenital anomalies (such as bladder exstrophy and myelomeningocele) can result in the damage or loss of the bladder –3. Consein vitro should be fully explored before use. In this study, we have isolated three DPSC clones from different patients. The clones were investigated by comparing their proliferation rates and potential to differentiate into three mesenchymal lineages , to determine the best clone as the candidate cell source for further tissue engineering research.Mesenchymal stem cells (MSCs) are one type of fibroblast-like cell population with potential extensive self-renewal and multilineage differentiation abilities . Compareβ-Catenin is a plasma membrane-associated protein that acts as an intracellular signaling transducer in the Wnt signaling pathway [β (GSK3β) is a proline-directed serine-threonine kinase, and its phosphorylation appears to be a critical step in directing β-catenin to the nucleus [β1 (TGF-β1), and vascular endothelial growth factor (VEGF), as well as hepatocyte growth factor (HGF), have been proven to play a vital role in SMC differentiation in vivo [in vitro model we established before.We have recently reported the feasibility of using human DPSCs as bladder SMC progenitors for the regeneration of SMCs . Althoug pathway . It has  pathway . Glycoge nucleus . Myogeni in vivo . Therefoin vitro to compare their capacities of differentiation.The pulp tissues were obtained from third molars (donors aged from 17 to 20 years) with the patient's informed consent and ethical approval by the South East Wales Research Ethics Committee of the National Research Ethics Service (permission number: 07/WESE04/84). The clonal populations of DPSCs were isolated using a fibronectin-based selection protocol as described previously  after etHuman SMCs were obtained as reported previously from the bladder of patients who underwent open procedures for their bladder, after patient consent and ethical approval by the South East Wales Research Ethics Committee of the National Research Ethics Service (permission number: 07/WESE04/84) . Brieflyβ1), as previously described [Differentiation of the A32 was induced by using conditioned medium (CM) collected from bladder SMC culture, supplemented with transforming growth factor beta 1 , SB216763 , and LiCl , as previously described [μM), SB216763 (20 μM), or LiCl (2.5 mM). An equivalent amount of dimethylsulfoxide was added to the control wells. After 14 days of incubation, the mRNA and protein levels were compared to the control group using qPCR and western blotting methods, respectively.The Wnt pathway was analysed by using the inhibitors including XAV939 (Wnt/escribed –28. Brieα-SMA (1 : 100), and antidesmin (1 : 50) at 4°C, overnight; PBS was used as the negative control. The cells were then washed with PBS 3 times and incubated with anti-mouse IgG Alexa Fluor-488 or Alexa Fluor-594 secondary antibodies for 1 h at room temperature. The nuclei were counterstained with DAPI (VectorLabs). The protein expressions were observed with a fluorescent microscope and analysed with ImageJ software.The cells were fixed with 4% PFA for 30 min and then incubated in 0.1% Triton X-100 for 10 min on ice and then blocked with bovine serum albumin (BSA) for 60 min at 37°C. After the blocking step, the cells were incubated with antimyosin (1 : 50), anti-The cells were washed and resuspended in PBS supplemented with 3% FBS that contained saturating concentrations (1 : 100 dilution) of the following reagents: FITC-conjugated anti-human monoclonal antibodies, anti-CD29-phycoerythrin (PE), anti-CD90-PE, anti-CD34-PE, anti-CD45-PE, anti-CD146-PE, or anti-STRO-1-allophycocyanin (APC) for 1 h at room temperature in the dark. As a negative control, PE- and APC-conjugated nonspecific mouse IgG1 were substituted for the primary antibodies. The cell suspensions were washed twice, resuspended in 3% FBS/PBS, and analysed with a flow cytometry cell-sorting Vantage cell sorter (Becton Dickinson). The data were analysed with a Mod-Fit 2.0 cell cycle analysis program (Becton Dickinson).Total RNA was extracted from the cells using an RNeasy Mini Kit (QIAGEN) according to the manufacturer's directions. The total yield of RNA per extraction was calculated using a NanoVue spectrophotometer  to measure the absorbance at 260 nm. A260/A280 ratios of 1.9-2.1 indicated extraction of high-quality RNA. cDNA was synthesised with 2000 ng RNA using MMLV reverse transcriptase (Promega). For qPCR, three separate cDNA samples were used and each measured in triplicate. Target-specific primers  were addβ , t-GSK3β , active β-catenin , myosin , α-SMA , desmin , and GAPDH  overnight at 4°C and then incubated for 2 h with a horseradish-peroxidase-conjugated anti-mouse IgG antibody or anti-rabbit IgG diluted 1 : 2000 . Protein bands were visualized on an X-ray film by using an enhanced chemiluminescence system . The relative protein expression intensities were quantified by densitometry using Quantity One analysis software.The total protein was extracted from the cells with lysis buffer containing protease inhibitors . The protein concentration was measured by a BCA-200 protein assay kit . Equal amounts of proteins were separated by 4-12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in TRIS-buffered saline with Tween (TBST) containing 5% fat-free milk for 2 h and probed with primary antibodies, p-GSK3P < 0.05 indicated statistical significance.Each experiment was performed at least three times, unless otherwise indicated. Data are reported as the mean ± SD (standard deviation) deviation from three independent experiments. The significance of the differences between the experimental and the control groups was determined by using one-way analysis of variance; Dental pulp cells were isolated from pulp tissue of extracted third molars from patients. Three clones of cells that adhered to fibronectin were selected, noted as A11, B11, and A32. The proliferation rate and differentiation potential of the three clones were analysed. A32 demonstrated a high proliferation capacity extending beyond 80PDs, whilst the other two clones (A11 and B11) exhibited less than 36PDs . Compareβ1. In this study, we evaluate whether DPSCs can generate a smooth muscle layer after an extended differentiation duration. Noninduced A32 were found to express α-SMA , total GSK3β (t-GSK3β), and active β-catenin in A32 in response to the induction of differentiation medium by western blotting. The protein level of p-GSK3β increased and maintained at the peak through 11 to 14 days  or GSK3 inhibitors (SB216763 and LiCl) significantly downregulated the expression levels of these growth factors  and B11 (from patient B). Investigation of the proliferation rate using PDs as an indicator demonstrated that two of the clones (A11 and B11) showed less than 36PDs and senesced within 85 days, while A32 had a high proliferation rate with over 85PDs that only started senescing after more than 300 days. Compared with the previous studies about the analysis of proliferation rate of MSCs from bone marrow [SMC-based tissue engineering provides a potential therapy for SMC pathology, including cardiovascular diseases, gastrointestinal diseases, urinary incontinence, and bladder dysfunction –31. A ree marrow , stem cee marrow –37, while marrow . The nicα-SMA and desmin were already present in noninduced human DPSC clone A32 at a low basal level, which indicates A32 may be more suitable to be induced into SMCs for bladder tissue regeneration. Additionally, this clone has the potential to differentiate into bladder SMC by the CM from cultured bladder SMC in combination with TGF-β1 [in vivo.The urinary bladder wall is mainly composed of the Detrusor smooth muscle layer which is made of smooth muscle fibers arranged in spiral, longitudinal, and circular bundles . The Deth TGF-β1 . In thisβ-catenin signaling plays a primary role in the regulation of proliferation and differentiation of stem cells [α and GSK3β. GSK3β originally isolated from muscles as a kinase promotes glycogen assimilation by phosphorylating and inactivating glycogen synthase. It is also involved in the maintenance and plasticity of the skeletal muscle mass, as well as playing an important role in skeletal muscle atrophy in vivo [. β-Catenin is a multifunctional protein which is located in both the nucleus and cytoplasm. It leads to myogenic specification and prevents adipogenic differentiation in adult stem cells [β and β-catenin are key factors in the canonical Wnt pathway. Activation of the Wnt signaling promotes the stabilization and accumulation of β-catenin in the cytoplasm by triggering GSK3β phosphorylation which prevents subsequent GSK3β-mediated β-catenin phosphorylation in association with Axin and adenomatous polyposis coli (APC) [β-catenin enters the nucleus and induces activation of target genes by binding with members of the T-cell factor (TCF) and lymphoid enhancer factor (LEF) transcription factor family [β/β-catenin contributes to the progressive nature of smooth muscle tissue remodelling [β/β-catenin signaling is involved in regulating the process of bladder SMC differentiation from human DPSCs by the SMC induction protocol. To confirm the effect of Wnt/β-catenin signaling and GSK3β on SMC differentiation, two types of pharmacological inhibitors were used. One is XAV939 which is a tankyrase inhibitor for inhibiting Wnt/β-catenin signaling [α and β isozymes of GSK3. Considering the high degree of sequence similarity, GSK3α and GSK3β share some similar functions. For example, the single loss of either GSK3α or GSK3β in mouse embryonic stem cells (ESC) did not negatively alter Wnt/β-catenin signaling, whereas GSK3α/β double knockout ESCs displayed hyperactivated Wnt/β-catenin signaling, resulting in dramatically skewed cell differentiation [α or GSK3β plays distinct roles in cardiomyocyte differentiation and cardiovascular development in mice [α and GSK3β have both common and nonoverlapping cellular functions, largely depending on the physiological context and the cell type studied. It has been reported GSK3β protein expression in human and mouse muscles was found to be three to four times higher than GSK3α, suggesting that GSK3β, rather than GSK3α, may be the predominant GSK3 isoform in the muscle [β as well as active β-catenin and also downregulated the expression of SMC markers  at the latter stage of differentiation at both mRNA and protein levels. It suggests that Wnt-GSK3β/β-catenin signaling promotes the human DPSCs to differentiate into bladder SMCs. Further analysis would be required by downregulation of GSK3α or GSK3β to investigate the role of GSK3α in regulating the process of DPSC differentiation into SMC.Canonical Wnt/em cells , 39. GSK in vivo , 41. β-Cem cells –44. GSK3li (APC) . The star family . It has odelling  and alsoodelling , as wellodelling . In thisignaling . The othignaling , 27. SB2ntiation . Neverthntiation . GSK3α o in mice –51. There muscle . Two typβ1, HGF, VEGF, PDGF-BB, bFGF, and EGF, play different roles in the process of SMC differentiation. It has been demonstrated that TGF-β1 evokes an important signal that induces expression of vascular SMC markers in a range of nonsmooth muscle precursor cell types, including multipotent embryonic fibroblast [β1 upregulated during the whole process of SMC differentiation, indicating that TGF-β1 also plays an important role in the bladder SMC differentiation from DPSCs. It has been shown previously that an increased expression of bFGF was associated consistently with SMC proliferation of the neointima formation [in vivo. Additionally, experimental inhibition of bFGF activation leads to the SMC marker expression in the model of BMMSC differentiation into SMC [β1, HGF, VEGF, and PDGF-BB, were all upregulated at 14 days of differentiation, indicating that these growth factors may induce the maturity of SMCs at the latter stage of bladder SMC differentiation from DPSCs. In order to further investigate whether Wnt-GSK3β/β-catenin signaling is involved in the mature process of SMC differentiation, we evaluated the expression of the growth factors above with or without two types of inhibitors at a later stage of SMC differentiation. We noticed that the expression of these growth factors was significantly downregulated with the Wnt/β-catenin inhibitor and GSK3β inhibitors at that stage, indicating that the Wnt-GSK3β/β-catenin signaling promotes the human DPSC differentiation into mature bladder SMCs by regulating the expression of these myogenic growth factors.Previous studies have identified that a variety of stem cells, including those from the embryo , 53, bonbroblast , neural broblast , and MSCbroblast . We hereormation  and atheormation  in vivo.into SMC . As one into SMC , which iinto SMC , 63, wasinto SMC . The expβ1 triggers phosphorylation of GSK3β, thus disrupting the APC/Axin/GSK3β/β-catenin complex. The stabilization and accumulation of β-catenin in cytoplasm translocates into the nucleus and enhances the expression of growth factors, including TGF-β1, HGF, VEGF, and PDGF-BB, which subsequently promote the SMC differentiation from DPSCs, for instance, clone A32.As summarized in in vitro using the bladder SMC induction protocol we established before. Additionally, we have found that the Wnt-GSK3β/β-catenin signaling is involved in SMC differentiation. Several growth factors  regulated by Wnt-GSK3β/β-catenin signaling promote the proliferation/differentiation during the process of bladder SMC differentiation from human DPSCs.In this study we characterized one of our DPSC clones (clone A32) with the best proliferation and differentiation abilities and demonstrated that the clone A32 possesses a potential to differentiate into a bladder smooth muscle cell layer-like phenotype


Basalt fiber-reinforced polymer (BFRP) composites are receiving increasing attention as they represent a low-cost green source of raw materials. FRP composites have to face harsh environments, such as chloride ions in coastal marine environments or cold regions with salt deicing. The resistance of FRPs subjected to the above environments is critical for the safe design and application of BFRP composites. In the present paper, the long-term durability of BFRP sheets and the epoxy resin matrix in a wet–dry cyclic environment containing chloride ions was studied. The specimens of the BFRP sheet and epoxy resin matrix were exposed to alternative conditions of 8-h immersion in 3.5% NaCl solution at 40 °C and 16-h drying at 25 °C and 60% relative humidity (RH). The specimens were removed from the exposure chamber at the end of the 180th, 270th and 360th cycles of exposure and were analyzed for degradation with tensile tests, scanning electron microscopy (SEM) and void volume fractions. It was found that the tensile modulus of the BFRP sheet increased by 3.4%, and the tensile strength and ultimate strain decreased by 45% and 65%, respectively, after the 360th cycle of exposure. For the epoxy resin matrix, the tensile strength, tensile modulus and ultimate strain decreased by 27.8%, 3.2% and 64.8% after the 360th cycle of exposure, respectively. The results indicated that the degradation of the BFRP sheet was dominated by the damage of the interface between the basalt fiber and epoxy resin matrix. In addition, salt precipitate accelerated the fiber–matrix interfacial debonding, and hydrolysis of the epoxy resin matrix resulted in many voids, which accelerated the degradation of the BFRP sheet. Fiber-reinforced polymers (FRPs) are increasingly being used for repair and rehabilitation of existing structures ,2. The gIn those applications, the long-term durability of the FRP composites is often one of the major advantages over the conventional materials ,9,10; duIn one such study, the durability of a glass FRP (GFRP)/carbon FRP (CFRP)-reinforced concrete beam subjected to salt attack immersion was examined; the chloride ions easily permeated into the epoxy resin through the existing voids and cracks without causing hydrolysis to the vinyl ester resin ,12. WangUnderstanding the durability of FRPs and their reinforced structures in a chloride-containing environment is necessary for their expanded use. Manuel et al.  studied Besides CFRPs and GFRPs, new high-performance composites have been developed , among wAs mentioned previously, the existing literature provides only a limited understanding of the durability of BFRP composites under the wet–dry cyclic chloride-containing environment. Because of the variation in exposure conditions and different material parameters, it is difficult to compare these different test results and draw general conclusions. As such, this study examines the durability of BFRP sheets and the epoxy resin matrix under a wet–dry cyclic condition in a chloride-containing environment. The residual tensile properties of the exposed BFRP sheets and epoxy resin matrix were determined after the designed cycle times, one of which can be used to assess the durability of the BFRP sheet under a wet–dry exposure in a chloride-containing environment.2, the average diameter was 13 µm, the density was 1.796 g/cm3, and the nominal thickness was 0.167 mm. The tensile strength, tensile modulus and elongation at break were 1700 MPa, 84 GPa and 2.6%, respectively. The parameters were provided by the manufacturer.The unidirectional BFRP sheets (BUF13-4.5-380) used in this study were provided by the Shijin Basalt Fiber Co., Ltd. . The weight of the basalt fiber sheets was 380 g/m3.The epoxy resin (HM-E8) used in this study was provided by Heroman chemical products Co., Ltd. . It was a two-component resin with thixotropic epoxy adhesive and epoxy resin binder mixed in the ratio of 2:1 by weight, and the density was 1.6 g/cmThe BFRP sheets were prepared in accordance with the guideline ASTM D 3039/D 3039 M-00 . The speThe dog-bone-shaped resin specimens for the uniaxial tensile test were prepared following ASTM D 638-99 . The epoThe BFRP specimens were prepared according to ASTM D 3039/D 3039 M-00 . Aluminuε1, ε2 and ε3 are the strains obtained from the test. In order to ascertain the reliability and accuracy of the test results, the change in in-plane bending (D1) and out-of-plane bending (D2) were guaranteed within 5%. The expression of D1 and D2 are given as: The strain can be obtained by the following formula:The tensile tests of the epoxy resin matrix were performed following ASTM D 638-99 . The tesThe residual tensile property was performed at the end of the 180th, 270th and 360th cycles of exposure. At least three specimens were used for each test, and the average values of these results were reported.−) was set as 3.5%. The process of a single wet–dry cycle was as follows: the specimens were immersed in 40 °C NaCl solution for 8 h, following by drying at 25 °C with 60% RH for 16 h. The temperature control precision was ±2 °C.The wet–dry cyclic accelerated aging was performed at the structural laboratory, Guangdong University of Technology , and the wet–dry cyclic system is shown in The surfaces of the BFRP sheet and the tensile fracture of the epoxy resin matrix were platinum coated for 10 min and observed using scanning electron microscopy .ε is the void volume fraction, and m, Z and A are mass, thickness and area per unit measure membrane, respectively. Based on the literature , the voiThe properties of the FRP composite are governed not only by the fiber and the resin matrix, but also by the interface between the fiber and epoxy resin matrix . Based oScanning electron microscopy (SEM) morphologies of the tensile fracture surface of the epoxy resin matrix are illustrated in 2+ exists in basalt fiber (about 10%) [In addition to the epoxy resin matrix, basalt fiber and the interface between the basalt fiber and epoxy resin matrix can also lead to degradation of the performance of the BFRP sheet. out 10%) , and theout 10%) :(5)Fe2++Therefore, the reaction may explain the degradation of BFRP sheets under the exposure environment. As the relative content of ferrous ion is low, the interface damage between the basalt fiber and epoxy resin matrix is another reason for the performance degradation of the BFRP sheet. Water diffusion through the interface into the BFRP sheets  and the The interface damage is illustrated in The SEM test results of the specimens exposed for 180 cycles are shown in The stress–strain curves of the epoxy resin matrix are shown in As a contrast, the stress–strain curves of the BFRP sheet are shown in To compare the performance degradation of the specimens under the effect of exposure, the normalized results of the specimens are shown in The wet–dry cycles with chloride-containing solution caused the hydrolysis of the epoxy resin matrix, and interfacial damage between the basalt fiber and epoxy resin matrix. Salt precipitation accelerated the fiber–matrix interfacial debonding during the wet–dry cycles, resulting in serious degradation of the tensile strength of the BFRP sheet. The longer the exposure time, the more serious the degradation found.Chloride ions accelerated the penetration and degradation of the epoxy resin matrix, resulting in the degradation of the tensile strength of the resin matrix. With the increase in the number of cycles, more water ingressed led to more plasticization, which was responsible for the reduction in the tensile modulus.The deformation was governed by the basalt fiber for the BFRP sheet. The decreased stress-transfer at the interface between the basalt fiber and resin matrix led to degradation of the tensile strength of the BFRP sheet, but the tensile modulus remained approximately constant as the cycling time increased.An experimental study has been conducted to investigate the long-term durability of the BFRP sheet and the epoxy resin matrix under a wet–dry cyclic condition in a chloride-containing environment. The residual tensile properties and void volume fractions were calculated, and SEM micrographs obtained. The following conclusions have been drawn from the experimental results and discussion:


Rationale: Effective targeting therapies are limited in Hepatocellular carcinoma (HCC) clinic. Characterization of tumor suppressor genes (TSGs) and elucidation their signaling cascades could shed light on new strategies for developing targeting therapies for HCC.Methods: We checked genome-wide DNA copy number variation (CNV) of HCC samples, focusing on deleted genes for TSG candidates. Clinical data, in vitro and in vivo data were collected to validate the tumor suppressor functions.Results: Focal deletion of GATA4 gene locus was the most prominent feature across all liver cancer samples. Ectopic expression of GATA4 resulted in senescence of HCC cell lines. Mechanistically, GATA4 exerted tumor suppressive role by orchestrating the assembly of a tumor suppressor enhancing module: GATA4 directly bound and potently inhibited the mRNA transcription activity of β-catenin; meanwhile, β-catenin was recruited by GATA4 to promoter regions and facilitated transcription of GATA4 target genes, which were TSGs per se. Expression of GATA4 was effective to shrink GATA4-deficient HCC tumors in vivo. We also showed that β-catenin inhibitor was capable of shrinking GATA4-deficient tumors.Conclusions: Our study unveiled a previously unnoticed tumor suppressor enhancing module assembled by ectopically expressed GATA4 in HCC cells and denoted a therapeutic opportunity for GATA4 deficient HCC patients. Our study also presented an interesting case that an oncogenic transcription factor conditionally functioned as a tumor suppressor when recruited by a TSG transcription factor. Here we report GATA4 as a functionally important HCC TSG. Ectopically expressed GATA4 orchestrated the assembly of a tumor suppressor enhancing module in HCC cells by directly binding and inhibiting the transcription activity of β-catenin to transcribe canonical Wnt pathway target genes. In the meantime, β-catenin was recruited by GATA4 to promoter regions to facilitate the transcription of TSGs which are GATA4 transcription targets. GATA4 thus created a tumor suppressor enhancing module between GATA4 itself and β-catenin. In vivo experiments showed that ectopic expression of GATA4 or administration of β-catenin inhibitors shrank GATA4-deficient HCC tumors. Our study revealed a previously unnoticed tumor suppressor enhancing module and shed light on a therapeutic opportunity for GATA4 deficient HCC patients. Our work also presented an interesting case that a typical oncogenic transcription factor functioned as a TSG when recruited by another TSG transcription factor.In HCC clinic, patients are limited by choice for effective treatment options. In order to identify functionally important TSGs and elucidate their signaling cascades in the hope of finding drug targets for HCC patients, we examined the DNA copy number variation (CNV) and focused our attention on deleted regions at whole genome level. Strikingly, we noticed a sharp peak of focal deletion in 8P23.1 chromosomal region across most HCC samples Figure A. These In order to validate its TSG function, we sought to ectopically express GATA4 in HCC cell lines with lower baseline expression and knockdown in those with relatively higher expression. For this purpose, we checked GATA4 expression level in liver cancer cell lines commonly used in cancer research community through western analysis, including two pairs of HCC/para-tumoral tissues as references for GATA4 expression in tumoral and normal liver tissues. We found comparable GATA4 protein level between tumor samples and cancer cell lines, which was consistently lower than that of para-tumoral tissues B. Among G12D transgenic mice G12D and knockout GATA4 simultaneously in hepatocytes of lsl-KrasG12D transgenic mice  inhibitors (CKI). Our western blot analysis showed no significant changes of p14ARF, p15INK4b, p16INK4a, p21, p27 or p53 levels before and after GATA4 expression in HepG2i cells F.Taken together, our data showed that GATA4 exerted its TSG function by inducing senescence of HCC cells through a non-canonical pathway.7 HepG2 cells to enrich endogenous protein with antibodies against GATA4 and β-catenin respectively. Reciprocal co-IP experiments solidly confirmed binding between endogenous GATA4 and β-catenin in HepG2 cells  in DOX treated HepG2i cells. Silver staining of the SDS-PAGE gel clearly revealed distinct protein species in GATA4 pull-down samples compared to IgG-enriched control Figure A. We thes Figure B. Furthes Figure A.GATA4 is a transcription factor, which constitutively locates in nucleus We further asked which domain of GATA4 mediated the interaction between GATA4 and β-catenin. For this purpose, we ectopically expressed in HEK293 cells FLAG tagged β-catenin together with Myc tagged full-length GATA4 and its truncated mutants deleted of N-terminal TAD domains, CTR region, ZF1, ZF2, or ZF1/ZF2 respectively D. Our coWe also tried to pin down the domain of β-catenin critical for interaction between β-catenin and GATA4. We therefore generated a series of β-catenin mutant, lacking N-terminal domain (NTD), armadillo repeats (ARM), and C-terminal domain (CTD) respectively E. HoweveHSPA12A, cJUN, CCND2, AXIN2 and C-MYC  and thereby inhibited the oncogenic function of β-catenin; 2) β-catenin enhanced transcription activity of GATA4 to exert tumor suppressor function. Given the fact that several high-efficiency systems for delivering genes-of-interest into target histocytes and tumor cells were developed or in clinical trialsSurprisingly, we inadvertently found that β-catenin enhanced transcriptional activity of GATA4 during our study. As shown in Figure We went on to validate whether β-catenin enhanced GATA4's transcription of TSG targets. We re-analyzed our RNA-seq data and found several liver cancer TSGs among GATA4-upregulated genes . We pick3 , which inhibited the oncogenic function of β-catenin; 2). β-catenin was recruited by GATA4 and thereby enhanced transcription activity of GATA4 to exert tumor suppressor function. Given the promising virial and non-virial systems to deliver gene-of-interest into target histocytes and tumor cellsβ-catenin has been well known as an oncogene through transcribing target genes of canonical Wnt signaling pathway. Tumor promoting function of β-catenin has been repeatedly validated: oncogenic mutations in CTNNB1 frequently found in HCC patients; therapeutic effect in preclinical tumor models has been reported with drug targeting β-catenin. In our current study, we discovered an unexpected role for β-catenin in HCC: in the presence of GATA4 expression, β-catenin was recruited by GATA4 to facilitate transcription of GATA4 target genes, among which are HCC tumor suppressor genes. Our current work therefore unveiled a previously unnoticed tumor suppressor enhancing module orchestrated by ectopically expressed GATA4 in HCC cells: 1). GATA4 inhibited transcriptional activity of β-catenin, thereof the expression of canonical Wnt target genes , shCTNNB1 (TRCN0000314920), and Luciferase shRNA . psPAX2, pMD2.G, pCAG-IRES-Neo and pLKO.1-puro were purchased from Addgene. The pLVX-TetOne-Puro plasmid was purchased from Clontech. The Top-Flash plasmid was a gift from Dr. Wei Wu's lab at Tsinghua University. Human SORBS1, DCN and CTNNB1 cDNA were kindly gifted by Dr. J. Han's lab at Xiamen University. Myc- or FLAG-tagged GATA4, β-catenin, TCF1, LEF1, all truncations and point mutations were constructed by standard molecular biology techniques.Doxycycline hyclate ; ICG-001 (Selleckchem); fetal bovine serum, DMEM and DMEM/F12 medium (Gibco); fibroblast growth factor (FGF) and epidermal growth factor (EGF) (PeproTech); Trizol reagent (TAKARA); protease and phosphatase inhibitor cocktail (Roche); Lipofectamine 3000 and B27 (Invitrogen); dual-specific luciferase assay kit (Promega); Cell Counting Kit-8 ; Western blotting substrate (Millipore); Cell Signaling Senescence β-Galactosidase Staining Kit (CST); silver staining Rapid silver staining kit's (Beyotime); BALB/c nude mice ; antibody against GATA4, Lamin B1, P21, FLAG, P15 and c-MYC (Abcam); HA and β-actin (Sigma); β-catenin, LEF1, TCF1, P14, P27, P53, p14/ARF, Caspase-9 and Caspase-3 (CST), P16/Ink4a (Epitomics); Cytokeratin (AE1/AE3) antibody  were purchased from the indicated manufacturers. SNU-387, SNU-449, PLC, NeHepLxHT, SK-Hep1, HepG2, HUH7 and HEK293 cells were obtained from ATCC.The HEK293 cells were transfected with two packaging plasmids (psPAX2 and pMD2.G) together with a pLVX-TetOne-Puro vector control or the same vector constructs for expressing GATA4, SORBS1, or DCN, or shGATA4, shLuciferase, shCTNNB1 respectively. Twenty-four hours later, cells were changed with new medium for another 24 hours. The recombinant virus-containing medium was filtered and used to infect cells in the presence of polybrene (8 μg/mL). The infected cells were selected with puromycin (0.5 μg/mL) for seven days before further experiments.3 cells were seeded in each well of 96-well plates and cultured overnight. DMEM containing 10% FBS medium was supplemented with 1 µg/mL DOX or 0.1 µg/mL ICG-001 for 1, 3, and 5 days. Proliferation activity was then determined using CCK8 cell counting kit following the manufacturer's protocol.For cell proliferation assays, 1×103 cells were plated in triplicate into 6 well plate containing culture medium with 10% FBS. Cells were grown for 14 days, then fixed with 4% formaldehyde in 1x PBS and stained with crystal violet. Colonies with the diameter larger than 200 µm were counted.For each cell line, 1×103 cells were seeded in Ultra-Low attachment 6 well plate in serum-free conditioned medium DMEM/F12 medium, 20 μl/mL B27 supplement, 20 ng/mL FGF and 20 ng/mL EGF. After 2~4 weeks, tumor spheres with the diameter over 100 µm were counted.For each cell line, 1×10GAPDH. Gene-specific primer sequences were as follows.Total RNA was extracted using Trizol reagent and subjected to real-time PCR analysis to measure mRNA levels of the indicated genes. Data shown are the relative abundance of the indicated mRNA normalized to that of AXIN2-Forward: AAATAACCCCTCAGAGCGATGAXIN2-Reverse: TTCCAGTTCCTCTCAGCAATCCCND2-Forward: ACTTGTGATGCCCTGACTGCCND2-Reverse: ACTTGGATCCGTCACGTTGCTNNB1-Forward: CACAAGCAGAGTGCTGAAGGTGCTNNB1-Reverse: GATTCCTGAGAGTCCAAAGACAGDCN-Forward: GCTCTCCTACATCCGCATTGCTDCN-Reverse: GTCCTTTCAGGCTAGCTGCATCHSPA12A-Forward: GGCTGGAGGTAGAAGGTGGAHSPA12A- Reverse: GCGTTATTCCTGTGTCCCCASORBS1-Forward: TATCAGCCTGGCAAGTCTTCCGSORBS1-Reverse: CCCGTCTGATTCCCTCTTCACTJUN-Forward: GCTGCTCTGGGAAGTGAGTTJUN-Reverse: TTTCTCTAAGAGCGCACGCAMYC-Forward: GGACCCGCTTCTCTGAAAGMYC- Reverse: GTCGAGGTCATAGTTCCTGTTGGAPDH-Forward: GAAGGTGAAGGTCGGAGTCGAPDH-Reverse: GAAGATGGTGATGGGATTTCWhole cell lysates were extracted by using the NP-40 lysis buffer , protein concentrations were determined by the Bradford assay. Soluble proteins (30~40 μg) were subjected to SDS-polyacrylamide gel electrophoresis. Coimmunoprecipitation and immunoblot analysis were then performed.6 HepG2i cells in 100 µL PBS and Matrigel (1:1). When tumors reached volume of around 90 mm3, the mice were randomly grouped and fed with either DOX containing or normal diet; or gavaged with 100 μL of 2,4-Dia suspended in 0.5% sodium carboxymethyl cellulose  or vehicle every other day. The body weight and tumor volume (= D×d2/2 (mm3), where D is the longest and d is the shortest diameter, respectively) were monitored as indicated in Figure 6-weeks-old male BALB/c nude mice  were used for in vivo animal experiments. The animals were housed in constant laboratory conditions of a 12 hours light/dark cycle and specific pathogen-free conditions. For xenograft study, mice were inoculated subcutaneously into the right-back with 5×105 cells were seeded in each well of 6 well plates and treated as indicated. The cells were cultured until the time of staining. For quantification of β-galactosidase staining positive cells, the blue positive cells in at least three randomly selected fields at 200×magnification under an inverted microscope were counted.β-galactosidase staining was performed using the Cell Signaling Senescence β-Galactosidase Staining Kit. Briefly, 2×10The related material, method and raw data are described in GEO website of accession number: GSE135579 (RNA-seq) & GSE135714 (ChIP-seq).G12D through tail vein injection. Liver tumor formation in KRASG12D/GATA4-/- mice compared to KRASG12D/TdTomato-/- mice 6 months post-infection.All mice were housed in a pathogen-free environment in Jinan University and all experimental protocols were approved by the Institutional Committee for Animal Care and Use at Jinan University. All animal work was performed in accordance with the approved protocol. We used recombinant lentivirus co-expressing Cre and CRISPR/CAS9 to infect transgenic mouse model: Lsl-KRASSupplementary figures, tables, and methods.Click here for additional data file.


Skin ulcers are a serious complication of diabetes. Diabetic patients suffer from vascular lesions and complications such as peripheral neuritis, peripheral vascular lesions, and collagen abnormalities, which result in skin wounds that are refractory and often develop into chronic ulcers. The healing of skin ulcers requires an inflammatory reaction, wound proliferation, remodeling regulation, and control of stem cells. Studies investigating diabetic cutaneous ulcers have focused on cellular and molecular levels. Diabetes can cause nerve and blood vessel damage, and persistent high blood sugar levels can cause systemic multisite nerve damage based on peripheral neuropathy. The long-term hyperglycemia state enables the polyol glucose metabolism pathway to be activated, increasing the accumulation of toxic substances in the vascular injured nerve tissue cells. Sustained hyperglycemia leads to dysfunction of epithelial cells, leading to a decrease in pro-angiogenic signaling and nitric oxide production. In addition, due to impaired leukocyte function in hyperglycemia, immune function is impaired and the immune response at relevant sites is insufficient, making diabetic foot more difficult to heal. The Wnt/β-catenin pathway is a highly conserved signal transduction pathway involved in a variety of biological processes, such as cell proliferation, apoptosis, and differentiation. It is considered an important pathway involved in the healing of skin wounds. This article summarizes the mechanism of action of the Wnt/β-catenin pathway involved in the inflammatory responses to diabetic ulcers, wound proliferation, wound remodeling, and stem cells. The interactions between the Wnt signal pathway and other metabolic pathways are also discussed. Diabetes mellitus (DM) is a systemic chronic metabolic disease with elevated blood glucose levels. The DM can cause multiple complications, including diabetic nephropathy, diabetic retinopathy, and diabetic cutaneous ulcers (DCUs). The DCU is one of the most common and serious complications of diabetes, with a quarter of patients developing a foot or skin ulcer. Each year, 50–70% of patients requiring amputations due to non-traumatic injuries are patients with DM . The preThe increasing age of China’s population has caused the prevalence of DCU to increase annually. Treatment of DCU is one of the most critical health issues that needs to be resolved urgently in clinical practice. In recent years, DCU studies have focused on treatment at the molecular level. It has been reported that in type 2 DM, nucleotide polymorphisms (SNPs) exist for TCF7L2 of the Wnt pathway . The Wntint-1,” was discovered by int-1, which is similar to MMTV gene sequences in tumors at integration sites. The wingless gene (Wg) of the Drosophila melanogaster is a homolog of int-1 and both together are known as Wnt . The interaction between the Wnt protein and its cognate receptor can be blocked by numerous soluble metabolites, including the Dickkopf protein (Dkk) or the Wnt inhibitor (WIF)  .The stable conduction of Wnt/β-catenin signaling pathway is dependent on the stabilization of β-catenin. Without the Wnt signal, free cytosolic β-catenin is phosphorylated by a complex of Axin, casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β). Phosphorylated β-catenin is ubiquitinated by β-transduced repetitive protein (β-TrCP) for proteasome-dependent degradation . When Wn2+ and Wnt/planar cell polarity (PCP) signaling pathways  and protein kinase C (PKC) and regulates cell motility and cell adhesion. In Wnt/Ca2+ signaling, Wnt ligand-receptor interactions result in the release of intracellular calcium, which acts as a secondary messenger to activate calmodulin-dependent protein kinase II (CaMKII). The CaMKII triggers the phosphorylation of transforming growth factor (TGF)-β-activated kinase 1 (TAK1), increasing the activity of lipoprotein kinase (LK), resulting in the dissociation of LEF1–β-catenin/DNA. This inhibits transcription of Wnt/β-catenin signaling activity /p38 microtubule-associated protein (MAP) kinase signaling pathway.There are four overlapping processes in DCU, including hemostasis, inflammatory reaction, an increase in wound surface, and remodeling of the wound surface . The maiPrevious studies have suggested that wound healing in patients with DM is reduced due to the following factors: 1. Glucose metabolism disorder and advanced glycation end products, which can interfere with the interaction between endothelial cells and leukocytes, and the inhibition of the function of mononuclear macrophages and endothelial cell proliferation . 2. Microvascular changes: manifest as extensive small vascular endothelial proliferation and capillary basement membrane thickening, causing a reduction in blood flow, local ischemia, hypoxia . 3. The The healing of DCU wounds is a complex process, involving multiple cells and molecules. The processes are intertwined and the interaction between molecules and cells is mutually restrictive.Inflammation is an important protective response that has a key role in the regeneration of damaged tissue and elimination of the trigger . For example, in the wound-healing stage of ulcers, the wound is invaded by numerous inflammatory cells. Phagocytic cell fragments and the release of growth factors aid in the prevention of infection by microorganisms and have an important role in wound healing . Poor inInterferon-γ (IFN-γ) and lipopolysaccharides (LPS) can effectively stimulate inflammatory factors, which lead to the significant upregulation of Wnt5a . The relAfter inflammation of the DCU wound, the tissue forms a proliferative phase by secreting fibroblasts, vascular endothelial cells, and keratinocytes. Fibroblast growth factors (FGFs), epidermal growth factors (EGFs), nerve growth factors (NGFs), and PDGFs regulate the activation and proliferation of the repair cells . The forIn vitro, the inhibition of Wnt5a in endothelial cells of DM patients restores insulin-induced eNOS phosphorylation and NO production, mediated by JNK. This suggests that the Wnt5a/JNK signaling pathway is involved in the endothelial dysfunction of DM  regulator in mammalian epithelial cells and hair follicles (HFs). The Wnt signaling pathways appear to have a central role in the regeneration of stem cell activity, although further study on this is required. The Wnt target gene products, Optimal wound healing models the process of epidermal development, including wound remodeling, trauma to the epidermis, disruption of the epidermal barrier, and activation of the immune cells. The wound remodeling period is an important period in wound healing. After the early extracellular wound matrix is formed, the collagen skeleton and proteoglycan filling in the matrix create scar tension, through apoptosis and maturation of the cells.On the 7th day after wound formation, fibroblasts show the contractile phenotype  myofibroblasts, resulting in shrinkage of the wound surface . During The Wnt/β-catenin is one of the most critical activation initiation signals for HF development. During the second stage, a large number of keratinocytes begin to proliferate and form “hair buds” on the epidermis. At this time, cyclin D1 is upregulated and plays a regulatory role in the cell cycle .Skin epidermal stem cells are regularly regenerated and differentiated, providing the body with an unlimited source of cells. The re-epithelialization of wounds is dependent on the proliferation of skin stem cells (SSCs) during DCU healing. Porcine adult bone marrow mesenchymal stem cells have been used to generate skin . This enThe advantage of self-sourced induced pluripotent stem cells (IPSCs) compared to other types of stem cells lies in that immune rejection can be minimized. The Wnt/β-catenin pathway has a role in the homeostasis of ASCs and is a focus in the regeneration of inflammatory tissue. Wnt signaling enhances the proliferation of epithelial cells .In contrast to a continuously regenerating epidermis, mature HFs progress through growth (growth phase), degeneration (degeneration phase), and rest (resting phase) cycles throughout their life. During the hair development cycle, the upper part of HF is permanent, while the lower part undergoes degeneration and is then regenerated by stem cells. Stem cells in HF can be divided into two groups: a set of outer layers located in the ridge, called the hair follicle stem cells (HFSCs), and the other is located in the secondary root below the bulge (sHG) . The HFSIn the developing epidermis, Wnt/β-catenin signaling induces the expression of HF, and the constitutive attenuation of epidermal Wnt/β-catenin signaling impairs the formation of HF, but does not affect the integrity of the interfollicular epidermis (IFE) . It is iFigure 2).During the DCU healing process, the Wnt/β-catenin signaling pathways work independently or together with other pathways to elicit an appropriate cellular response. It is becoming increasingly clear that signaling pathways do not operate in isolation, but are strongly intertwined. Although many signaling pathways have been studied extensively, the role of some signaling pathways in DCU is unknown and requires investigation . The healing of wounds is regulated at cellular and molecular levels. Although pharmaceuticals that target the Notch and Hedgehog signaling pathways have been tested in clinical trials, pharmaceuticals regulating Wnt are currently untested in a clinical setting. The Wnt clinical trials could improve the treatment of DCU and become a new target for developing new drug treatments. The Wnt pathway interacts with the Notch signaling, Hippo and TGF-β signaling pathways. These interactions significantly increase the complexity of Wnt signaling studies, which may be a source of adverse effects in the development of pharmaceuticals. Therefore, a comprehensive approach for the preclinical phase of drug discovery is required. The role of Wnt signaling in the epidermis is diverse, due to a combination of phase and context-dependent interactions with other signal inputs. Wound healing is a complex process, and the high glucose environment of DM skin increases the complexity. For effective DCU treatment, the pathogenesis of DCU must be understood. The Wnt/β-catenin signaling pathway is involved in the wound-healing process; however, the specific mechanism is unknown. It has been reported that there is a dysfunction in the Wnt/β-catenin signaling pathway in DM refractory wounds due to a decrease in Wntl expression in high glucose environments. However, the details are unclear (The inflammatory responses involved in the healing of DCU through the Wnt/β-catenin signaling pathways, the proliferative phase, and the remodeling phase of the wound have been discussed in this article ( unclear . It has  unclear . HoweverHZ was responsible for the literature review and the writing. XN was responsible for corrections. XS, JZ, YC, QY, CS, and JY were responsible for proofreading.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.