Glycation occurs as a non-enzymatic reaction between amino and thiol groups of proteins, lipids, and nucleotides with reducing sugars or α-dicarbonyl metabolites. The chemical reaction underlying is the Maillard reaction leading to the formation of a heterogeneous group of compounds named advanced glycation end products (AGEs). Deleterious effects have been observed to accompany glycation such as alterations of protein structure and function resulting in crosslinking and accumulation of insoluble protein aggregates. A substantial body of evidence associates glycation with aging. Wnt signaling plays a fundamental role in stem cell biology as well as in regeneration and repair mechanisms. Emerging evidence implicates that changes in Wnt/β-catenin pathway activity contribute to the aging process. Here, we investigated the effect of glycation of Wnt3a on its signaling activity. Methods: Glycation was induced by treatment of Wnt3a-conditioned medium (CM) with glyoxal (GO). Effects on Wnt3a signaling activity were analyzed by Topflash/Fopflash reporter gene assay, co-immunoprecipitation, and quantitative RT-PCR. Results: Our data show that GO-treatment results in glycation of Wnt3a. Glycated Wnt3a suppresses β-catenin transcriptional activity in reporter gene assays, reduced binding of β-catenin to T-cell factor 4 (TCF-4) and extenuated transcription of Wnt/β-catenin target genes. Conclusions: GO-induced glycation impairs Wnt3a signaling function. Aging-associated disabilities are generated by an accumulation of randomly occurring molecular damages during life time, which affect the organism at multiple levels such as metabolism, immune response, repair, and regeneration as well as altered inter- and intracellular communication. Typical alterations detectable in aged tissues are increased concentrations of reactive oxygen species (ROS) and concomitant increased DNA-damage, lipid and protein oxidation ,2. OftenAGEs are generated by an initial non-enzymatic reaction of amino groups with reactive carbonyl groups of monosaccharides known as the Maillard reaction , therebyIn addition, AGE modification itself can induce signaling mainly through the receptor for advanced glycation end products (RAGE), a multi-ligand transmembrane receptor containing immunoglobulin domains ,25. RAGEThe canonical Wnt/β-catenin signaling cascade acts as a pivotal regulator of developmental and pathogenesis pathways including cell stemness maintenance/differentiation, carcinogenesis, and aging-related phenotypes ,33. MoreBased on that knowledge, we hypothesized that Wnt ligands as secreted, cysteine-rich proteins  are susc2 atmosphere at 37 °C.Human embryonic kidney (HEK)-293 and Neuro-2a cells were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% (v/v) fetal bovine serum (FBS)  and 100 U/ml penicillin, 100 mg/ml streptomycin (Sigma) in a humidified 5% COg for 10 min. The abundance of the Wnt3a protein in CM was confirmed by Western blotting and luciferase reporter gene assays. The conditioned medium was stored at 4 °C for further experiments. Only the second batch of conditioned medium was applied in the experiments. Before GO-treatment, CM was diluted 1:2 with fresh complete DMEM.Wnt3a-secreting cells (L-M(TK-)Wnt3A) and the corresponding empty vector control cell line were cultured in high glucose DMEM as mentioned above to produce Wnt3a CM and control CM . BrieflyGlyoxal (GO) and methylglyoxal (MGO) were purchased from Sigma-Aldrich . For glycation, conditioned media were pre-incubated with the indicated concentrations of GO or MGO at 37 °C for 3 h and subsequently added to either HEK-293 or Neuro-2a cells for another 18 h. In a second approach, medium was first incubated with 1 mM or 2 mM GO at 37 °C for 5 h. Then GO was removed by dialysis using membranes with a molecular weight cutoff of 4 kDa . Dialysis was conducted overnight in 2 liters of dialysis buffer ) at 4 °C. For glycation of purified recombinant human Wnt3a protein , 0.5 μg protein was incubated with 2 mM GO for 5 h at 37 °C. Monoclonal anti-carboxymethyllysine (anti-CML26) antibody was obtained from Acris/OriGene Technologies GmbH .5 cells/well. The next day, cells were co-transfected with 500 ng Topflash/Fopflash (pGL3-OT/OF) luciferase constructs  [3PO4 pH 7.8, 0.2 % (v/v) Triton X-100) and 20 μl of each sample lysate was separately subjected to Firefly- and Renilla-luciferase measurement using a Mithras LB 940 Multimode Microplate Reader .Assays were performed as previously reported . In detare, USA)  and 50 nProtein A-sepharose CL-4B  beads were pre-coupled with anti-FLAG M2 antibody  overnight at 4 °C. Cells were lysed in immunoprecipitation IP buffer , 1% (v/v) Triton X-100) with freshly added protease  and phosphatase  inhibitors and lysates were then subjected to immunoprecipitation with anti-FLAG M2 pre-conjugated beads for additional 2 h. After 3× washing, precipitated proteins were eluted with 2× sodium dodecyl sulfate (SDS) sample buffer and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting with the indicated antibodies. To investigate the effects of dialysis on GO-treated conditioned media HEK-293 and Neuro-2a cell lysates were generated after 12 h incubation with dialyzed GO-treated Wnt3a CM and control CM. Cells were solubilized in radioimmunoprecipitation assay (RIPA) buffer  sodium deoxycholate, 0.1% (w/v) SDS, 1% (v/v) NP-40) containing protease inhibitor and phosphatase inhibitor cocktails (see above). Western blots were performed using the indicated antibodies anti-β-catenin (C14) , anti-phospho-β-catenin (Ser33/37) , anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH)  according to the manufacturers’ recommendations. Anti-Wnt3a antibody was obtained from R&D Systems .t) values of the target genes were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Differential expression was calculated according to the 2−ΔΔCt method. The following primer pairs were used for the detection of canonical Wnt target genes: sp5 fwd: 5’-GGGGAGACTCAGCAGACG-3’, rev: 5’-TGGGTCCCTATGTCCGAA G-3’; axin2 fwd: 5’-CAAGCCTGGCTCCAGAAG-3’, rev: 5’-GCATCCTCCGGTATGGAAT-3’; cyclin D1 fwd: 5’-GCTGTGCATCTACACCGACA-3’, rev: 5’-TTGAGCTTGTTCACCAGGAG-3’; GAPDH fwd: 5’-AGCCACATCGCTCAGACAC-3’, rev: 5’-GCCCAATACGACCAAATCC-3’.Total RNA extraction and cDNA synthesis were conducted according to the manufacturer’s instructions using the NucleoSpin RNA II kit  and the High-Capacity cDNA Reverse Transcription Kit . Real-time PCR was performed using Applied Biosystems StepOnePlus system with BRYT Green dye  as described previously . Each PCStatistical analyses were performed using SigmaPlot software  with results presented as the mean ± standard deviation (SD). P-values of <0.05 were considered statistically significant.Glyoxal is formed in large amounts by lipid peroxidation  or at loTo test if the activity of Wnt3a is altered by GO-treatment, Topflash/Fopflash (TOP/FOP) reporter gene assays in HEK-293 and Neuro-2a cells were performed. The Topflash reporter plasmid contains three TCF/LEF response elements whereas in the Fopflash plasmid these sites are mutated. Activation of the Wnt/β-catenin pathway induces binding of β-catenin to TCF/LEF and thereby activates transcription of the Firefly luciferase reporter gene. First, HEK-293 or Neuro-2a cells were transiently co-transfected with Topflash or Fopflash and Renilla luciferase  plasmids. Wnt3a CM and control CM were pre-incubated with different concentrations of GO  for 3 h. The GO-treated media were subsequently diluted 1:2 with fresh medium and added to the reporter plasmid-transfected cells. GO-treatment of Wnt3a CM induced a dose-dependent reduction of the Wnt3a-stimulated transcriptional activity, both in HEK-293 A and NeuNext, we wanted to exclude that the GO, which is still present in the Wnt3a CM, may modify the Frizzled/ low-density lipoprotein-related protein (LRP) receptor complex at the cell surface and thereby block receptor-mediated signal transduction. Therefore, GO-pretreated cell culture media were subjected to dialysis to remove excess GO. As shown in In the absence of a Wnt ligand, excess β-catenin is phosphorylated by casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK3β) within the β-catenin destruction complex and subsequently degraded by the ubiquitin-proteasome pathway ,48. In tIn line with the inhibition of transcriptional activity and the reduced amount of total β-catenin detected in response to GO-treatment of Wnt3a CM, we assumed that β-catenin-binding to TCF-4 was attenuated. To test this, Neuro-2a cells were transiently transfected with N-terminal 3xFLAG-tagged TCF-4 and harvested 24 h later. TCF-4 was immunoprecipitated with Protein A-sepharose-CL-4B bound anti-FLAG M2 antibody and analyzed for co-precipitated endogenous β-catenin by Western blotting. Application of GO (0.5 mM)-treated Wnt3a CM resulted in the reduced co-immunoprecipitation of β-catenin .sp5 [axin2 [cyclin D1 [Finally, we performed quantitative RT-PCR to evaluate effects of glycated Wnt3a CM on the mRNA expression of known Wnt/β-catenin target genes including sp5 , axin2 [5 [axin2 , and cycyclin D1  in HEK-2Protein glycation is a spontaneous, non-enzymatic reaction of reducing sugars or α-DCs with preferentially lysine, cysteine, and arginine residues in proteins, finally resulting in a set of different modifications called AGEs. In consequence, protein structure and function are altered and often lead to the accumulation of damaged macromolecules . This stIn this study, in vitro glycation of Wnt3a in cell culture supernatants isolated from Wnt3a-secreting L-M(TK-) cells  was indusp5 (specificity protein 5) [axin2 [cyclin D1 [Taken together, our data indicate that glycation of Wnt3a impairs its signaling activity. Consistently, we observed that addition of glycated Wnt3a CM resulted in increased phospho-β-catenin and reduced total β-catenin levels, and consistently a reduced amount of β-catenin binding to TCF-4. This led to a reduced expression of endogenous Wnt target genes as shown for otein 5) , axin2 [) [axin2  and cyclyclin D1  by quantyclin D1 ,56. Axinyclin D1 .All our findings correlate with a reduced signaling activity as a consequence of Wnt3a glycation (see model in Future studies have to confirm our in vitro results in vivo to justify that Wnt ligands indeed can effectively be glycated in different processes during aging, such as impaired repair and stem cell regeneration, or may contribute to the pathogenesis of diseases such as diabetes, osteoporosis, or neurological disorders . Neverth


The effect of paraffin wax encapsulated microcrystalline cellulose (EMC) particles on the mechanical and physical properties of EMC/epoxy composites were investigated. It was demonstrated that the compatibility between cellulose and epoxy resin could be maintained due to partial encapsulation resulting in an improvement in epoxy composite mechanical properties. This work was unique because it was possible to improve the physical and mechanical properties of the EMC/epoxy composites while encapsulating the microcrystalline cellulose (MCC) for a more homogeneous dispersion. The addition of EMC could increase the stiffness of epoxy composites, especially when the composites were wet. The 1% EMC loading with a 1:2 ratio of wax:MCC demonstrated the best reinforcement for both dry and wet properties. The decomposition temperature of epoxy was preserved up to a 5% EMC loading and for different wax:MCC ratios. An increase in wax encapsulated cellulose loading did increase water absorption but overall this absorption was still low (<1%) for all composites. In the polymer science field, epoxy polymers offer great versatility and an ample range of enhanced properties, such as stiffness, specific strength, dimensional stability, chemical resistance, and strong adhesion within the matrix . Howeverd-glucose units which are capable of forming strong hydrogen bonds leading to the formation of crystal fibers [Cellulose comes from renewable resources which are abundant and readily available in nature. Cellulose provides a stable backbone of β (1→4) linked l fibers . Other al fibers ,7,8. Thel fibers ,10. Howel fibers ,12,13,14The efficiency of cellulose reinforced composites depends on the ability to transfer stress from the matrix to the cellulose fiber ,15. ThisOne key hurdle with cellulose-based composites is the strong attraction between fibers resulting in unwanted agglomeration. The interface energy and excess of hydroxyl groups result in an increased agglomeration within the composite causing stress concentrations during fiber loading ,17. CurrRecently, a wax encapsulation technique has been developed specifically for microcrystalline cellulose ,29. In tMW: 342.3 g/mol; melting point: 260–270 °C; density: 1.27–1.6 g/cm3 (20 °C)), was purchased from VWR Chemical Co. . It was stored in sealed a container before use to avoid moisture uptake from the environment.Paraffin wax was purchased from Gulf Oil Corporation, Houston, TX, USA . The matrix epoxy polymer (EPON 828)  was purchased from Fisher Scientific . All materials were stored in sealed containers and placed in a refrigerated room (5 °C). Diethylenetriamine  curing agent was purchased from VWR Chemical Co. . MCC with an average particle size of 90 μm  was used to observe the morphology of un-coated MCC and MCC after wax encapsulation. The samples were put on a transparent glass slide and another glass slide was used to separate the sample particles in order to observe individual particles. Then micrographs of each sample were taken using different magnifications.After encapsulation, the EMC particles were added and mixed with epoxy resin prior to cure. EMC loading rates of 0%, 1%, 3%, and 5% (wt % of epoxy resin) were applied at room temperature and mixed with the polymer until fully homogenized. The samples were then obtained by pouring the mixtures into aluminum dishes (10 cm diameter) after DETA was added as a curing agent (10% by weight) to EPON 828 and fully homogenized . The mixMt) was determined using the following equation:Wt is the weight of the sample after soaking and W0 is the weight of the sample before soaking and after drying in the oven.Water absorption of the composite specimens were performed according to ASTM D1037 . First tFlexure tests performed for both dry and wet samples (before and after 24 h immersion in water absorption test). Flexure tests were conducted using a three point bending protocol at room temperature and at 40% relative humidity. A Z010 Zwick/Roell testing machine  was used with a support distance of 40 mm and testing speed of 1 mm/min (ASTM D790-10) . Six speThe cross-section morphology of EMC/epoxy composites after tensile tests were observed using a Zeiss Evo 40XVP scanning electron microscope  to better understand the surface properties of the interaction or bond between Epon 828 and wax or EMC particles. The samples were mounted on aluminum stubs using carbon tape. The samples were then coated with a thin layer of gold to prevent charging before the observation by SEM. The accelerating voltage was 20 kV.Fourier transform infrared spectroscopy (FT-IR) measurements were performed using a Perkin–Elmer Spectrum 400 instrument  fitted with a single reflectance ATR diamond probe. The samples  were measured immediately after manufacture to avoid samples absorbing moisture from the atmosphere.In order to study the thermal stability and changes in degradation patterns of MCC, neat EPON 828 and their composites, thermogravimetric analyses (TGA) was carried out using a TGA Q5000 instrument  under nitrogen (20 mL/min) from room temperature to 700 °C and at a heating rate of 15 °C/min.t-test was used to detect the significant differences on the mechanical properties between the composites and the control (pure epoxy).The data of water absorption and mechanical tests were analyzed using SAS . Analysis of variance  was used to examine differences of water absorption among the wax to MCC ratios and MCC additions. A paired Generally, the MCC morphology changed after encapsulation. Epoxy composites absorbed more water after EMC addition. Water absorption increased as EMC loading increased. Interestingly, for those samples that had the same loading but different ratios of wax to MCC, the water absorption decreased with decreased wax coverage. However, most results were not significantly different, except for the loading of 3% wax and MCC ratios of 1:3 and 1:4 and the loading of 5% wax and MCC ratios of 1:3 and 1:4 . For exaWater moisture uptake occurred as cellulose loading increased and the wax to MCC ratio decreased (less wax) from 1:2 to 1:4. It was found that lower wax:MCC ratios  might decrease the epoxy modulus , which hScanning electron microscope (SEM) images were used to observe the fractured surface morphology of the EMC/epoxy composites . It was −1 for pure MCC is typical of β-glucosidic linkage. The absorption at 912 cm−1 was attributable to the unreacted epoxide group characteristic of epoxy resin. Its disappearance in the neat cured epoxy and all composites indicated that all epoxide groups reacted after cure. The strong absorptions between 1000 and 1250 cm−1 were assigned to C–O–C (ether) or C–O (H) of cellulose. The absorption at 2925 cm−1 was attributable to saturated hydrocarbon groups (C–H). Furthermore, the absorption around at 2850 and 2925 cm−1 were attributable to wax [−1 was due to hydroxyl groups.To assess the degree of cure in the matrix and understand the chemical interactions between exposed cellulose and epoxy, characteristic bands were assigned based on a priori data from the literature ,39. The e to wax ,41. Addi−1.In EMC-epoxy composites, the epoxide group reacted with –NH2 groups from the amine or hydroxyl groups on the MCC surface ,43. It iThe thermal stability and changes in degradation patterns of the raw material and the composites were evaluated by thermogravimetric analysis (TGA). Wax encapsulated microcrystalline cellulose (EMC) allowed for the homogenous dispersion of microcrystalline cellulose (MCC) in the epoxy matrix without much agglomeration. The wax coverage was not a complete sheath, resulting in adequate cellulose exposure for crosslinking between cellulose and epoxy matrix. In summary, the present wax encapsulation method is simple, cost effective, and may not require additional capital during scale up to a manufacturing process. The EMC particles exhibited many advantages such as limited agglomeration, improved hydrophobicity, and homogenous dispersion in non-polar matrices. The EMC could be employed in the epoxy matrix and provide successful reinforcement at only 1% loading. The results indicated that 1% of EMC loading with a 1:2 ratio of wax to MCC was good for both dry and wet property reinforcement, which suggested that there could be an optimum crosslink density between the cellulose and epoxy matrix. The thermal resistance of the epoxy polymer was also not lowered after addition of EMC. The epoxy cellulosic composites utilizing biomass materials could be beneficial for the polymer and wood-related industries by incorporating wasted materials into new environmental friendly products. Furthermore, the cellulosic epoxy composites could enlarge the applications of epoxy polymers, such as in packaging, automotive, and electrical fields.


BRI3 (brain protein I3) is one of the Wnt/β-catenin pathway target genes as indicated by the results of serial analysis of gene expression (SAGE) and microarray analyses performed in our laboratory. The Wnt/β-catenin signaling pathway is an evolutionarily conserved pathway, which has important functions in early vertebrate development, axis formation, cellular proliferation, and morphogenesis. Previous studies showed that BRI3 expression is upregulated at both mRNA and protein levels upon β-catenin activation by various approaches, such as lithium treatment and overexpression of Wnt ligands in Huh7 (hepatocellular carcinoma) cell lines. Moreover, with regard to the previous literature, BRI3 was found to have a very important role in the TNFα-mediated cell death pathway. In this study, we screened a human liver cDNA library by yeast two-hybrid assay using BRI3 protein as bait, with the aim of finding novel interaction partners of BRI3. Library screening by yeast mating resulted in the identification of three candidate positive clones. Among these, IFITM3 and MGAT1 proteins were confirmed as interaction partners by using cotransformation in yeast cells and coimmunoprecipitation from mammalian cell lines. Considering the poor functional characterization of BRI3 to date, identification of novel BRI3-interacting proteins is an essential first step in determining the action mechanism of BRI3 with respect to the Wnt/β-catenin pathway. BRI3 (brain protein I3) was originally identified asa 125-amino-acid transmembrane protein that isoverexpressed in TNFα-treated L929-murine fibrosarcomacells . The blocking of new BRI3 proteinsynthesis by using BRI3-antisense RNA resulted inincreased resistance of these cells to TNF-induced celldeath with magnitude greater than 1000-fold. Althoughthe exact action mechanism of BRI3 within theTNFinduced cell death pathway still remains unknown, it ishypothesized that BRI3 synthesis might act as a negativecheckpoint of this pathway .BRI3 has been selected among the potentialWnt/βcatenin signaling pathway targets based on serial analysisof gene expression (SAGE) screening and an equivalentmicroarray screeningWnt signaling is an evolutionarily conserved pathwayin various organisms from worms to mammals and playsimportant roles in several biological processes suchas development, differentiation, cellular proliferation,morphology, motility, and cell fate. Wnt proteins constitutea family of secreted cysteine-rich glycoproteins thatexhibit distinct expression patterns in embryo and adultorganismsIf the Wnt/β-catenin signaling pathway is notactivated, cytoplasmic β-catenin levels are kept lowthrough continuous proteasome-mediated degradation,which is controlled by a multiprotein complex containingglycogen synthase kinase 3β (GSK-3β), adenomatouspolyposis coli (APC), and axin. In the absence of a Wntsignal, β-catenin is present in the axin complex. In thiscomplex, cytosolic β-catenin, but not the cadherin-boundβ-catenin, is continuously phosphorylated, ubiquitinated,and degraded by proteasome .The activation of the Wnt/β-catenin signaling pathwayis initiated by binding of a Wnt ligand to the Frizzledreceptor (Fz) and low-density lipoprotein receptor-relatedprotein (LRP) 5/6 coreceptor. In this case, Dishevelled(Dsh) inhibits the GSK-3β-dependent phosphorylationof β-catenin in response to the Wnt signal. Consequently,β-catenin is dissociated from the destruction complexand starts to accumulate in the cytosol. The accumulatedβ-catenin is then translocated into the nucleus, binds to theT-cell factor/lymphoid enhancer factor (TCF/LEF) familyof transcription factors, and activates the expressions ofseveral cell cycle- and differentiation-related target genessuch as axin, c-myc, and cyclin D1The Matchmaker GAL4-Yeast Two-Hybrid System(Clontech) was used together with the pretransformedhuman liver cDNA library (Clontech). A fresh and largecolony of the bait strain (AH109 [pGBKT7/Bri3]) wasinoculated into 50 mL of SD/-Trp liquid medium andincubated at 30 °C with shaking (230–250 rpm) until theOD600 reached 0.8. The cells were centrifuged at 1000 × gfor 5 min and then resuspended in 5 mL of SD/-Trp. In asterile 2-L flask, a 1-mL aliquot of the library strain wascombined with 5 mL of bait strain. Then 45 mL of 2XYPDA liquid medium was added to the flask and incubatedat 30 °C for 22 h with slow shaking (50 rpm). After 22 hthe cells were centrifuged. The 2-L flask was rinsed twicewith 50 mL of 0.5X YPDA. The rinses were then combinedand used to resuspend the pelleted cells. The cells wereagain centrifuged for 10 min and all pelleted cells wereresuspended in 10 mL of 0.5X YPDA liquid medium.The mated culture was spread onSD/-Ade/-His/-Leu/Trp plates (200 µL per 150-mm plate). The plates wereincubated at 30 °C for 5–6 days.The yeast host strains used in this study wereobtained from Clontech (USA): AH109 and Y187 .The identification of the cDNA clone in the prey vectorwas done by yeast colony PCR and sequencing of the PCRsamples. For this purpose, single and fresh colonies werepicked with sterile pipette tips and resuspended in 3 µL ofNaOH (25 mM) in separate PCR tubes. Liquid nitrogenwas used to quick-freeze the samples. Then the sampleswere placed into the PCR machine and boiled at 95 °Cfor 10 min. Master mix was prepared using thepACT2FpACT2R primer pair and distributed to the tubes. For thePCR reaction, the following cycling conditions were used:94 °C for 5 min, 30 cycles , 72 °C for 10 min, hot-start at 94 °C.Clontech’s YeastMaker Yeast Transformation System wasused according to the manufacturer’s protocol for alltransformations of yeast cells. A PEG/LiAc (polyethyleneglycol 3350/lithium acetate)-based method was appliedfor the preparation and transformation of competentyeast cells. For the cotransformation of the bait and preyvectors, 0.2 µg of each vector was used together with 5 µLof herring testes carrier DNA (10 mg/mL).HEK293T cells were transfected with plasmids carryingthe tagged versions of the genes. First, a 1:1 suspensionof protein G agarose bead slurry was washed withicecold lysis buffer three times. Anti-HA antibody dilutedin 1X PBS was incubated with 30 µL of protein G agarosebeads for 4 h at room temperature by swinginghead-overtail. After the incubation, the antibody-bead complexeswere washed 3 times with Co-IP lysis buffer at 4 °C byspinning, and then 48 h after transfection adherent cellswere washed with ice-cold 1X PBS and then lysed inicecold Co-IP lysis buffer supplemented with 1X proteaseinhibitor cocktail and 1 mM PMSF. The cells on the platewere harvested using a cell scraper and transferred to anEppendorf tube. Complete lysis was assured by pipettingthe solution several times and incubating on ice for 30 min.The cell lysate was centrifuged at 12,000 rpm for 15 min at4 °C; 30–40 µL of input was obtained and the remainingsupernatant was added to the antibody-bead complex inEppendorf tubes. The tubes were allowed to swingheadover-tail at 4 °C in a cold room overnight. The next day,the antibody-bead-protein complexes were washed threetimes with Co-IP lysis buffer and once with 1X PBS. Thefinal supernatant was removed and the tubes were storedat –80 °C for later use in SDS-PAGE analysis.Huh7 cells grown on 18-mm coverslips inside 12-wellculture plates were used for imaging with confocalmicroscopy. The cells were transfected with the plasmidsexpressing the fluorescent-tagged versions of our proteinsof concern using the Turbofect transfection reagent(Thermo Scientific) according to the manufacturer’sinstructions. Forty-eight hours after transfection, thegrowth medium was aspirated and the cells were washedwith 1X PBS. Thereafter, 250–300 µL of ice-cold 4%paraformaldehyde was added to the cells in each well.The cells were incubated at room temperature for 20min without shaking. Then 4% paraformaldehyde wasaspirated and the cells were washed with 1X PBS threetimes. Next, 250 µL of DAPI solution (1 µg/mL) was addedto the cells. The cells were incubated with DAPI for 1–2min and then washed with 1X PBS three times. Usingforceps, the coverslips inside the wells were placed ontothe slides with 3 µL of mounting medium. After drying fora few minutes, nail polish was applied to intersection areasaround the coverslips and the samples were analyzed withthe confocal microscope.The candidate transcriptional targets of the canonicalWnt/β-catenin pathway were determined previously inour laboratory by using genome-wide microarray analysesand SAGE techniquesYeast two-hybrid assay was performed as a first stepin order to determine the interaction partners of the BRI3protein. For this purpose, a pretransformed human livercDNA library was used. Yeast mating was performedbetween the MATa strain (AH109) transformed with thepGBKT7/BRI3 bait vector and the MATα strain (Y187)pretransformed with the human liver cDNA library. Theestimated number of independent clones screened by thismating was calculated to be 2.6 × 106. The mated culturewas allowed to grow on SD/-Ade/-His/-Leu/-Trp agarplates, so that high stringency was used in order to detectthe activation of the reporter genes ADE2 and HIS3. Thepositive clones obtained from yeast mating were restreakedto single colonies on SD/-Ade/-His/-Leu/-Trp agar plateswith X-α-Gal in order to confirm the phenotype. As aresult of two-hybrid interactions, in addition to ADE2 andHIS3 reporter genes, MEL1, which encodes the enzymeα-galactosidase, can also be expressed. Yeast coloniesthat express MEL1 turn blue in the presence of thechromogenic substrate X-α-Gal. Therefore, those singleblue colonies were analyzed primarily for being candidatepositive colonies Figure .The yeast colonies selected in the blue/white screeningwere subjected to colony PCR analysis in order to identifythe corresponding cDNA inserts. PCR products thatcorresponded to single and clear bands were sequencedand the identities of those cDNA clones were revealedby performing a BLAST search of the raw outputs overvarious databanks in order to find significant matches.A list of all candidate proteins determined as putativeinteraction partners is given as supplementary material(Table S1). Several candidates from this list have abundantprotein expression levels in the liver cDNA library andare mostly characterized as “sticky” proteins in yeasttwohybrid screenings with respect to the previous literature;therefore, these proteins were eliminated as being falsepositives. Eventually, three candidate cDNA clones were determined forfurther analysis. The vectors containing the cDNA insertof interest were isolated from the corresponding yeastcolonies. Then the bait vector together with the vectorcontaining the candidate cDNA clone were cotransformedinto the yeast cells for confirmation. The transformed yeastcells were spread on nutrient selective media(SD/-Ade/His/-Leu/-Trp) and tested for their ability to grow intocolonies. As a result, cotransformation yielded coloniesfor two out of three selected cDNA clones Figure .uhTs, IThe BRI3 protein has two isoforms. The a-isoformis 125 amino acids in length. On the other hand, theb-isoform of BRI3 is a 98-amino-acid polypeptidesharing the same N-terminus but has a totally distinctC-terminus compared to the a-isoform  resulted in significantupregulation of BRI3. Supporting data were obtained fromlithium treatment of Huh7 cell lines, luciferase reporterassay, overexpression of Wnt ligands, and ChIP assay byusing the anti-β-catenin antibodyBRI3 has been poorly characterized so far, as can bejudged from the current literature. Its function and actionmechanism are largely unknown. Therefore, we aimed toprovide clues about the functional relevance of BRI3 forthe Wnt/β-catenin pathway. As a first step in our study, weintended to discover novel interaction partners of the BRI3protein in order to shed light on the action mechanismof BRI3. Yeast two-hybrid assay was employed for thispurpose. A human liver cDNA library was screened usingBRI3 protein as bait. Following the two-hybrid libraryscreening by yeast mating, colonies were selected onhighstringency growth media (-4 dropout media lacking Ade/His/Leu/Trp amino acids) Figure . IdentifWith the purpose of verifying the interactions, furtherexperiments were performed using the two isoforms ofBRI3, which result from an alternative splicing event.Alignment of the amino acid sequences of the two BRI3isoforms suggest that the N-terminals are the same;however, the BRI3 a-isoform has a distinct C-terminuscompared to the shorter BRI3 b-isoform Figure .Coimmunoprecipitation was performed in order totest for the interaction of these BRI3 isoforms with thecandidate binding partners obtained from the yeasttwohybrid assay. A strong positive interaction was determinedfor the a-isoform of BRI3 with candidate proteins MGAT1and IFITM3 Figure . HoweverFor further confirmation, a colocalization assaywas performed in Huh7 cell lines by expressing thefluorescent- tagged proteins in order to visualize theirsubcellular localizations. The results indicate an intensecolocalization of BRI3 with MGAT1, especially in theperinuclear area of Huh7 cells, which might be inside anorganelle such as the Golgi apparatus or ER Figure .In factMGAT1 is known to code for an enzyme essentialfor the synthesis of hybrid and complex N-glycans. Thefinding of MGAT1 as an interacting partner of BRI3 canbe regarded as promising in the sense that MGAT1 is alsoone of the genes that exhibit differential expression levelsin response to β-catenin activation, as was demonstratedin the initial SAGE analysis. Furthermore, our recentexperiments indicated that MGAT1 is upregulated atboth mRNA and protein levels in response to β-cateninactivation by various approaches. Thus, MGAT1 can bedefined as a novel transcriptional target of theWnt/βcatenin signaling pathwayAccording to the previous literature, IFITM3 hasbeen identified as a new molecular marker in humancolorectal tumors and it has been stated that IFITM3gene expression is controlled by Wnt/β-catenin signalingin mouse and human intestinal epitheliumIn this study, we aimed to determine the novelinteraction partners of BRI3 by yeast two-hybrid assay.In the course of this work, we have identified andconfirmed MGAT1 and IFITM3 as binding partners ofBRI3. Furthermore, all these three proteins are regulatedby Wnt/β-catenin signaling. The functional significanceof these novel interactions will be among the mostimportant subjects of future experiments.


The relative content in the ECP radicals of mono- and diphenol was determined by the MALDI-TOF mass spectrometry method. The organic epoxy fraction, according to gas chromatograpy-mass spectrometry (GC-MS), contains 50–70 wt % diglycidyl ether of bisphenol A. PEO resins obtained in the present work have reduced viscosity when compared to other known phosphazene-containging epoxy resins while phosphazene content is still about 50 wt %. Resins with an epoxy number within 12–17 wt %, are cured by conventional curing agents to form compositions with flame-retardant properties, while other characteristics of these compositions are at the level of conventional epoxy materials.Phosphazene-containing epoxy oligomers (PEO) were synthesized by the interaction of hexachlorocyclotriphosphazene (HCP), phenol, and bisphenol A in a medium of excess of epichlorohydrin using potassium carbonate and hydroxide as HCl acceptors with the aim of obtaining a product with lower viscosity and higher phosphazene content. PEOs are mixtures of epoxycyclophosphazene (ECP) and a conventional organic epoxy resin based on bisphenol A in an amount controlled by the ratio of the initial mono- and diphenol. According to  Out of all the varieties of synthetic polymers, epoxies, developed over 70 years ago, won a special place in industry and everyday life, not necessarily in terms of overall manufacturing volume, moreso in their specific role . ClassicHowever, polymers based on unmodified epoxy resins often have low and unstable performance characteristics, in particular flammability and relatively low heat resistance . A rather effective way to increase the heat resistance of epoxy matrices is with structural modification, for example, with compatible oligomers of higher functionality, which are incorporated into the three-dimensional network structure formed during curing. Examples of such resins are triglycidyl-p-aminophenol (TGPAP), tetraglycidyl-4,4′-methylenedianiline (TGMDA), and epoxidized novolacs ,4.The solution of the flammability problems of epoxy polymers is a more complex issue and involves trade-offs. Thus, a traditional and effective way to reduce the flammability of epoxy systems is to replace bisphenol A based epoxy resins with their brominated analogs. However, brominated epoxy resins, as well as other halogen-containing compounds, emit toxic gases when they come into contact with flames, which limits their use, for example, in civil aviation and transport, for environmental reasons ,6. One of the promising ways of modifying epoxy polymers to increase heat and fire resistance is the introduction of organometallic compounds, in particular phosphorus compounds, known as universal flame retardants for a large number of polymeric materials. Refusal of halogen-containing flame retardants in favor of phosphorus-based ones is a modern global trend .However, the introduction of low-molecular, non-reactive compounds into the material, including red phosphorus and its inorganic compounds  worsens the mechanical properties and transparency of polymers . OrganicThe combination of the above factors leads to the fact that epoxy resins are not always able to satisfy the growing needs of high-tech industries, especially in the aerospace industry, automotive industry, electrical engineering, electronics, etc.A possible way to solve the problems described above, including the flammability of epoxy resins, is the use of modifiers based on phosphazenes.The main chain of organophosphazenes consists of alternating atoms of phosphorus and nitrogen, and at the phosphorus atom there are organic radicals introduced by the substitution of halogen in halogenphosphazenes. The nature of organic substituents, usually introduced by the reaction of nucleophilic substitution of chlorine, can vary widely and determines the properties of the final polymer or oligomer. The unique properties of various organophosphazenes cause the ever-growing interest of researchers in phosphazene chemistry . Compare(1)The synthesis of organophosphazenes with reactive epoxy groups for addition to the epoxy component ,41,42,43(2)The synthesis of organophosphazenes with reactive amine groups for use as a curing agent or its component ,50,51,52Researchers have long been trying to combine the exceptional properties of organophosphazenes as highly effective flame retardants with the function of structural modifiers, such as polyfunctional epoxy resins. There are two main synthetic approaches that allow to obtain functional phosphazenes capable of forming covalent bonds with epoxy matrices:Functional epoxyphosphazenes are highly effective flame retardants that not only do not reduce mechanical properties, since they are well compatible with the epoxy matrix, but can also improve them, probably due to the formation of a special three-dimensional polymer network, in the nodes of which phosphazene cycles are located .Currently, the majority of methods for the synthesis of functional epoxyphosphazenes described in the literature are of primarily scientific interest due to the complexity of scaling and the large number of intermediate stages ,41,42,43In recent years, phosphazene-containing epoxy oligomers (PEO) with reduced flammability on the base of cyclic chlorophosphazenes have been synthesized and characterized . The mosThe main problem in the synthesis of PEO is the high functionality of HCP, which requires the use of a more than 10-fold molar excess of diphenol to achieve a more complete replacement of the chlorine atoms in it and to avoid gelation. As for the industrial use of epoxyphosphazenes, the main limiting factor complicating the preparation of the formulation and worsening its processing properties is that most epoxyphosphazenes are solids with a softening temperature of 80–100 ° C and high melt viscosity. High average functionality can also influence processing properties.n = 4 and 5 and a conventional organic epoxy monomer. These PEOs in comparison with pure epoxyphosphаzenes contain organic epoxide, which is in fact an active diluent that lowers the viscosity and average functionality to an acceptable level for further processing. However, the viscosity of such PEO concentrates at ambient temperature is still more than 200 Pa·s, which is a fairly high value, close to the processing limit. Epoxidation of a mixture of HAP and excess of diphenol with epichlorohydrin produces PEO, which contains fractions of the usual bisphenol A based epoxy resin and epoxycyclophosphazene (ECP) oligomers with a variable ratio . In 55,,55,56, sThe content of ECP in a mixture with the organic epoxide may be increased with simultaneous reducing of the content of residual chlorine, the functionality of HCP and its molecular weight, which was realized in our previous work  by replan. The average content of epoxy groups in these PEO is 13%–14% by weight. Compositions based on diglycidyl ether of bisphenol A (DGEBA) and methyltetrahydrophthalic anhydride, containing 15% of the obtained oligomers in the epoxy component, compared to pure DGEBA, were characterized by more than 30% higher glass transition temperature and flexural strength, and at 75% content of PEO were non-combustible and had limiting oxygen index (LOI) of 30 [According to matrix-assisted laser desorption/ionization time-of-flight MALDI-TOF mass spectrometry, the main components of the oligomer formed are phosphazene-containing di-, tri- and tetraepoxides, the ratio of which in the reaction mixture can be varied by alternation of the value of I) of 30 . HoweverThus, the development of new scalable methods for obtaining epoxyphosphazene-containing epoxy resins based on available starting materials with reduced viscosity and improved technological properties is of great scientific and practical interest and may contribute to accelerating the widespread use of phosphazene-containing resins as components of high-tech flame-retardant polymer composite materials.In order to exclude phenolate synthesis in the known method  Figure  and to oIn the present work PEO was synthesized by direct interaction of HCP, phenol and bisphenol A (BPA) in epichlorohydrin (ECH) medium as reagent and solvent in the presence of KOH with a role of both HCl acceptor and epoxy-forming reagent  e method . Potassie method .A 100 mL three-necked flask, equipped with a reflux condenser, a mechanical stirrer, and a thermometer, was charged with 1 g (0.0028 mol) of HCP, 2.68 g (0.0144 mol) of bisphenol A, 0.54 g (0.0058 mol) of phenol, and 65 mL of epichlorohydrin. The reaction mixture was heated to 50–55 °C and thermostated at this temperature for 30 min until all solids were completely dissolved. After that, 2.32 g (0.0414 mol) of potassium hydroxide was added and the process was conducted for 2 h at a temperature of 60 °C. At the end of the synthesis, the hot solution was filtered off and excess solvent was distilled off. The resulting mixture of epoxy oligomers was dried in vacuo at 85 °C. The reaction product is a slightly colored viscous liquid. The yield was 3.96 g (71%).A 100 mL three-necked flask, equipped with a reflux condenser, a mechanical stirrer, and a thermometer, was charged with 1 g (0.0028 mol) of HCP, 0.54 g (0.0058 mol) of phenol, 0.95 g (0.0069 mol) potassium carbonate, and 65 mL of epichlorohydrin. The reaction mixture was heated to 60 °C and the process was conducted for 2 h at this temperature. The reaction mixture was then cooled to 50 °C, 3.28 g (0.0144 mol) of bisphenol A was charged and incubated at this temperature for 15 min until the bisphenol A was completely dissolved. After that, 2.32 g (0.0414 mol) of potassium hydroxide was added and the process was conducted for 3 h at a temperature of 60 °C. At the end of the synthesis, the hot solution was filtered off and excess solvent was distilled off. The resulting mixture of epoxy oligomers was dried in vacuo at 85 °C. The reaction product was a slightly colored viscous liquid. The yield was 4.18 g (75%).31P and 1H NMR spectra were measured in chloroform-d solutions with a Bruker AV-400 spectrometer  operating at 162 and 400 MHz, respectively. The signals due to the deuterated solvents were used as internal references. The chemical shifts of the signals were calculated relative to the signals of tetramethylsilane (1H) and phosphoric acid (31P), which were used as references. The spectra were processed with the help of the MestReNova Lab software package .The MALDI-TOF mass spectrometric analysis was carried out on the Bruker Auto Flex II instrument .The gas chromatography-mass-spectrometry (GC-MS) study was carried out on a VARIAN-3800 CP/4000 MS chromatograph-mass spectrometer . Separation of the substances was carried out using a VF-5ms chromatographic capillary column with a length of 30 m, an internal diameter of 0.25 mm, and a fixed-phase layer thickness of 0.25 μm. The carrier gas was helium. The injector temperature was 280 °C. The volume of the injected sample was 1 μL. The temperature of the column thermostat changed from 50 °C (1 min) to 280 °C (10 min) at a rate of 10 °C/min. The temperature of the ion source and the interface was 230 and 280 °C. The mass spectra of the substances were registered in the electron impact regime in the mass range 33–1000 Da. The processing of the obtained data was carried out with the usage of computer software “Varian MS Workstation”, the identification of the substances was carried out using the NIST 11 mass-spectra library.The gel-permeation chromatography (GPC) was carried out on Shimadzu LC-20 Prominence  chromatograph equipped with refractometric and UV detector  and a PSS column , tetrahydrofuran (THF) was used as an eluent (1 mL/min). Molecular mass values were estimated with the use of a polystyrene calibration curve. Elemental analysis was carried out by spectrophotometry.2O) divided by the total molecular weight of epoxy resin. Thus, the theoretical content of epoxy groups was calculated by the formula:M are the molecular weights of the oxirane group and the whole epoxy resin molecule, respectively, and n is the number of oxirane groups in the epoxy resin molecule. The experimental epoxy group content was determined by the method of reverse acid-base titration [V is the amount of NaOH solution for titration of a sample containing epoxy resin in mL; V0 is the amount of NaOH solution for titration of blank sample without epoxy resin in mL; 43 is the molecular weight of the oxirane group; N is the concentration of NaOH solution, mol⋅L−1; and g is the mass of epoxy resin sample in g.In the present paper, epoxy group content (in wt %) is the weight of oxirane groups  with the epoxy group content of 24.6%–25.1% and epoxy equivalent of 171–175 which is referenced as DGEBA and with ERISYS® Resorcinol diglycidyl ether RDGE  with the epoxy group content of 34.4%–36.4% and epoxy equivalent of 118–125.The viscosity of epoxy oligomers was evaluated on a Reotest-2 rotational viscometer with a working cone-plane unit. The viscosity of the PEOs obtained in present and earlier papers was compared with conventional low molecular weight bisphenol A based epoxy resin D.E.R.31P NMR spectroscopy and MALDI-TOF spectrometry  together with insignificant amounts of tetraaryloxy-substituted compounds (AB2 system with doublet at δp = 20–22 ppm and triplet at δp = 5–8 ppm).According to Laser mass spectrometry data confirm the presence in the phosphazene fraction of mainly pentaaryloxy-substituted HCP, containing residues of phenol and diphenol in various proportions in the substituents near phosphorus atom , Table 1The content of compounds with different ratios of mono- and diphenolic aryloxy radicals as substituents near phosphorus atom can be controlled by varying the initial ratio of HCP:phenol:bisphenol A, as follows from The quantitative composition of PEOs was determined from the relative intensity of the peaks on the MALDI-TOF spectra. On the example of the initial molar ratio of HCP:phenol:bisphenol A equal to 1:3:5 the content of individual compounds in the phosphazene fractions formed by methods A and B  is compaMore homogeneous is the composition of PEOs obtained according to method A : It contm/z = 960 and 1150 in It is noteworthy that there are no compounds with four and five epoxy groups in the B synthesis product, but they appear in an amount up to 10 wt % of aryloxyphosphazenes with unsubstituted OH groups of bisphenol A radicals .Phenylglycidyl ether (PGE) is present in a small amount only in the product synthesized according to method B (5%–10%). This indicates the preferential interaction of phenol not with epichlorohydrin, but with HCP at the initial stages of the process.The results of the analysis of the electron impact mass spectra of the fractions  isolated4.As a result, the majority of compounds fixed on mass spectra (peaks 3–8 in A similar decomposition of the isopropylidene group with the formation of a double bond was observed earlier under conditions of the synthesis bisphenol A based hydroxyaryloxycyclotriphosphazenes at 170 °C :The main component of the organic fraction of the resulting epoxides is diglycidyl ether of bisphenol A (DGEBA), the highest yield (~70%) of which is achieved in the synthesis of method A with the simultaneous feeding of starting reagents.The compounds’ formulas given in the sixth column of It is noteworthy that in the reaction mixture there is practically no monoglycidyl ether of bisphenol A (MGEBA) with a molecular weight of 284, which corresponds to peaks with an insignificant intensity in the mass spectra of fraction 5. The main compound in fraction 5 for both synthetic schemes is the compound with a molecular weight of 268—monoglycidyl ether of 4,4′-dihydroxydiphenol-1,1-ethenyl.As for compounds with one chlorohydrin group and the central ethenyl group (peak 8 on the chromatogram), the lack of a tendency to their dehydrochlorination is apparent due to both the heterogeneity of the process (KOH is insoluble in the reaction medium) and its insignificant duration (2 h).From The properties of different PEO obtained in this work and other PEOs ,56,57 thThe phosphazene component contents found from GPC data and calculated from the phosphorus content value are close to each other and are about 50%–60% for phenol-bisphenol A based product and are up to 45% for resorcinol-based product. The values of the weight average molecular weight according to GPC exceed the values obtained by the MALDI-TOF method, which can indicate both the limited applicability of the standard polystyrene calibration for these systems and the fact that high molecular weight particles may not be fixed by the MALDI-TOF method due to their limited volatility. However, the tendency of an increase in the average molecular weight with an increase in the content of the phosphazene component is confirmed by both GPC and MALDI-TOF, regardless of the nature of the starting reagents. It should be noted that the molecular weight values of the low molecular weight organic fraction determined by GPC and GC-MS are close and correspond to the theoretical assumption that its main component is diphenol diglydyl ether. From elemental analysis and GPC data it follows that with a comparable content of undesirable chlorine, the PEO obtained in this work is characterized by the both highest content of phosphorus (up to 5.4%) and phosphazene component (up to 61%).The usage of phenol as additional reagent lead to reduced average fuctionality of the product of 1.9–2.2, which is closer to commercial DGEBA or RDGE resins when compared to 2.2–2.5 for the mixtures based on only bisphenol A or resorcinol. When compared to resorcinol-based phosphazene-containing epoxy oligomers, the values of viscosity of PEO obtained in this work are slightly higher. However, the viscosity of PEO obtained in this work at a ratio of HCP:phenol:bisphenol A = 1:4:4 and industrial bisphenol A based epoxy resin  are comparable . At the The phosphazene-containing epoxy oligomers obtained in the present work have an epoxy group content within 15–16 wt %, phosphorus content within 4.6%–5.4% and epoxyphosphazene component content of 50%–60%. These phosphazene-containing epoxy oligomers may be cured by conventional curing agents to form materials with reduced flammability, while other characteristics of these compositions are at the level of commercially available epoxy materials . The vis


However, the mechanisms of RON/RONΔ160-mediated gastric cancer growth and metastasis remain vague. We therefore examined the actions of RON, RONΔ160, and β-catenin in gastric cancer cells and tissue samples, and their effects on cell growth in vitro and in vivo. We found that in gastric cancer samples and cell lines, there was positive correlation between RON/RONΔ160 and β-catenin levels, and that they formed a RON/RONΔ160-β-catenin complex which was translocated to the nucleus. Hypoxia led the binding of hypoxia-inducible factor-1α to the RON/RONΔ160-β-catenin complex, which increased nuclear translocation and expression of downstream oncogenic signaling molecules. Overexpression of RON/RONΔ160 promoted the proliferation and migration of gastric cancer cells, which were also enhanced by hypoxia. Suppression of RON using siRNA or anti‑RON monoclonal antibody diminished gastric cancer cell and tumor growth in vitro and in vivo. These findings establish a link between the receptor tyrosine kinase RON and β-catenin and provide insight into the mechanism by which they contribute to gastric cancer progression.Aberrant accumulation of the receptor tyrosine kinase recepteur d’origine nantais (RON) has been verified in gastric adenocarcinoma. Upregulation of RON and its splice variant RONΔ160 contribute to the growth and migration in gastric cancer cells  Receptor tyrosine kinases (RTKs) are high-affinity cell surface receptors that participate in various signaling pathways regulating cellular proliferation, survival, apoptosis, and migration ,2. RTKs in vitro and in vivo [in vitro and in vivo [Recepteur d'Origine Nantais (RON), also known as Macrophage-stimulating protein receptor (MST1R), is a member of the c-Met RTK superfamily . RON is  in vivo ,18. More in vivo . We therin vitro and in vivo.Once activated by its ligand macrophage-stimulating protein , RON initiates multiple downstream signaling cascades that affect cell adhesion and motility, growth, and survival ,21. ThesPrevious studies indicate that RON is highly expressed in gastric cancer tissues , but levTo explore the interaction of RON/RONΔ160 and β-catenin at a cellular level, we analyzed the transcript and protein levels of RON and β-catenin and the downstream effector molecules in two gastric cancer cell lines and one normal human gastric mucosal epithelial cells. As in gastric cancer tissues, RON and β-catenin were highly expressed in KATOIII cells. This was accompanied by up-regulation of c-Jun, survivin and AKT, and the downregulation of c-Myc and Cyclin D1. In MGC-803 and SGC-7901 cells, which express lower levels of RON, expression of downstream effectors was also reduced . NotablyActivated β-catenin enters the nucleus and influences the expression of numerous transcription factors and, in turn, multiple cellular progresses . In the Nuclear translocation of RON may occur in serum-starved and hypoxic cancer cells ,34, and Hypoxia promotes tumor growth and increases the risk of metastasis. For example, it causes the separation of β-catenin and TCF-4 but promotes the interaction of β-catenin and HIF-1α, while reducing expression of c-Myc and Cyclin D1 ,26. In tBecause RON/RONΔ160-β-catenin signaling mediates hypoxia-induced signaling, we assessed their effects gastric cancer cell growth under hypoxic conditions. Overexpression of RON or RONΔ160 stimulated MGC-803 cell proliferation and colony formation, and this effect was increased under the condition of hypoxia . Upregulin vivo, xenograft experiments were performed. The experimental protocol is shown in in vitro data.To evaluate the tumor-promoting action of RON/β-catenin signaling on gastric cancer in vivo. We found that in mice receiving the antibody, the tumor volume (117±16 mm3 vs 1144 ± 97 mm3) and tumor weight (1.04 ± 0.42 mm3 vs 0.14 ± 0.03 mm3) were notably reduced as compared to mice receiving saline    . Moreove0.004 g) . In addi0.004 g) . These rGastric cancer is a major cause of cancer death worldwide . CurrentThe receptor tyrosine kinase RON is a member of c-MET proto-oncogene family and a novel prognostic marker and therapeutic target for gastroesophageal adenocarcinoma; 56.1% of gastric carcinomas exhibit high expression of both RON and RONΔ165, leading to a poor survival rate . In the Aberrant β-catenin expression is associated with most types of cancer, including hepatocellular, colorectal, lung, and gastric cancers . Upon acTumor cell microenvironments often induce activation of Wnt/β-catenin signaling and facilitate the adaptation of gastric cancer cells to hypoxia, leading to gastrointestinal tumorigenesis ,43. An ein vitro proliferation and migration experiments and in vivo xenograft assay showed that suppressing RON using siRNA or an anti-RON monoclonal antibody observably restrained the development and progression of gastric cancer, and that simultaneously knocking down RON and β-catenin had an even greater inhibitory effect. Thus, while β-catenin may not be indispensable, it clearly enhances the pathogenic effect.Although RON/RONΔ160 and β-catenin appear to facilitate gastric cancer progression by enhancing the cell proliferation and migration ,45, it rIn summary, our findings revealed that direct interaction between RON and β-catenin and their contribution to the pathogenesis of gastric cancer and that the variant RONΔ160 exerts similar effects. Moreover, under the hypoxic condition, like those seen in solid tumors, the RON/β-catenin complex binds HIF-1α, which further enhances the tumor growth. We therefore suggest that future investigation of that RON as new therapeutic target for anti-cancer immunotherapy is warranted.A total of 30 samples of gastric cancer tissue (14 cases), paracancerous tissue (11 cases) and normal gastric tissue (5 cases) were collected from First Affiliated Hospital, Zhejiang University School of Medicine. Written consent was obtained from all patients. The recombinant plasmid pcDNA3.1-RONΔ160 and blank plasmid pcDNA3.1 were purchased from Thermo Fisher Scientific . Female Balb/c nude mice (5-6 weeks) were purchased from Vital River  and maintained in a room under controlled light (12 h/day) and temperature (22 ± 2°C) conditions.2/95% air. For transfection of RON or RONΔ160, cells at 70-80% confluence, and plasmid-transfection reagent mixture was added to the plate for 20 min. Twenty-four hours later, the cells were collected for further investigation. For knockdown assays, siRNAs targeting RON or β-catenin were transfected into cells at 30-50% confluence using LipofectamineTM 2000 regent . All siRNAs were purchased from Thermo Fisher Scientific . For hypoxic treatment, the cells were cultured at 1% O2 in a modulator incubator.The KATOIII, SGC-7901, and MGC-803 human gastric cancer cell lines were obtained from the American Type Culture Collection . All gastric cancer cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum  at 37°C in a humidified incubator under 5% CO3 cells/well in 96-well plates and grown for 24, 48, or 72 h in complete RPMI-1640 medium. The live cell fractions were then determined using the CCK-8 kit according to the manufacturer’s instructions. The absorbance (OD) at 450 nm was measured using a multiscan plate reader.Gastric cancer cell proliferation was assessed using a Cell Counting Kit-8 . Untreated cells (Blank) and cell transfected with siRNA N.C, RON siRNA, β-catenin siRNA, RON siRNA + β-catenin siRNA, and RON siRNA + β-catenin were plated at a density of 3 x10Cells were collected using trypsin without EDTA, washed twice with ice-cold PBS, then resuspended in 500 μl of binding buffer containing 5 μl of Annexin V and incubated for 15 min at room temperature in the dark. Five minutes before measurement, 5 μl of propidium iodide (PI) were added into each sample, and the percent apoptotic cells were determined using flow cytometry .5 cells/chamber in the upper chambers on 8.0-μm, fibronectin-coated, polycarbonate membranes  and cultured in fresh serum-free medium. The lower chambers contained 600 μl of medium with 10% FBS. After incubation for 24 h, the upper chambers were removed, and the cells fixed for 30 min with ethanol. The cells were then washed twice with PBS and stained with 0.1% crystal violet for 20 min. Cell migrating through the membrane were counted using a microscope.Migration assays were performed using 12-well plates with Transwell inserts. Cells were plated at a density of 1x10Total RNA was isolated from cells and tissues using Trizol Reagent  according to manufacturer’s instructions, after which 1 μg of mRNA was used as template for reverse transcription PCR with a First-Strand cDNA Synthesis Kit . The PCR reaction was performed using SYBR Green  with the following primers: RON, 5’-CGCGGATCCGGCGCTCTTGGCTGAGGTCAAG-3’ (forward) and 5’-GGAATTCGGCACTATCTGCTCCACCTCCCC-3’ (reverse). β-actin, 5’- CTACAATGAGCTGCGTGTGG-3’ (forward) and 5’- CTACAATGAGCTGCGTGTGG-3’ (reverse). β-actin served as an internal control. Relative gene expression levels were calculated using the 2-∆∆Ct method.Total cell lysates were extracted as described previously , after wIn brief, cells were grown on cover glasses for 24 h then washed for twice with ice-cold PBS buffer. After fixing in 3% paraformaldehyde for 30 min at 4°C, the cells were washed with 50 mM NH4Cl and permeabilized with 0.1% Triton100 for 15 min at room temperature. The cells were then incubated with anti-RON, anti-β-catenin or anti-HIF-1α antibodies for 1 h at room temperature and labeled using Texas red-conjugated secondary antibodies. The immunolabeled cells were examined using Carl Zeiss LSM5 EXITER laser scanning confocal microscope. The immunofluorescence quantification for nuclear translocation was performed by manual counting (3 fields per section).6 gastric cancer cells were subcutaneously injected into the armpit area (5 mice per group). Ten days later, the xenografted mice were intratumorally injected with 50 nM RON or/and β-catenin siRNAs twice/weekly. Another xenografted mice were intratumorally injected with 50 nM β-catenin siRNAs twice/weekly followed by intraperitoneal injection of 20 μg/ml anti-RON antibody (Zt/g4) once/weekly. Tumor size was measured every 3 days. The mice were sacrificed and the tumor was excised 28 days after the initial injection. Tumors were photographed and the homogenized for protein and RNA extraction. Tumor size was calculated using the formula: 0.5 × length × width2. Monoclonal antibodies Zt/g4 specific to RON sema domain was kindly supplied by Professor Yao .Balb/c nude mice were randomly divided into case and control groups and allowed to adapted for 1 week. Thereafter, 2 x 10Three independent experiments were performed. All statistical analyses were done using GraphPad Software, Prism 5.0. Numerical data are presented as the mean ± SEM. Values of P<0.05 are considered significant.Ethical approval for the present animal study and the usage of human tissues were received from the Zhejiang University School of Medicine Ethics Committee . National Institutes of Health guide for the care and use of laboratory animals was followed by all authors when carried out the animal study.All patients provided written informed consent for the publication of all associated data in this study.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.


Amphetamine (AMPH) and methamphetamine (METH) are widely abused psychostimulants, which produce a variety of psychomotor, autonomic and neurotoxic effects. The behavioral and neurotoxic effects of both compounds (from now on defined as AMPHs) stem from a fair molecular and anatomical specificity for catecholamine-containing neurons, which are placed in the brainstem reticular formation (RF). In fact, the structural cross-affinity joined with the presence of shared molecular targets between AMPHs and catecholamine provides the basis for a quite selective recruitment of brainstem catecholamine neurons following AMPHs administration. A great amount of investigations, commentary manuscripts and books reported a pivotal role of mesencephalic dopamine (DA)-containing neurons in producing behavioral and neurotoxic effects of AMPHs. Instead, the present review article focuses on catecholamine reticular neurons of the low brainstem. In fact, these nuclei add on DA mesencephalic cells to mediate the effects of AMPHs. Among these, we also include two pontine cholinergic nuclei. Finally, we discuss the conundrum of a mixed neuronal population, which extends from the pons to the periaqueductal gray (PAG). In this way, a number of reticular nuclei beyond classic DA mesencephalic cells are considered to extend the scenario underlying the neurobiology of AMPHs abuse. The mechanistic approach followed here to describe the action of AMPHs within the RF is rooted on the fine anatomy of this region of the brainstem. This is exemplified by a few medullary catecholamine neurons, which play a pivotal role compared with the bulk of peripheral sympathetic neurons in sustaining most of the cardiovascular effects induced by AMPHs. Both acute and chronic AMPHs intake/administration determines behavioral, purely motor, and vegetative alterations; each effect relies on a quite specific constellation of reticular nuclei, which often overlap. This calls for a constant reference to the functional anatomy of the brainstem reticular formation (RF), which stands as the seminal brain area to comprehend the neurobiology of AMPHs. Short-term effects of AMPHs include intense euphoria, increased heart rate, hypertension, hyperthermia, excitation, alertness and wakefulness  and mostly methamphetamine (METH) are widely abused psychostimulants, which possess a phenylethylamine structure. In the present review article, we focus on both compounds, which, from now on are referred to as AMPHs meant “Since the present review is an attempt to relate the effects of AMPHs with specific NE nuclei of the brainstem, a preliminary synthetic overview of the neuroanatomy of these nuclei appears to be mandatory. This will make it easier to orient within the brainstem when referring to the site-specificity of the effects induced by AMPHs.Catecholamine-containing nuclei are mainly housed within the lateral extent of the RF . A very via a volume transmission. Altogether, these features synergize to produce extremely widespread effects following AMPHs activation of NE nuclei.Neurons belonging to the LC region profusely send their axons to the entire CNS, providing the main source of NE to the brain, and mostly, to the whole cerebral cortex , as demonstrated since the early ‘80s  or prefrontal cortex abolishes hyper-locomotion induced by AMPHs  potentiates D1 receptor-dependent AMPHs-induced sensitization in the ventral striatum, to an extent, which is not replicated by DAT inhibition  is involved in the behavioral effects of AMPHs. In line with this, METH releases ACh in adult mice (Dobbs and Mark, Ch6 ACh neurons are not involved in METH-induced drug-seeking behavior (Dobbs and Cunningham, p-tyrosine with the VMAT inhibitor reserpine, completely blocks AMPHs-induced ACh release both in vivo and in striatal slices (Cantrill et al., 2+ accumulation, which in turn facilitates DA exocytosis (MacDermott et al., via ACh release (Drew et al., 2+ increase and subsequent nitric oxide-synthase activation have been linked to AMPHs-induced neurotoxicity (Pubill et al., in vivo and striatal synaptosomes. Studies focusing on the effects of AMPHs-induced ACh release on NE system are missing so far, which warrants additional studies to test ACh-NE interplay following AMPHs.Although molecular targets responsible for AMPHs-induced monoamine release are well known, the molecular mechanisms through which AMPHs release ACh are not fully established yet. A stream of interpretation indicates a close functional relationship between DA system and ACh release. This stems from evidence showing that administration of D1 and D2 receptor antagonists blunts AMPHs-induced ACh release within the striatum, hippocampus and frontal cortex (Ajima et al., MF wrote the article. FL contributed to important intellectual content. FB, CB and LR contributed to the literature review and made artwork. FF was the coordinator of the article. He participated in drafting the article and also in critically revising the article.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.


Local knockdown of APC, at the site of sciatic nerve crush injury enhanced evidence for electrophysiological, behavioural and structural regeneration in vivo. This was accompanied by upregulation of β-catenin. Collectively, the APC-β-catenin-LEF/TCF transcriptional pathway impacts intrinsic mechanisms of axonal regeneration and neuronal plasticity after injury, offering new options for addressing axon regeneration.Molecules and pathways that suppress growth are expressed in postmitotic neurons, a potential advantage in mature neural networks, but a liability during regeneration. In this work, we probed the APC (adenomatous polyposis coli)-β-catenin partner pathway in adult peripheral sensory neurons during regeneration. APC had robust expression in the cytoplasm and perinuclear region of adult DRG sensory neurons both before and after axotomy injury. β-catenin was expressed in neuronal nuclei, neuronal cytoplasm and also in perineuronal satellite cells. In injured dorsal root ganglia (DRG) sensory neurons and their axons, we observed paradoxical APC upregulation, despite its role as an inhibitor of growth whereas β-catenin was downregulated. Inhibition of APC in adult sensory neurons and activation of β-catenin, LEF/TCF transcriptional factors were associated with increased neuronal plasticity  Despite misconceptions that their regrowth is robust, the axons of peripheral neurons experience only slow and incomplete recovery following injury by trauma or disease. Hesitant early outgrowth, limited intrinsic growth properties, misdirection and a hostile microenvironment all conspire to render ‘permanent’ neurological deficits in patients. New approaches to engage a more robust growth potential in neurons are required.2. In turn, their knockdown has been associated with heightened growth in both naive and preconditioned adult neurons.Manipulating the intrinsic properties of neurons and providing them with an accelerated growth pattern at the outset of regeneration is a newer approach toward improving these outcomes. However, to identify candidate proteins and pathways with the potential to significantly alter growth in otherwise stable, connected adult neurons, lessons from tumour biology are of interest. Among the repertoire of oncology molecules, including those robustly expressed in adult neurons, some attenuate cellular growth as ‘tumour suppressor’ molecules. PTEN (phosphatase and tensin homolog deleted on chromosome ten) and Rb1 (retinoblastoma) are examples that act as ‘brakes’ to neuronal regenerative regrowth3. It has, for example, been linked to neurogenesis, neuron positioning, axon and dendrite development, synaptogenesis and myelin compaction5. Additional, roles of Wnt signaling have been identified in axon guidance of corticospinal tract (CST) axons6 and in neuroprotection and regeneration after optic nerve injury7.The Wnt signaling pathway, widely expressed, is of importance in peripheral and central neurons8. APC operates to impede the key transcriptional molecule of the Wnt pathway, namely β-catenin by participating in its destruction complex, forming a structural scaffold to phosphorylate β-catenin. Phosphorylated β-catenin is subsequently degraded by the proteasome9. Loss of wild type APC expression results in the nuclear accumulation of β-catenin, which interacts with TCF/LEF transcription factors to cause aberrant gene transcription and cell proliferation. For example, over-expression of β-catenin results in enhanced tumour cell proliferation, increased cell motility and resistance to apoptosis11.Specific molecules within this pathway include the tumor suppressor gene adenomatous polyposis coli (APC), frequently mutated in colorectal cancer14. For example, conditional ablation of β-catenin in neural stem cells and their progeny impair dentate gyrus neurogenesis16. In later stages it has roles in the mature central nervous system, including regulation of the neuronal cytoskeleton and synaptic differentiation13, axon guidance and neuronal cell survival17. β-catenin phosphorylation at residue Y654 and Y142 increased axon growth and branching through TCF4/β-catenin-dependent transcription in hippocampal neurons in vitro12. Wnt/β-catenin is activated in the spinal cord in models of neuropathic pain19. Galectin-3 knockout mice expressed higher levels of β-catenin, associated with enhanced neuronal survival, axon regeneration and recovery of motor function in reinnervated hind limbs after traumatic nerve lesions20. Moreover, β-catenin is required for Schwann cell proliferation in vitro, a key partnering step in nerve regeneration20.β-catenin is important for neurons during development influencing neural proliferation, neuronal differentiation, dendrite formation and participating in the initial formation of the neural plate and neural crest21. As expected through its interactions, APC influences proliferation, apoptosis, cell adhesion, and migration7. APC is strongly expressed in both the developing and adult nervous system22. APC has been detected in neurites of neuroblastoma cells23, dorsal root ganglion neurons24and cortical neurons23. Roles identified to date include cell cycle control of neuronal PC12 (pheochromocytoma) cells25, apoptosis of neural crest cells26, neurite formation25, and receptor accumulation in synapses27.Given the critical roles of β-catenin in the developing and adult nervous system it is an expectation that APC might impact them28. In this work we examined this pathway from a differing perspective through knockdown of the APC inhibitory interaction with β-catenin. Not previously examined, we confirm expression of key elements of the pathway in adult peripheral neurons. Moreover, by manipulating this pathway we identify an impact of APC knockdown on neuronal regrowth both in vitro and during regrowth in vivo. Inappropriate activation of APC signalling in injured adult neurons appears to offer a new hitherto unrecognized regenerative ‘brake’.Collectively these findings provide a rationale to evaluate whether the activation of the β-catenin signal transduction pathway through APC manipulation promotes axon regeneration in adult neurons. Some work, however, has also suggested otherwise, identifying no impact of pre-injury β-catenin DRG knockdown on regeneration after nerve crush29. There were increases in total APC protein expression in both DRG and sciatic nerve 3 d following injury   Fig. . After sns) Fig. . APC wasns) Fig.  in the pns) Fig. .Figure 11, is very prominently expressed in slower growing nonpeptidergic IB4 binding neurons30.APC immunocytochemistry reactivity and protein expression by immunoblotting were also identified in dissociated and cultured DRG neurons after axotomy at 3d  significantly increased neurite outgrowth in dissociated DRG sensory neurons . ICG-001-treated cells lowered neurite outgrowth . We confirmed 80% knockdown of APC protein in DRG cultures after 18–20 h treatment with APC siRNA when compared to injured scramble sequence siRNA controls Fig. . Mean towth Fig.  comparedwth Fig. . Whereaswth Fig. , combinewth Fig. . These awth Fig. .Figure 534. This reciprocal relationship, involving β-catenin degradation by the ubiquitin-proteasome system35 was confirmed above in adult sensory neurons. β-catenin associates with the transcription factors of the lymphoid-enhancing binding factor/T cell factor (LEF/TCF) family to stimulate cell proliferation. To examine this aspect of APC/β-catenin signalling, we further knocked down APC expression in sensory neuronal cells using siRNA (50nmol) as above and examined protein expression. Western blot analysis and immunohistochemistry confirmed APC knockdown  applied to the crush site immediately after 15 min, with repeated injections over 5 days post crush injury  In vivo, local knockdown of APC, at the site of a sciatic nerve crush injury is associated with indices of improved functional and structural recovery. Overall, the findings support important roles for reciprocal and meaningful APC-β-catenin signalling during injury and recovery following peripheral nervous system injury.The major findings of this work were (i) primary sensory neurons and axons express the reciprocal signaling partners APC and β-catenin; APC rises and β-catenin declines following axotomy injury; (ii) knockdown of APC in adult sensory neurons increases their neurite outgrowth and increases β-catenin nuclear expression; (iii) rises β-catenin and LEF-TCF expression are associated with increased neurite outgrowth following nerve injury in the setting of APC knockdown; inhibition of β-catenin activity blocks enhanced APC siRNA related outgrowth 38.While peripheral neurons exhibit some capacity to regenerate, in contrast to severe limitations in the CNS, the extent of recovery remains limited and the timetable is prolonged. It is a misconception that peripheral nerve injuries are capable of robust or full regeneration: functional deficits following major nerve trunk injuries or axonal disease remain severe and persistent. Given these issues, there is significant interest in unraveling a range of molecules and pathways that impact the growth properties of postmitotic neurons. Transcription factors can activate or repress target gene expression by binding multiple promoter regions with diverse impacts on a large number of genes. Inducing the expression or activity of transcription factors and coordinately blocking the function of transcriptional repressors provides an efficient way to enhance the regenerative potential of neurons11, dendritogenesis, synapse formation, neurite outgrowth, neural regeneration, and cell survival through prevention of apoptosis39. Previous reports showed that the role of the Wnt/β-catenin pathway in neurite outgrowth has been examined in other neuronal or neuronal-like cells40. β-catenin inhibition with small molecules in PC 12 cells deregulates β-catenin/TCF/LEF signalling and inhibits neuronal differentiation and outgrowth32. These interactions are complex however. For example, APC deficient cortical neurons exhibit exuberant axon branching in vitro41. APC has been shown to be involved in cell cycle control of neuronal-like PC12 cells, and apoptosis of neural crest cells23. In our work, we identify paradoxical rises of APC protein in DRG after injury, when enhanced growth is advantageous. Moreover, the primary sensory neurons expressing APC were of a variety of cell sizes but there was particularly intense expression involving small caliber neurons that colocalized with IB4-expression. Usually, IB4 small positive neurons have lower levels of the expression of the heavy neurofilament subunit in transverse section of DRG ganglia. In vitro this distinction is usually less prominent when the neurons are dissociated. IB4-binding-positive neurons are nociceptive, innervate the epidermis and demonstrate attenuated growth properties both in vitro and in vivo. Following nerve injury, the epidermal terminals of IB4 neurons retract, a finding potentially linked to skin hyperalgesia. Our previous studies30 have shown that sensory neurons expressing glial cell-derived neurotrophic factor coreceptor α1 (GFRα1), which is largely overlapping with IB-4 neurons, had lesser baseline growth and plasticity. Similarly, Tucker et al.42 showed that IB-4 neurons have impaired neurite growth, although they are capable of growth after exposure to GDNF and laminin. Our results are in line with these findings and suggest that APC is prominently  expressed in IB4-positive neurons after nerve injury and may serve as a mechanism of neurite growth inhibition. It is interesting that this population also expresses a higher level of PTEN, an additional inhibitor of growth1. Why two growth ‘brakes’ should be similarly upregulated in this population of neurons is interesting and not known at this time. Notably, the injury-related upregulation of APC was also expressed in Schwann cells and in axons regenerating in the proximal stump of the injury site. Our results are consistent with upregulation of APC proteins and downregulation of β-catenin protein in vivo, which may promote apoptosis through enhancement of the β-catenin destruction complex involving proteasomal degradation of ubiquitinated proteins39.In this work, we addressed the role of the transcriptional repressor APC, a tumour suppressor molecule, and its partner β-catenin, during axon regeneration following peripheral nerve injury. We identified a role for this canonical pathway as regulated by APC. β-catenin-TCF/LEF transcriptional factors are essential in neuronal fate specificationet al.43 have also shown that Wnt/β-catenin signalling is required for adult hippocampal neurogenesis under physiological conditions. In post mitotic neurons, overexpression of β-catenin together with cadherins, participates in dendritogenesis, synaptogenesis and synaptic formation44. There is also a role for Wnt/β-catenin signalling in regulating synaptic plasticity and axonal growth. Similarly, the N-and C-terminal domains of β-catenin enhancement mediate the cell-cell adhesion or promote axon branching in CNS neurons in vivo45. In our work, APC siRNA enhanced levels of intracellular β-catenin that accumulated in the cytoplasm and nucleus where it is expected to form a complex with T-cell factor or lymphoid enhancer factor (LEF/TCF) proteins; this was associated with robust increases in neurite outgrowth. It is remarkable that the impact of APC was substantially greater in preconditioned neurons, indicating persistent expression of this block to regrowth previously considered optimal for neurons. However, pharmacological inhibition of β-catenin blocked the impact of APC knockdown. In separate work, blocking Wnt/β-catenin signalling was shown to abolish neurogenesis in adult hippocampal progenitor cells in vitro and suppress neurogenesis in vivo43. Notably, β-catenin disruption also showed increased neuronal cell death, suggesting that the Wnt/β-catenin/LEF pathway controls not only the generation but also the survival of newly generated neurons46.In contrast, the Wnt/β-catenin signal is activated in tumour cells when APC is non-functional. This drives high levels of β-catenin accumulation in the cytoplasm that translocate into the nucleus, form a complex with TCF/LEF factors and activate Wnt target genes involved in cell survival, proliferation, and differentiation. Activated Wnt signalling plays a central role in intrinsic regeneration in the adult retina, and activation of the signalling promotes retinal neuron regeneration after injury. Lie in vivo improved indices of peripheral nerve regeneration following sciatic nerve crush. The findings may differ from those of Saijilafu et al.28 who did not identify an impact of β-catenin knockdown on regrowth of sensory axons after a nerve crush in mice. This paper identified key roles for PI3K in supporting peripheral nerve regeneration, operating through activation of pAkt, inactivation of GSK3 and induction of Smad1. Their approach, involved DRG electroporation and siRNA knockdown of β-catenin 2 days before crush and 5 days before harvesting and evaluation. It is possible that early knockdown within DRGs may be transient and if carried out early, might not achieve persistent knockdown during early regrowth. Our approach was to repeatedly apply siRNA through the first week of regenerative regrowth when it is possible that β-catenin expression is more important.Here we also note that siRNA-mediated APC protein suppression 19. Despite this caveat we did identify evidence of enhanced regeneration in our model after APC knockdown. Regenerating axons had evidence of greater maturity with higher conduction velocities. The numbers of myelinated axons distal to the injury zone were substantially greater in APC knockdown nerves. While the findings suggested a direct neuronal and axonal impact, we cannot exclude impacts on local Schwann cells and myelination independently. While conduction velocities were higher, mean axon calibers were similar, but this likely reflects repopulation by added numbers of smaller caliber regenerating axons not captured by the conduction studies (which emphasize the fastest conducting fibers). As in previous work, a non-viral siRNA approach applied locally to a site of axon damage provided knockdown of the inhibitory protein locally and in the ipsilateral DRG. Additional work in vivo could include longer term studies such as assessment of skin reinnervation.While not shown here, crush injury characteristically is associated with an earlier (approximately 14 days) decline in the sensitivity of the hind paw to thermal and mechanical sensation. While this usually improves somewhat by 28d, in the present work, limbs exposed to local APC knockdown had a remarkable gain in the sensitivity of the limb to both sensory modalities. In the case of mechanical sensitivity, it is possible that our results parallel findings of allodynia identified as mediated by β-cateninin vitro and nerve regrowth in vivo. As in other systems, this canonical signaling pathway appears to be a key player in neuronal regrowth.In summary, the present study provides evidence that APC/β-catenin signalling pathway impacts the regeneration of neurons after injury in the adult mammalian peripheral nervous system. Manipulation of this pathway influences neuronal plasticity ad libitum. Animal care and all experimental protocols were reviewed and approved by the University of Calgary and University of Alberta Animal Care Committees and followed the guidelines of the Canadian Council of Animal Care guidelines. Experiments were designed to minimize the number of animals required and to minimize animal suffering as per Canadian Council on Animal Care guidelines.Adult male Sprague–Dawley rats (180–200 g) and CD-1 mice (18–20 g) were obtained from Charles River Laboratories, Quebec, Canada. All animals were housed on 12-h light/dark cycle with food and water Before surgery, rats or mice were anesthetized with Isofluorane . Under sterile conditions, the left sciatic nerve was cut or crushed at the mid thigh level, the skin wound was sutured with 3–0 silk suture and the rats or mice were caged individually for three days. For sham injury, the left-side skin was cut at the mid thigh level, and the muscle was displaced to expose the sciatic nerve, but the nerve was not injured. Buprenorphine jello was provided once a day after the injury for analgesia.in vitro using a modification from the method of Lindsay47. Briefly, 35 mm dishes were coated with 0.1% poly-L-lysine  overnight and next morning washed 3 times with double distilled water and coated with 0.01% laminin (Sigma) for 1 h at 37 °C. L4-L6 DRGs were removed from the rats and placed into L15 medium (Invitrogen), where the axon roots and dural tissue were manually removed. The DRG were washed three times in cold L15, then DRGs were incubated for 1.5 hours at 37 °C in L15 containing 1 mg of collagenase per ml. After multiple pipetting, the solution was centrifuged at 800 rpm for 5 minutes, followed by three washes in L15. Cells were resuspended in 1 ml of L15, and passed through a 70 micron mesh. Then the cell solution was loaded gently onto 2 ml of 15% BSA in L15 and centrifuged at 900 rpm for 10 minutes. The pellet cells were washed once in L15, and resuspended in DMEM-F12 (Invitrogen) medium containing 1% N2 supplement (Invitrogen), 10 µM cytosine β-D-arabinofuranoside hydrochloride (Sigma), 0.5–0.8% BSA, 0.2 ng/ml NGF (Cedarlane Laboratories) and 1% penicillin/streptomycin. Finally, cells were plated immediately on 4-well plates, previously coated with poly-L-lysine. The plate was maintained in an incubator at 37 °C with 5% CO2. For in vitro inhibitor studies, neuronal cultures were incubated with 12 µM concentrations of the β-catenin pharmacological inhibitor (ICG-001), were treated with culture medium, and the carrier DMSO treated cells served as control. In separate experiments APC siRNA (Qiagen), or scrambled siRNA were transfected using HiPerFect Transfection reagent (Qiagen) according to manufacturer’s instructions. Briefly, per well of a 4-well plate, 5 µl transfection reagent and 50nmol siRNA (2.5 µl) were each added to 50 µl DMEM without supplements. The two solutions were combined and the complexing reaction was allowed to continue for 20 min at room temperature. One hundred microlitres of the siRNA-transfection reagent complex were added to each well. Cultures were kept for 18–20 h then fixed and processed for immunocytochemistry.Before tissue harvesting, rats were anesthetized with Isoflurane (Abbot Laboratories) and then killed three days following the conditioning lesion or sham surgery. Primary adult DRG neurons were dissociated and maintained Tissue samples from rats were fixed in modified Zamboni’s fixative , 0.5% picric acid, and 0.1% phosphate buffer) overnight at 4 °C. Tissues were then washed in PBS three times, cryoprotected in 20% sucrose/PBS, and left at 4 °C overnight. After embedding in optimum cutting temperature (OCT) compound (Miles), 14-μm-thick sections were placed onto poly-L-lysine-coated slides. The slides were probed for APC , β-Catenin , and double labelled with anti-NF-200 , anti-S100  or unconjugated IB4 lectin (Vector Laboratories) followed by goat anti-IB4 , and incubated at 4 °C for 24 h then rinsed three times in PBS. Secondary antibodies were anti-mouse IgG CY3 conjugate  and Alexa Fluor 488 goat anti-rabbit IgG conjugated  for 1 h at room temperature. Finally, cover slips were mounted on the slides with bicarbonate buffered glycerol (pH 8.6) and the slides were viewed with a multiphoton microscope (Nikon A1R). Negative controls included omission of primary antibodies on parallel sections  for 30 minutes at RT. Neurons and neurites were stained with 1-hour incubation of primary antibodies including mouse anti-neurofilament 200 (NF200) , rabbit anti-APC  and rabbit anti-β-catenin , incubated overnight at 4 °C. After washing with PBS twice for 15 minutes each, secondary antibodies [CY3 sheep anti-mouse  and Alexa Fluor 488 goat anti-rabbit ] were applied following PBS at room temperature for 1 h. The samples were then washed in PBS and mounted using DAPI vectashield (Vector laboratories) to label the nuclei and imaged using a Zeiss Axioskope fluorescent microscope with digital camera and Axiovision imaging software . Adobe Photoshop software was used to merge images. For neurite extension assays, the NF200-labeled DRG neurons was photographed and the pictures were analyzed by MetaExpress software to measure the total neurite growth, the number of neurites per cell body, and the mean cell body size based on fluorescent intensity (of NF200) above background; this is set by program parameters based on the level of background. Number of primary branches were quantified by the MetaExpress program . For analysis between 40 and 60 neurons were analysed per condition and per culture day .1. Briefly, total RNA samples from non-injured and injured sciatic nerve tissue from mice was extracted using an TRIzol Reagent  according to the manufacturer’s instructions One μg of total RNA was treated with DNAse  and processed to complementary DNA (cDNA) synthesis using the Super Script II Reverse Transcriptase (Invitrogen). Primers used were as follows: APC1 F 5′-TGACCTTTGGAGATGTTGCCA-3′; APC1, R 5′-CCGCAAAACACTTGCAATAACC-3′ RPLPO: F, 5′-AAGAACACCATGATGCGCAAG-3′; R, 5′-TTGGTGAACACGAAGCCCA-3′.Quantitative reverse transcription-polymerase chain reaction was performed according to previous descriptionsCT) with an exponential growth of polymerase chain reaction product during the linear phase was recorded. Relative expression values were generated using the comparative CT method (2CT−∆∆), where all genes of interest were standardized to the expression of RPLPO.Products were labelled using SybrGreen I fluorophore (Invitrogen). The cycle number at which the fluorescence signal crossed a fixed threshold (threshold cycle Sciatic nerve and DRG (L4-L5) samples were prepared in RIPA lysis buffer (Sigma), containing a protease inhibitor cocktail. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Separated proteins were transferred onto PVDF membrane in Tris-glycine-methanol buffer for 2 h at 4 °C. Membranes were blocked for 1 h at 25 °C in phosphate-buffered saline with 0.1% Tween 20 supplemented with 5% non-fat milk. They were then incubated overnight with the respective antibodies: APC , β-Catenin , LEF , TCF  and tubulin or actin  as a loading control in 2% bovine serum albumin in Tris-buffered saline (TBS). The membranes were then incubated for 1 h at 37 °C with a goat anti-rabbit and a goat anti-mouse immunoglobin antibodies conjugated to horseradish peroxidase and diluted, respectively, at 1:10,000 and 1:10,000 in TBST containing 0.1% Tween 20 and 5% non-fat milk. Immunoreacting bands were detected using the enhanced chemiluminescence (ECL) detection reagents. Quantification of bands was through Adobe Photoshop and the band densities were normalized with those of the loading control.in vivo injection, 4.05 µl of siRNA (1.35 µM) were mixed with 20 µl HiPerfect Transfection reagent and 35.95 µl saline. An injection of 40 µl of siRNA preparation was administered to each mice crush site, and remaining 20 µl transdermally into the hind paw on the crush side for 7days.The control (scramble) siRNA or APC siRNA were transfected using HiPerfect Transfection reagent (Qiagen) according to manufacturer’s protocol. For 48. In brief, a radiant heat source was applied individually to the middle of the hindpaw and the latency (in seconds) to withdrawal was measured. Three separate trials were performed for the withdrawal response. Mechanical and thermal testing was performed on identical days with an interval of at least 1 h between the two tests. Analyses were done with the observer blinded to the nature of the treatment group.The experimental mice underwent mechanical (Von Frey filaments), hindpaw grip strength and thermal testing at Day 0 (before crush injury), and 28 days following sciatic nerve crush, There were 5-min intervals provided between a total of 3 trials performed during the same day. Mechanical sensitivity was measured using an automated Von Frey apparatus . A filament with a progressively-increasing force (2 g/s) was applied to the plantar surface of the mice through the wired mesh until a withdrawal reflex was noted. Three separate trials were conducted and the mean latency (time to withdraw) and amount of threshold force were calculated. For testing the recovery of thermal sensation, we used the Hargreaves apparatus49. Motor sciatic nerve conduction recordings were carried out using stimulation at the sciatic notch and knee with recording over interosseous foot muscles in mice. Sensory sciatic nerve conduction recordings were carried out by stimulation of the toe digital nerves with recording at the knee. Near-nerve temperatures were maintained at 37 °C ± 0.5 °C.Motor and sensory electrophysiology were carried out under isoflurane anesthesia, as previously described50. Transverse sections at 1-µm thickness were made through the approximate center of the crush area and stained with toluidine blue for light microscope morphometry.Ipsilateral, sciatic nerves distal (~10 mm) to the crush site were harvested at 28 days after sciatic crush. Mice underwent sciatic crush then daily application, for 6 days, of APC or scrambled siRNA. Sciatic nerves were fixed in glutaraldehyde (2.5%) buffered in cacodylate (0.025 M) overnight, washed, and stored in cacodylate buffer (0.15 M), then post-fixed in osmium tetroxide (2%), washed in graded alcohols then embeddedStatistical analysis using one-way ANOVA with Tukey post‐hoc analysis was performed for neurite extension data. When only two experimental groups were compared, a Student’s t-test was used.Supplementary Information