The importance of Wnt/β-catenin signaling in cancer stem cells (CSCs) has been acknowledged; however, the mechanism through which it regulates the biological function of CSCs and promotes cancer progression remains elusive. Hence, to understand the intricate mechanism by which Wnt controls stemness, the specific downstream target genes of Wnt were established by analyzing the genetic signatures of multiple types of metastatic cancers based on gene set enrichment. By focusing on the molecular function of Wnt target genes, the biological roles of Wnt were interpreted in terms of CSC dynamics from initiation to metastasis. Wnt signaling participates in cancer initiation by generating CSCs from normal stem cells or non-CSCs and augmenting persistent growth at the primary region, which is resistant to anti-cancer therapy. Moreover, it assists CSCs in invading nearby tissues and in entering the blood stream, during which the negative feedback of the Wnt signaling pathway maintains CSCs in a dormant state that is suitable for survival. When CSCs arrive at distant organs, another burst of Wnt signaling induces CSCs to succeed in re-initiation and colonization. This comprehensive understanding of Wnt target genes provides a plausible explanation for how Wnt allows CSCs variation during cancer progression. Conversely, expression of a non-degradable β-catenin expands the CSC population [+ normal stem cells and triggers neoplasia by transforming these cells into CSCs [Wnt signaling is a highly complex and evolutionarily conserved pathway that maintains pluripotency during embryonic development and regulates homeostasis in somatic stem cells from various tissues . In recepulation . In the pulation . Similarpulation  and the pulation  and livepulation . Althougnto CSCs . In addinto CSCs . Hence, nto CSCs ,3,4. Subnto CSCs . CSCs thnto CSCs ,6. In th® System, Available online: http://www.ingenuity.com, Redwood City, CA, USA). Subsequently, we interpreted these biological functions in terms of initiation, persistence, maintenance, resistance to anticancer therapy, invasion of neighboring tissues, and metastasis of CSCs , receptor tyrosine kinase (RYK) or receptor tyrosine kinase-like orphan receptor (ROR), but not through β-catenin . The can of CSCs . The ful of CSCs  and the + cell populations [+ cells and Wnt activation was also confirmed in localization analyses of LGR5+ cells [+ cells decreases tumor formation in human organoids [+ cell populations and contributing to tumor initiation [Cancer initiation is associated with various intrinsic and extrinsic factors, including direct DNA damage by genotoxic compounds, viral infection, and inherent and acquired genetic mutations. Tumorigenesis is strongly associated with the presence of CSCs, which cause heterogeneity of cancer cells according to proposed hierarchical models . Althougulations . The pos5+ cells , which w5+ cells . A recenrganoids . Moreoverganoids , creatinitiation .+ cells exhibit the stem-like characteristics and the ability to overcome quiescence with sufficient niche signals [min/+ mice leads to significant reduction in DCLK1 protein levels, which consequently attenuates crypt hyperplasia and tumorigenesis in APCmin/+ mice without affecting downstream NF-kB signaling [min/+ mice attenuates intestinal adenoma and adenocarcinoma, whereas DCLK1 overexpression facilitates intestinal tumorigenesis in this model [+ stem cells. Long-lived DCLK1+ tuft cells were also involved in regeneration upon chemical or microbial-induced injury and likely contributed to colon cancer as a tumor-initiating population with persistent Wnt activation [DCLK1, the transcription factor, is regulated by Wnt signaling through the LEF-binding site at its promoter region  and has  signals ,45. Alth signals ,47,48. D signals , and β-cignaling . In addiis model . Wang et+ or CD44+/CD24− breast CSCs exhibit higher TCF/LEF-dependent transcriptional activity and treatment with Wnt3a further increases relative numbers of CSCs, whereas Wnt ligand knockdown decreases them [+/CD133+ liver CSCs decrease following the blockade of β-catenin-dependent transcription [Other CSC biomarkers include cluster of differentiation 44 (CD44), cluster of differentiation 133 (CD133), and aldehyde dehydrogenase (ALDH), which are positively regulated by Wnt/β-catenin signaling. Accordingly, compared with non-CSCs, ALDHses them . Similarcription . HoweverBoth CSCs and non-CSCs can grow persistently, whereas only CSCs adapt to the surrounding environment to avoid cell death. Accordingly, small subpopulations of CSCs of patients have been maintained for years in differentiated culture systems, and non-CSCs can be de-differentiated to CSCs using cytokine supplements under appropriate culture conditions ,54 and nhigh/CD133high CSC populations, and KLF5 overexpression enhances colony-forming ability and resistance to anticancer drugs, such as cisplatin and 5-fluorouracil, reflecting increased anchorage-independent growth [high/CD133high cells [+ stem cells suppresses their proliferation and survival in association with nuclear localization of β-catenin and the generation of abnormal apoptotic cells in intestinal crypts [Kruppel-like factor (KLF5) is a zinc-finger transcription factor that is critical for maintaining stem cell integrity and regulating the cell cycle . The KLFt growth . Regulatgh cells . In micel crypts .Endothelin1 (EDN1) is primarily expressed in vascular epithelial cells, where it maintains vascular tone. EDN1 is reportedly secreted by many solid tumors, and it induces persistent growth and survival by promoting cell proliferation and suppressing apoptosis. Although the mechanisms that lead to pathological EDN1 levels are unclear, β-catenin suppression has shown to be closely associated with reduced EDN1 mRNA expression . MoreoveAchaete-scute homolog 2 (ASCL2) is an intestinal stem cells transcription factor that contains a basic helix-loop-helix domain and a downstream Wnt signaling target . ASCL2 oAs a member of the activator protein-1 superfamily, Fos-related antigen-1 (FRA1) positively regulates transcription and post-transcriptional processes. Accordingly, elevated FRA1 mRNA levels are associated with aberrant β-catenin accumulation in lung , brain , breast The transcription factor Myc proto-oncogene protein (MYC) favors cell growth and proliferation and suppresses cell cycle arrest ,74. ThesCyclin D1 (CCND1) is a traditional Wnt/β-catenin target gene, and time-lapse photography and quantitative image analyses have shown that CCND1 facilitates continued cell cycle progression . This stChemo-resistance has also been associated with the ATP-binding cassette subfamily B member 1 (ABCB1), which is an ATP-dependent drug efflux pump that transports molecules across cell membranes. ABCB1 overexpression and its product P-glycoprotein are responsible for multiple drug resistance (MDR). The basal promoter of ABCB1 has several β-catenin/TCF4/LEF1-binding sites, suggesting that the canonical Wnt/β-catenin pathway regulates ABCB1, as shown in early colorectal , neuroblAlthough the origin of CSCs remains controversial, re-acquisition of CSC phenotypes in differentiated non-CSCs has been reported in multiple studies, and EMT is considered a major driver. EMT is a crucial process for morphogenesis during embryonic development and was initially recognized by developmental biologists. However, studies conducted in the last decade have shown that EMT can be aberrantly rebooted in adult tissues under pathological conditions, such as fibrosis and cancer in terms of poor wound healing and cancer invasiveness, respectively. EMT also contributes to the acquisition of invasion potential during breast carcinogenesis and chemotherapy. Specifically, during EMT, morphologic and phenotypic changes increase cancer cell motility, dissemination, invasiveness, and dedifferentiation ,94. Mult+ ovarian CSCs exhibit high MT1-MMP expression, and MT1-MMP knockdown specifically inhibits CSC invasiveness without affecting non-CSCs [Matrix metalloproteinase-7 (MMP7) is a well-known β-catenin target gene. Accordingly, ectopic LEF1 expression increases β-catenin binding to TCF-binding sequences and activates the MMP7 promoter . Moreovenon-CSCs . These eIn a previous study, inhibition of hyaluronan (HA) synthase-2 (HAS2), a biosynthetic enzyme of hyaluoran which is known as one of MMP7 regulators, was shown to decrease MMP7 expression under the condition of HAS2 inhibition and consequently inhibit the invasion ability of colon cancer cells . HAS2 ex+ populations tend to expand when Wnt signaling is activated by APC mutation. In contrast, CD44+ populations are depleted by abrogation of the TCF4/β-catenin complex following genetic disruption of the TCF4/β-catenin-binding site [+ populations frequently bound to CXCR4/CXCL12 [+ CSCs with TCF/β-catenin-dependent expression.HA biosynthesis is activated as a consequence of increased HAS2 expression following Wnt signaling activation. Thereupon, the extracellular matrix components enter into HA-rich condition. As CD44 has HA binding site in its extracellular domain, HA binds to CD44 and transfers signals that mediate cellular responses to microenvironments, thereby increasing its motility . This HAing site . Similar4/CXCL12 , suggest+ glioma CSCs that co-express the CXCR4 [+ subpopulations of CD133+ pancreatic CSCs can evade primary tumors and disseminate into the blood stream [Moreover, the direct regulation of Wnt on CXCL12 has been confirmed in previous studies . Specifihe CXCR4 , and oned stream . Variousd stream ,122. Col+/CD24−/low or ALDH1high/CD24−/low expressing CSCs have been discovered in patients with metastatic breast cancer [Both cancer cell and CSC populations are heterogeneous, and CSC subpopulations with higher metastatic potency are likely to be disseminated into nearby tissues. In addition, migration to distant sites through the blood stream leads to the colonization of secondary organs and outgrowth. Circulating disseminated CD44t cancer . Similart cancer  and recut cancer  cancer. t cancer .Dickkopf-related protein (DKK1) is a Wnt downstream molecule and a negative feedback component that likely plays essential roles in the regulation of late competency. Upon activation, Wnt reportedly binds TCF in the promoter region of DKK1 and induces its expression . Li et aMoreover, one study has indicated that Wnt/β-catenin signaling controls metastatic colonization of target organs and that human lung adenocarcinomas enhance the competence of lung adenocarcinoma cells to colonize bone and brain tissues using distinct Wnt signaling pathways through LEF1 and HOXB9 . During Claudin1 (CLDN1) is a tight junction protein (TJP) that regulates the permeability of epithelial barriers through which small ions and neutral solutes enter . Recentl+ CSC [+ CSCs are recently documented as a subpopulation that forming outgrowths at distant metastatic sites by exhibiting constitutive reprogramming [+ CSCs [+ CSCs are more frequently observed from the secondary organ outgrowth site with elevated β-catenin accumulation level, while CD44v6+ CSCs are hardly observed from the primary tumor site [+ populations was significantly decreased under the conditions of TCF4 loss [+ CSCs compared to that of CD44v6− counterpart, and the LEF/TCF-dependent transcriptional activity is activated in CD44v6+ CSCs as well [+ CSCs [Metastatic CSCs has recently emerged which refers the specific subpopulations of CSCs that colonize better at the distant organs than other cell populations ,143. The+ CSC ,118,144.gramming . The spemor site . In addiCF4 loss . Accordi as well . Moreove [+ CSCs . From thIn pre-metastatic niches, various components of secondary microenvironments can stimulate CSC colonization by activating Wnt signaling. For example, the extracellular matrix protein tenascin C is commonly found in stem cell niches and supports Wnt-mediated outgrowth of breast cancer micrometastases by increasing the expression of Syndecan 4, which has been implicated as a co-receptor of the Wnt receptor FZD7 ,146,147.+ T cells to regulatory T cells, thereby inducing apoptosis of CD8+ cytotoxic T cells [+ cytotoxic T cells [Lastly, Wnt signaling activation facilitates immune surveillance evasion through Cyclooxygenase-2 (COX2), which is directly upregulated by Wnt through its LEF/TCF promoter-binding site  and prod T cells . Through T cells . CollectWnt signaling is considered as a major contributor to CSC biology. In this review, we summarized and characterized Wnt signaling mechanisms that regulate CSCs from initiation to metastasis. Initially, continuously upregulated Wnt signaling converts normal stem cells and differentiated cells into CSCs. Subsequently, abnormally hyperactivated Wnt signaling allows entry into the early stages of metastasis and then facilitates persistent growth, invasion, migration, and homing. Negative feedback of Wnt signaling pathway then induces CSC dormancy, and subsequent hyperactivated Wnt signaling is central to re-initiation and colonization of metastatic sites. Although Wnt signaling inhibitors have been developed in multiple studies, their use is limited by the involvement of Wnt signaling in homeostasis and development, leading to potential side effects. Moreover, Wnt signaling is dynamic throughout the process from initiation to metastasis, complicating the timing of therapeutic interventions that target Wnt. Therefore, further comprehensive studies on the downstream mechanisms of Wnt are required to develop novel therapeutic agents.


Dishevelled (DVL) assembles Wnt signalosomes through dynamic head-to-tail polymerisation by means of its DIX domain. It thus transduces Wnt signals to cytoplasmic effectors including β-catenin, to control cell fates during normal development, tissue homeostasis and also in cancer. To date, most functional studies of Dishevelled relied on its Wnt-independent signalling activity resulting from overexpression, which is sufficient to trigger polymerisation, bypassing the requirement for Wnt signals. Here, we generate a human cell line devoid of endogenous Dishevelled (DVL1– DVL3), which lacks Wnt signal transduction to β-catenin. However, Wnt responses can be restored by DVL2 stably re-expressed at near-endogenous levels. Using this assay to test mutant DVL2, we show that its DEP domain is essential, whereas its PDZ domain is dispensable, for signalling to β-catenin. Our results imply two mutually exclusive functions of the DEP domain in Wnt signal transduction – binding to Frizzled to recruit Dishevelled to the receptor complex, and dimerising to cross-link DIX domain polymers for signalosome assembly. Our assay avoids the caveats associated with overexpressing Dishevelled, and provides a powerful tool for rigorous functional tests of this pivotal human signalling protein. Summary: A physiological complementation assay in Dishevelled null-mutant human cells establishes an essential function of the DEP domain of Dishevelled in its binding to Frizzled for signal transduction to β-catenin. The Wnt/β-catenin cascade is an ancient signalling pathway that specifies numerous cell fates during embryonic development and tissue homeostasis of animals, and its aberrant activation leads to various cancers, most notably colorectal cancer . Its keyDVL is an ancient signalling hub protein found in all animals and in humans. It functions as a pivot in transducing Wnt signals either to β-catenin to effect nuclear Wnt responses, or to various alternative cytoplasmic effectors that elicit ‘non-canonical’ Wnt responses, typically in post-mitotic cells, specifying cellular features such as planar cell polarity (PCP) . AnimalsThe DIX domain has a unique activity in undergoing dynamic and reversible head-to-tail polymerisation resulting in meta-stable filaments, which enables Dishevelled to assemble signalosomes . These ain vitro binding assays based on recombinant protein suggested that this motif can interact directly with the PDZ (The PDZ and DEP domains have both been implicated in the binding to Frizzled receptors. Frizzled proteins are distantly related to the G-protein coupled receptor (GPCR) family  cells whose three DVL paralogs were deleted by CRISPR–Cas9 technology . These DVL TKO cells are unable to stabilise β-catenin in response to Wnt and to activate β-catenin-dependent transcription, but these responses can be restored by re-expression of DVL2 at near-endogenous levels. Using this assay to test various mutants of DVL2, we demonstrate that its DEP-dependent binding to Frizzled is essential for Wnt-dependent signalling to β-catenin, whereas its PDZ domain is dispensable for β-catenin responses. This establishes two mutually exclusive functions of the DEP domain, switching between binding to Frizzled and cross-linking Dishevelled polymers for signalosome assembly. Our complementation assay opens up further studies of the function of this pivotal signalling protein, and might also be potentially adaptable for the study of non-canonical Wnt responses.We generated DVL TKO HEK293T cells using CRISPR–Cas9 technology, selecting individual cell clones that fail to express any of the three DVL paralog proteins, as judged by western blotting A, and coFig. S1). These results corroborate the epistatic relationship between Dishevelled and LRP6 in human cells, establishing LRP6 as a key effector of Dishevelled during signal transduction to β-catenin.As Dishevelled signals through LRP6 , we wondFinally, we also noticed that DVL TKO cells proliferate more slowly than their parental control D, consisWe chose DVL2 to restore Wnt/β-catenin responses in DVL TKO cells as this is the predominant DVL paralog in many mammalian cells e.g. . Dvl2 isBy contrast, β-catenin-dependent transcription is clearly Wnt3a-dependent if DVL TKO cells are supplied with low levels of DVL2–GFP by means of the pBABEpuro vector, which produces ∼10× induction of the β-catenin-dependent signalling activity, corresponding to ∼50% of the transcriptional response mounted by the parental HEK293T cells B. This iMovie 1). It is thus likely that the growth of the stably transfected DVL TKO cells depends on their ability to limit the expression of the DVL2–GFP rescue transgene. There seems to be negative selective pressure to ensure that the cellular concentration of DVL2–GFP is kept to near-physiological levels .The rate of proliferation of the DVL TKO line stably expressing DVL2–GFP is indistinguishable from that of its parental wild-type (wt) control D, indicaXenopus Fz7 interacted with the purified PDZ domain from Dvl1 . For comparison, we also tested these mutants after transient transfection of these cells, i.e. under conditions of overexpression . Described below is the use of the β-catenin-dependent reporter to assess the rescue activity of DVL2 as, of the five read-outs monitored  spectroscopy, which is capable of detecting low-affinity binding in the high micromolar Kd range. We purified 15N-labelled minimal wt or mutant PDZ domains of DVL2 after bacterial expression, and acquired 1H–15N correlation spectra, alone or after incubation with a well-validated C-terminal peptide from Dvl1 , confirming that the ligand binding of this mutant is severely compromised, as previously shown , confirming that the overall structural fold of the PDZ domain is not affected by any of these point mutations. We also tested a Lipoyl (Lip)-tagged peptide spanning the KTxxxW motif from FZD5 (GKTLESWRRFTS) for binding to Lip–PDZ, but failed to see any spectral changes  previously tested for interaction with Dvl1 PDZ  that are expected to block ligand access, based on structural information on this cleft and its interaction with cognate physiological ligands . To conf1 DVL-C; . As expe1 DVL-C; A, as pre1 DVL-C; . Project1 DVL-C;  confirme1 DVL-C; B. In conly shown . Howeverly shown C, which  changes D. An equDvl1 PDZ . Our faiOE assays, despite its relatively high expression levels; however, V334E and DARA responded to Wnt in the same way as wt DVL2–GFP . Thus, the DEP domain is essential for the recruitment of Dishevelled to Frizzled, whereas the PDZ and DIX domain are dispensable.To confirm that the L445E and K446M mutations block the binding of DVL2 to Frizzled, we tested their effects on full-length DVL2–GFP in a recruitment assay based on co-overexpression with SNAP–FZD5 in HEK293T cells see also , alongsiFig. S4), thus forming a β-sheet that connects two DEP monomers. Some of the residues in this β-sheet have hydrophobic side-chains  that mediate association of two dimers into a tetramer. Each of these residues also functions in the monomer to mediate DEP binding to Frizzled , but which occupies a relatively peripheral position within the Frizzled-binding DEP finger .To resolve this, and to determine whether DEP tetramerisation is required for Wnt-dependent signalling in DVL TKO cells, we aimed to substitute residues that are involved selectively in tetramerisation but not in Frizzled binding. The best candidate is I447 whose side chain contributes two key hydrophobic interactions to the DEP tetramerisation interface , as previously shown . By contrast, the dimerisation-defective DEP mutants are both proficient in restoring low levels of FZD6 and FZD7, like the PDZ cleft mutants, the polymerization-deficient DIX mutants, and a mutant whose YHEL motif downstream of the DEP domain was substituted to AHEA . Thus, the cumulative structural and biophysical evidence argues strongly against the notion that the Dishevelled PDZ domain binds directly to Frizzled, consistent with our findings that this domain is also dispensable for Wnt-dependent signal transduction to β-catenin. In other words, the physiological role of the Dishevelled PDZ domain is yet to be demonstrated; its main function might be to downregulate signalling through binding to negative Wnt regulators such as Dapper and Naked  sustains development to viable flies without a detectable defect in signalling to Drosophila β-catenin  are viable . It thus seems that normal development in flies requires only a fraction of the wt function of its Dishevelled DEP domain.One of our most striking observations was that the expression levels of wt and signalling-competent DVL2 mutants were kept low, to a level similar to that of endogenous DVL, indicating that these levels are subject to negative selective pressure. This can be explained by our finding that cell cycle progression is blocked if DVL2 is left to accumulate to high levels, although we have not investigated the molecular basis for this phenomenon. Conversely, the expression levels of our inactive DVL2 mutants were universally high, possibly as a result of positive selective pressure; clearly, absence of Dishevelled slows down the rate of cell proliferation D, and th-catenin . LikewisDrosophila suggested that Dishevelled functions below LRP6 ; SNAP–FZD5 ; LRP6–GFDVL  or LRP6 ; clones were screened by immunoblotting, and deletions were confirmed by genomic DNA sequencing. To generate stable DVL2-expressing cell lines, DVL TKO cells were transfected with 200 ng DVL2–GFP pBabePURO:PEI (1:3), and selected with 2 µg/ml puromycin.To generate DVL TKO cells, HEK293T cells  targetin4 cells were seeded, and cells were counted every 24 h ; results were analysed by two-way ANOVA with repeated measures, and are shown as mean±s.e.m.HEK293T cells were cultured and transfected as described , and sin417-511, DVL2 PDZ265-361, FZD5 (GKTLESWRRFTS) and FZD8 peptide sequences (GKTLESWRALCTR) were tagged N-terminally with 6×His and Lip followed by a linker (ENLYFQS) encoding a cleavage site for tobacco etch virus (TEV) protease. Lip–Fz7 -RIL cells (Stratagene) at 37°C, grown overnight at 24°C, and induced with isopropyl β-D-1-thiogalactopyranoside at OD 0.8. Lip–DEP, Lip–PDZ and Lip–FZDs were purified on NiNTA beads (Qiagen) essentially as described . Backbone resonance frequencies were obtained for 500 µM 13C,15N-labelled protein, using unmodified Bruker pulse programmes for HNCACB, CBCA(CO)NH, HN(CA)CO and HNCO. BEST-TROSY (1H-15N correlation spectra were acquired for purified 15N-PDZ with 128 complex and 1024 points (1.1 and 0.7 Hz/point in the processed data) in t1 and t2, respectively, and 8 transients per t1 point. In each case, 100 µM PDZ protein was incubated with 3× molar excess of DVL-C .Minimal wt and mutant escribed , and useST-TROSY  or fast-


Compared to epoxy nanocomposite containing 2.5%wt of Nanomer I.28E organoclay (m-clay), it was found that at 2.5%wt d-clay loading, significant enhancements of ~14%, ~6% and ~26% in tensile strength, tensile modulus, and fracture toughness of epoxy nanocomposite can be achieved, respectively. Effect of DNA as clay modifier on thermal performance of epoxy nanocomposite containing 2.5%wt d-clay was evaluated using TGA and cone calorimetry analysis, revealing significant decreases of ~4000 kJ/m2 and ~78 kW/m2 in total heat release and peak of heat release rate, respectively, in comparison to that containing 2.5%wt of m-clay.Deoxyribonucleic Acid (DNA) has been recently found to be an efficient renewable and environmentally-friendly flame retardant. In this work, for the first time, we have used waste DNA from fishing industry to modify clay structure in order to increase the clay interactions with epoxy resin and take benefit of its additional thermal property effect on thermo-physical properties of epoxy-clay nanocomposites. Intercalation of DNA within the clay layers was accomplished in a one-step approach confirmed by FT-IR, XPS, TGA, and XRD analyses, indicating that  Layered silicate clays have been widely utilized to equip the pristine polymers with value-added properties, such as considerable mechanical strength, thermal durability, and gas impermeability169Tg) of epoxy polymers. To reduce the plasticisation effects, strong interactions e.g., covalent bondings between clay modifiers with polymer matrix are inevitably required to be established at interface18Tg of epoxy-clay nanocomposites. A common strategy to surmount the plasticisation effects at interface is coupling the hydroxyl groups of surface and edges of clay with polymer matrix through silane compoundsLayered silicate clay materials inherently have a hydrophilic nature and as such their compatibility with most industrial polymers is poor and consequently incorporation of unmodified clay into polymers, not only, does not improve performance of the polymers, but also potentially could deteriorate intrinsic properties of the parent polymers111315et al. coated montmorillonite with protein biopolymers extracted form soy plant using the pH change, leading to the exfoliation of clay layers into biopolymerset al. reported a biocompatible epoxy/clay nanocomposite with enhanced mechanical properties for tissue engineering applications using modified bentonite with an oil derived from a specific plantet al.in situ polymerization of dopamine within clay layers. In this approach, cationic amine groups of polydopamine were exchanged with clay cations, and the hydroxyl groups of polydopamine were tasked to enhance the interfacial interactions through forming hydrogen bondings with an epoxy polymer.During the past decades, there has been a significant interest in use of sustainable and renewable materials instead of conventional hazardous substances for development of high-performance materials2526One of the most promising renewable materials, recently employed to enhance thermal performance of textile fabrics is DNA derived from fishing industrial waste323332353638−1 and 3420 cm−1 are ascribed to H−O−H stretching vibration bands of water molecules bonded to the Si−O surface on the clay. The stretching bands of Al−OH and Fe−OH are also appeared at below 916 cm−1. The peak at 1635 cm−1 observed for p-clay can be attributed to the –OH deformation of water. The Si−O stretching vibration bands are observed around 1100 cm−1. After modification of clay with DNA, obvious new peaks at around 1230 cm−1, 1680 cm−1, and 3200 cm−1 were appeared in FTIR spectrums of d-clay and DNA, as indicated in −1 in FTIR spectrum of DNA. Moreover, an obvious peak at 1450 cm−1 denotes presence of C = C stretching bonds in nucleobase of DNA structure for both d-clay and DNA samples. As shown in + cations were removed from d-clay structure. d-spacing was investigated using XRD analysis. As shown in d-spacing from 1.2 nm for p-clay to 1.9 nm for d-clay. This increment in d-spacing of d-clay facilitates monomers penetration within clay layers. However, in comparison to the d-clay, m-clay has higher d-spacing which is due to having modifier based long alkyl chains.Wa), immersion wetting (Wi), and spreading wetting (Ws). The work of dispersion (Wd) is the sum of these three aforementioned wetting terms which can be expressed as follows:Organophilicity of clays depends on wetting of modified clay by epoxy resin, which plays a significant role in dispersion quality in the matrix. The process of wetting of clays by epoxy resin consists of three types of wetting including adhesion wetting (γLV) and contact angle between epoxy and nanoclay (θ°). Wa, Wi, and Ws are spontaneous when θ° < 90°41et al.Wd could prove such observations. The highest Wd was observed for the d-clay. Compared with m-clay, a considerable increase ~27% could be observed for d-clay i.e., Wdincreases from 132.47 mN/m to 168.52 mN/m after 3600 s. This is arising from functional groups introduced to d-clay, being able to have physico-chemical interactions with epoxy resin, leading to strong repulsive forces to keep particles apart; consequently, the tendency to form agglomeration is supposed to be less in d-clay, compared to m-clay.Wetting and dispersion could be determined by the epoxy surface tension , τ and τc are, respectively, the shear stress and yield stress. The K and n are the flow consistency index and the flow index, respectively. Flow index determines the flow behavior. In other words, n < 1 for shear thinning behavior and n > 1 for shear thickening behavior could be observed in the nano-suspensions. The Herschel–Bulkley’s model parameters were calculated and presented in Where c is observed for suspensions reinforced with the same content of d-clay. Another extra reason behind such trend could be related to the temporary formation of hydrogen bonding between hydroxyl resulting in initial resistance toward shear stress with functional groups of DNA carbohydrates. Such phenomenon is more obvious at high contents of d-clay. To put it differently, compared with nanosuspension filled with 5 wt% m-clay, the addition of the same content of d-clay to epoxy shows an increase of ~23 Pa in τc. It could be deduced that role of DNA as reactive modifier in increment of viscosity as well as τc would be more effective in higher contents because interfacial interactions lead to decrease the possibility of agglomeration formation, which causes more d-clay to be involved in formation of network. Moreover, the same increasing trend is observed for flow consistency, whereas a decreasing trend could be detected for flow index.As illustrated in n e.g., 0.76 and 0.72 are observed for the nano-suspensions containing 2.5 and 5 wt% d-clay, respectively, whereas dispersion of m-clays into epoxy resin could not induce the same shear-thinning performance. In other words, higher shear-thinning could be only seen when clay is modified with DNA based modifier. It is argued that although modification of clay with DNA could increase viscosity of nano-suspensions, we subscribe to the view that a significant decrease in the entanglement density of epoxy molecules could be obtained. It could imply that aligned d-clay could act like slippery agents, schematically presented in 47As viscosity behavior of the nano-suspensions also depends on nanoclay dispersion levels into epoxy matrix, another prerequisite condition for viscosity discussion is the relation of dispersion level with flow index. As discussed in literatures45d-spacing in the nanocomposite. As can be seen for EP-D2.5 sample, this nanocomposite system shows no peak in its XRD pattern, demonstrating that initial d-spacing of dry d-clay which was ~1.9 nm, is completely expanded so that its 2θ° becomes <2° . This finding reveals the present of exfoliated structures in this nanocomposite. In contrast, EP-D5 system has an obvious XRD peak reflection in 2θ° = 3.1° which implies formation of the induced intercalated clay structures into epoxy nanocomposite at higher d-clay content with a basal d-spacing of ~2.8 nm.In order to verify the hypothetical considerations discussed in DSC and rheological analyses, nano/micro-structures of epoxy nanocomposites containing d-clay were examined by XRD and TEM analysis. Details of nanocomposites structure were investigated by TEM observations as illustrated in 5051The mechanical properties of epoxy nanocomposites containing various concentrations of m-clay and d-clay are shown in et al.et al.et al.et al.On the contrary, as evidenced by DSC and rheological analysis presented earlier, m-clays do not interact effectively and covalently with epoxy resin compared to d-clays. When it comes to moduli, it is argued that the moduli of nanocomposites could be improved by adding either m-clay or d-clay. It means that although interfacial adhesion could enhance nanocomposite properties, moduli are mostly controlled by some factors such as: (i) high aspect ratio of a single clay platelet, (ii) higher stiffness of fillers, and (iii) restriction of polymer chain mobility11E’) versus temperature for various epoxy systems. Moreover, as presented in δ, which is the ratio of the loss modulus to the storage modulus, gives insight into polymer chains movement in relation with the strength of the epoxy system. From temperature corresponding to the maximum value of tanδ, glass transition temperature (Tg) can be obtained. Moreover, crosslink density of the epoxy systems could be evaluated using following equation5657ve is the estimation of crosslink density, and R is the universal gas constant. Er is storage modulus corresponding to the Tr where Tr is Tg + 30, and Eg is also defined as storage modulus corresponding to the Tg−30.Where T < Tg which is glassy region of epoxy system. However, the storage modulus of EP-M2.5 system become lower than that of the EP at T > Tg. In other words, the EP has higher storage modulus at its rubbery region in comparison to EP-M2.5. This is because of the formation of plasticity effect on epoxy matrix at the interface with m-clay, leading to a reduction in ability of load transfer from matrix to the m-clay, causing lower Tg of EP-M2.5 in comparison to the EPTg of the EP decreases from 172 °C to 169 °C when incorporating m-clay into epoxy matrix, which is in agreement with other reports2259Tg of EP-D2.5 shows 6 °C and 9 °C increases, respectively compared to the pure EP and EP-M2.5. These increments not only do denote that no plasticity effect at interface of d-clay with epoxy is present, but also show that d-clay can establish a strong interface with epoxy, leading to the restriction of segmental chains motion. Moreover, the EP-D2.5 shows 0.35 mmol/m3 increment in the crosslink density while a significant reduction was observed for the crosslink density of EP-M2.5, compared to the EP system. This means that delaminated/exfoliated d-clay layers provide a higher surface available to be encountered with epoxy matrix, being able to have chemical bondings with the matrix, whereas a high crosslink density at interface of m-clay with epoxy matrix is effectively hindered by inducing the plasticity effect.As it can be seen from d-spacing could lead to epoxy monomer diffusion. As epoxy monomer is diffused, elastic force applied by epoxy molecules cross-linking inside the clay galleries leads to exfoliation of clay layers i.e. swelling of clay galleries occurs49d-spacing resulting from long alkyl chain quaternary ammoniums, its interactions with epoxy molecules still remains as weak as van der Waals forces, which can act like flaws in composites causing their premature failures and delamination, under mechanical loadings. Moreover, the higher chance of formation of agglomerates in m-clay could intensify such devastating effects and as such the properties of such composites will be similar to the micro-particle filled compositesTi and Tmax , char yield at 850 °C, and total activation energy required for thermo-oxidative degradation , in evaluation of thermal properties using TGA analysis. Activation energy for each degradation step was calculated using Kissinger methodWe have investigated the effect of DNA as clay modifier and natural flame retardant on the thermal properties of epoxy-clay nanocomposites. In order to have a comprehensive evaluation, thermal performance of nanocomposites was examined by TGA and cone calorimetry to compare the thermo-oxidative degradation and flammability properties. β is heating rate, and C is constant. By plotting E value can be obtained using the slope, which their plots are presented in Where Ti, Tmax,1, and Tmax,2 are observed, respectively, for nanocomposite incorporated with 2.5 wt% d-clay when compared to that incorporated with 2.5 wt% m-clay. As shown in Etotal of EP-D2.5 is ~172 kJ/mol which is ~10 kJ/mol higher than that of the EP-M2.5. These TGA results show a significant additional thermal stability effect on epoxy nanocomposites containing d-clay, resulting from DNA intercalated within clay layers.As expected, addition of 2.5%wt clay regardless of its modifier type enhances all thermal characteristics of epoxy matrix. Regarding the effect of DNA modifier on the thermal properties, 16 °C, 6 °C, and 19 °C increases in tPHRR) were extracted and summarized in Combustion behaviours of samples were also examined by cone calorimetry as a useful tool in evaluating the flame retardancy performance under a forced-flaming combustion. 2. The PHRR value of EP-M2.5 is 1298 kW/m2 which is only 224 kW/m2 lower than that of pure sample. While d-clay exhibits a notable flame retardant effect on epoxy system and the addition of 2.5%wt d-clay into the epoxy matrix results in a 322 kW/m2 reduction in PHRR. Moreover, tPHRR of pure EP increases from 71 s to 87 s and 96 s for the EP-M2.5 and EP-D2.5, respectively. This shows that EP/D2.5 requires 9 s longer to reach its PHRR, in comparison to EP/M2.5. The mechanism behind this observation stems from intrinsically flame retardancy of DNA modifier, possessing phosphate groups which can act as a barrier in formation of char. Moreover, DNA can release ammoniac and carbon dioxide gas under heating conditions and reduce flammability of the system2 lower than that of the EP-M2.5. These remarkable reductions in PHRR and tPHRR values of EP-D2.5 could also result from the insulation barrier effect of a cohesive and compact char layers on postponing the oxygen diffusion and the escape of volatile decomposition compounds produced during the combustion2The results demonstrate that pure EP exhibits a high PHRR of 1542 kW/mWe have compared the PHRR and THR values as well as mechanical performance of the epoxy-clay nanocomposite containing 2.5 wt% d-clay with the published reports in literatures in which epoxy nanocomposites contain both low and high nanofiller loadings. As shown in tPHRR values of epoxy composites containing neat clay, neat fish DNA were also obtained and their results are presented in -DNA2.5 sample), the highest improvement in both PHRR and THR were observed which are ~60.3% and ~51.1% decreases in PHRR and THR, respectively. This clearly confirms that DNA is an effective agent for improvement of flame retardency of epoxy polymer systems.Moreover, in order to evaluate the contribution of DNA in overall flame retardency of the epoxy systems, PHRR, THR, and Nonetheless, despite the role of DNA in improving the flammability performance of clay-epoxy nanocomposites, DNA to a great extent is incompatible and unprocessable with epoxy resin only which extremely hinder fabrication of epoxy-DNA composites with appropriate mechanical performance. Therefore, to take advantages of DNA performance, it should be grafted on a proper platform such as clay prior to incorporating into epoxy to provide composites with both improved mechanical and flame properties. In other words, both DNA and clay are set to cooperate with each other considerably through various mechanisms: (i) DNA can contribute to uniform clay dispersion as well as clay-matrix interactions leading to greater mechanical and flame properties, (ii) DNA agent can yield further improvement in flame properties via its functional groups having phosphorous and nitrogen, (iii) the reduction in mechanical properties which may be caused by DNA agent can be compensated by well dispersed clay. As a result, both flame and mechanical properties can be enhanced.The results of epoxy droplet contact angle revealed ~44% increase in work of dispersion which is a critical factor in determination of epoxy resin compatibility and reactivity, and ~17% further decrease in penetration of epoxy droplet into DNA modified (d-clay), both in comparison to a commercially modified clay e.g., Nanomer I.28E.The dispersion levels of d-clay into epoxy matrix studied by XRD and TEM analyses confirmed the outstanding role of DNA as a modifier and its remarkable influence on the well dispersed structures including intercalated/exfoliated structures arising from intra-gallery polymerization.The rheological behaviours of epoxy-clay nanosuspensions, as another evidence for dispersion and interaction, proved the possibility of interactions between d-clay and epoxy monomers leading to formation of a network, which possesses high viscosity level and being resistance to shear rates. It was concluded that d-clay attached to epoxy chains could act as slippery agents to promote shear thinning behaviour.Tg of epoxy/d-clay system versus ~1% decrease for epoxy/m-clay system, both compared to pure epoxy system, demonstrates that plasticity effect of nano-clays on Tg of epoxy nanocomposite was eliminated as a result of the effective interfacial interactions.Inclusion of d-clay into epoxy resin led to a significant improvement in tensile strengths, moduli and fracture toughness compared to composites containing m-clay. This phenomenon results from improved clay-matrix interfacial adhesion, better dispersion and more effective role of d-clay in consumption of crack energy through various mechanisms such as crack arresting, deviation, and pull out procedures as confirmed by SEM micrographs. Observation of ~3% increase in Contribution of DNA molecules to the considerable improvement of thermal stability and fire resistancy of epoxy-clay systems was approved by TGA and cone calorimetry results. This improvement is as a result of the formation of condensed char layers during combustion due to the release of effective suppressant agents during the decomposition of DNA structures.Waste DNA from fishing industry has a great potential for recovery and re-use as a source of flame retardant materials in nanocomposites while improving strength. Herein, for the first time we have shown that fish DNA can be used in modification of clay nanomaterials for preparation of epoxy nanocomposites with significantly improved mechanical and flammability properties. Based on the results obtained in this study, the following detailed conclusions can be drawn for the proposed application:Epoxy resin  and diethylenetriamine as curing agent were obtained from Sigma-Aldrich and used as received. The used pristine clay was sodium montmorillonite and the organoclay was a commercial product under the name of Nanomer I.28E, which were supplied by Nanocor Co., USA. DNA powder from herring sperm was supplied from Sigma-Aldrich and stored at below 8 °C. All the solvents used in this study were of analytical grade.3 solution. The obtained DNA-modified clays (d-clays) at various pHs were then vacuum dried at 60 °C prior to use. By measuring differences between initial pristine clay weight with various d-clays weight, amount of intercalated DNA at each pH can be calculated, which is presented in To intercalate the DNA structures into clay layers, as-received DNA (2.00 gr) was dispersed into 200 ml DI water by stirring for 1 h, followed by adding 1.0 M HCl aqueous solution to adjust the pH to 2, 3, 4, and 5. The resultant solutions were stirred for further 3 h at 60 °C. In a separate beaker, pristine clay (2.00 g) was dispersed into 200 ml boiling water and stirred for 2 h before sonicated for 1 h in an ultrasonic bath. The dissolved and pH adjusted DNA solutions were added to the clay/DI water suspension and further stirred for 6 h to allow for the complete cation exchange process. The final mixtures were then filtered and washed several times with abundant DI water until no chloride detected by adding 0.1 N AgNOTo take the full advantage of solvent properties in increasing the layers spacing in clay, fabrication of polymer nanocomposites were conducted according to the “slurry-compounding” processα source at a power of 180 W (15 kV × 12 mA) and a hemispherical analyser operating in the fixed analyser transmission mode. Survey spectra were acquired at a pass energy of 160 eV. The atomic concentrations of the detected elements were calculated using integral peak intensities and the sensitivity factors supplied by the manufacturer. XRD patterns were obtained using a PANalytical X’Pert Pro Diffractometer with Cu Kα radiation (λ = 1.54184 Å), operated in 2°–10° (2ϑ°) at 45 kV and 30 mA with a step size of 0.033. The spreading of an epoxy droplet on compacted discs of clay, provided with a compaction pressure of 20 MPa, was analysed using KSV Model CAM101 Contact Angle Meter  equipped with an Olympus DP70 high resolution microscope at ambient temperature. A 4 μL droplet of epoxy was poured onto compacted discs with diameter and thickness of 13 mm and 4 mm, respectively; and the amount of epoxy droplet penetrated to each clay substrate was evaluated by digital image analyser.FT-IR spectra were recorded with KBr pellets containing the samples on a FTIR spectrophotometer of Bruker Optics. X-ray photoelectron spectroscopy (XPS) analysis was performed using an AXIS Nova spectrometer  with a monochromated Al K2, according to the ISO5660 standard.DSC analyses were performed using a TA Q200 DSC instrument in high purity nitrogen atmosphere. The samples were heated up to 150 °C at the heating rate of 10 °C/min. From the exotherms obtained, the heat of reaction and the peak temperature were determined. Rheological evaluations were carried out using a TA DHR 3 rheometer with cone–plate geometry. A cone with a diameter of 40 mm and a tilt angle of 2° were utilized, and gap width was fixed to be 49 μm. The range of shear rate, used in this experiment, was chosen to be between 0–1000 1/s. The nanosuspensions were located between the cone and plate and soaked for five minutes. Dynamic mechanical properties of the epoxy-clay nanocomposites were examined using a TA Instruments Q800 in the cantilever bending mode. The instrument was calibrated before use and the samples were prepared according to ASTM E1640 before being mounted on a single cantilever clamp. The DMA analysis were carried out from 25 °C to 250 °C at a heating rate of 2 °C/min and the frequency value of 1 Hz. TGA tests of various modified clay were carried out using a Perkin–Elmer TGA instrument at the heating rate of 10 °C/min under a steady nitrogen flow of 60 ml/min. While, TGA analyses of polymer nanocomposites were operated at various heating rates under an air flow of 100 ml/min. Flammability of the polymer nanocomposites were examined by cone calorimeter  and measurements were performed at an incident heat flux of 35 kW/m1C) and critical strain energy release rate (G1C) according to following equations:The fracture surfaces of tensile samples were examined using a scanning electron microscope (SEM) operated at 25 kV. The fracture surfaces were gold-coated prior to microscopy observations. Transmission electron microscope (TEM) samples with specimens of approximately 80 nm in thickness were prepared using a Leica Ultracut UCT ultramicrotome at room temperature. Microtomed sections were imaged by a Philips TEM at 300 kV in bright field mode. Tensile tests were performed on dog-bone samples according to ASTM D638 Type I by using an Instron universal testing machine; cross-head speed 5 mm/min with a 30 kN load cell. Moreover, according to ASTM D 5045, fracture toughness was measured using the compact tension specimen see  with dimAnd PQ, B, W, a, E, and ν are the maximum load, the thickness, the width, crack length, Young’s modulus, and Poisson’s ratio, respectively. According to literatures, Poisson’s ratio is considered 0.35 for DER 332 epoxy resinWhere How to cite this article: Zabihi, O. et al. Fish DNA-modified clays: Towards highly flame retardant polymer nanocomposite with improved interfacial and mechanical performance. Sci. Rep.6, 38194; doi: 10.1038/srep38194 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Caudal type homeobox 2 (CDX2) is expressed in intestinal epithelial cells and plays a role in gut development and homeostasis by regulating cell proliferation. However, whether CDX2 cooperates with the mammalian target of rapamycin complex 1 (mTORC1) and Wnt/β-catenin signaling pathways to stimulate cell proliferation remains unknown. The objective of this study was to investigate the effect of CDX2 on the proliferation of porcine jejunum epithelial cells (IPEC-J2) and the correlation between CDX2, the mTORC1 and Wnt/β-catenin signaling pathways. CDX2 overexpression and knockdown cell culture models were established to explore the regulation of CDX2 on both pathways. Pathway-specific antagonists were used to verify the effects. The results showed that CDX2 overexpression increased IPEC-J2 cell proliferation and activated both the mTORC1 and Wnt/β-catenin pathways, and that CDX2 knockdown decreased cell proliferation and inhibited both pathways. Furthermore, the mTORC1 and Wnt/β-catenin pathway-specific antagonist rapamycin and XAV939 ]-4H –thiopyranopyrimidin-4-one) both suppressed the proliferation of IPEC-J2 cells overexpressing CDX2, and that the combination of rapamycin and XAV939 had an additive effect. Regardless of whether the cells were treated with rapamycin or XAV939 alone or in combination, both mTORC1 and Wnt/β-catenin pathways were down-regulated, accompanied by a decrease in CDX2 expression. Taken together, our data indicate that CDX2 stimulates porcine intestinal epithelial cell proliferation by activating the mTORC1 and Wnt/β-catenin signaling pathways. CDX2, a caudal-related homeobox gene coding for a homeodomain transcription factor, is an essential regulator of gut development and homeostasis. CDX2 conditional knockout mice failed to form a mature endoderm in their intestinal epithelia  and inacDifferences exist with regard to the effect of CDX2 on cell proliferation in different observations. Studies on cancer cells, including human colorectal carcinoma cells HT-29) , human c , human The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is responsible for translation and in the control of cell growth and metabolism. The Wnt/β-catenin signaling pathway is indispensable to cell polarity, proliferation, differentiation and migration. Both the expression level and the level of phosphorylation of the relevant functional proteins in both pathways are important parameters for measuring the state of cell proliferation ,12. Yet,To further elucidate the effect of CDX2 in normal intestinal epithelial cell proliferation and the relationship between CDX2, and the mTORC1 and Wnt/β-catenin pathways, we constructed two models, an overexpression model and a knockdown model of CDX2 in the porcine jejunum epithelial cell line, IPEC-J2. Antagonists specific to the mTORC1 or Wnt/β-catenin pathways were used to validate the results.Immunofluorescence microscopy, real-time PCR and Western blot were performed to assess CDX2 overexpression in IPEC-J2 cells. Compared with the control group, CDX2 fluorescence signaling a, mRNA aThe MTT -2,5-diphenyltetrazolium bromide) assay and cell counting were used to evaluate the effect of CDX2 overexpression on IPEC-J2 cell proliferation. OD values d and cel2448), p-S6K1 (Thr389), p-S6 (Ser235), p-4EBP1 (Thr70) and eIF4E were increased in the overexpression group , p-S6K1 (Thr389), p-S6 (Ser235), p-4EBP1 (Thr70) and eIF4E were decreased in the knockdown group , p-S6K1 (Thr389), p-S6 (Ser235), p-4EBP1 (Thr70) and eIF4E decreased. Similarly, the levels of Axin2 and GSK-3β increased, and the levels of β-catenin, Cyclin D1 and c-Myc decreased  demonstrated that CDX2 occupies hundreds of genomic sites in ISCs and thousands of additional sites in VCs . MoreovemTOR mRNA and protein expression [In consideration of the conflicting role of CDX2 on cell proliferation and to obtain further insight into its stimulating effect, we measured the expression of proteins in the mTORC1 and Wnt/β-catenin pathways. Firstly, the present study found that phosphorylated proteins in the mTORC1 pathway were up-regulated in the overexpression group and were down-regulated in the knockdown group, which suggested that CDX2 might activate the mTORC1 pathway in IPCE-J2 cells. However, few studies have shown that mTORC1 activity may be decreased by CDX2. A study on CDX2 heterozygous knockout mice demonstrated CDX2 haploinsufficiency led to an increase in mTOR kinase activity  and CDX2pression . The mTOpression . Given tSimilarly, no conclusions about the activating or inhibiting effect of CDX2 on the Wnt/β-catenin pathway can be drawn. In human colon cancer cells, CDX2 limited cell proliferation and the Wnt/β-catenin signaling pathway by binding β-catenin and disrupting the β-catenin-TCF complex . In contOur results suggest that both the mTORC1 and Wnt/β-catenin pathways play a part in CDX2-mediated cell proliferation. While it is necessary to investigate which pathway is more important and if there is a synergistic effect on cell proliferation, it is noteworthy that treatment with rapamycin alone, XAV939 alone, or with a combination of both caused a significant decrease in the proliferation of CDX2 overexpressing IPEC-J2 cells, with a synergistic effect in the cells treated with both drugs. Furthermore, rapamycin treatment led to a more significant reduction in cell proliferation than the XAV939 treatment. Moreover, CDX2 expression was reduced in each treatment, which is in agreement with previously published data that identifies CDX2 as a direct Wnt target during hindgut differentiation . Taken tStudies have shown that the mTORC1 and Wnt/β-catenin pathways positively regulate each other. Inoki et al., (2006) reported that GSK3 (glycogen synthase kinase) participates in the activation of TSC2 (tuberous sclerosis s) to inhibit mTORC1 activity . β-catenIn summary, the present study illustrates the promoting effects of CDX2 on IPEC-J2 cell proliferation, activating the mTORC1 and Wnt/β-catenin pathways. Moreover, the study uncovers the mutual regulation among the mTORC1 and Wnt/β-catenin pathways and CDX2 .2. Medium was changed every two days [IPEC-J2 cells, kindly provided by Dr. Yu-long Yin (University of Chinese Academy of Sciences), were cultured in DMEM/F12 medium  supplemented with 5% fetal bovine serum , 1% penicillin/streptomycin, 5 μg/L Insulin-Transferrin-Selenium  and 5 μg/L epidermal growth factor  at 37 °C in 5% COtwo days . TreatmeCDX2 and an unrelated negative control siRNA were designed and synthesized, and then purified via high performance liquid chromatography   . 24 h pr4 cells/mL into 96-well culture plates  and fixed with 4% paraformaldehyde for 30 min. Cells were then permeabilized with 0.3% Triton X-100 for 15 min and blocked in a protein solution  for 20 min. The primary antibody, anti-CDX2 , was incubated with the cells for 2 h at room temperature. Secondary staining was performed with Cy3-conjugated antibodies  and incubated at room temperature for 1 h. Nuclei were stained with 4’6-diamidino-2-phenylindole  for 5 min at room temperature. Fluorescence signals were observed with a fluorescence microscope . Three independent experiments were performed.pcDNA3.1+ and CDX2-pcDNA3.1+ were stably transfected into cells. Transfected cells were seeded at 1 × 10n = 6) was determined. Specific primer pairs used are detailed in Ct−ΔΔ method. The experiment was performed in triplicate.Using a Stratagene Mx3005P qPCR system  and the SYBR Green Real-Time PCR Master Mix , mRNA expression -2,5-diphenyltetrazolium bromide  for 4 h, crystals were then dissolved in DMSO. Optical density (OD) values were evaluated in an ELISA reader  at a wavelength of 490 nm. Three independent experiments with 20 samples per treatment were conducted to confirm the result.Cells were seeded in 96-well plates at 1 × 105 cells/mL. After washing twice with PBS, cells were detached with 0.25% trypsin (Sigma) for 5–8 min at 37 °C, and then blocked with an equal volume of growth medium. The number of viable cells was determined using a automate cell counter . Three independent experiments were conducted.Cells were seeded in 6-well plates at 1 × 10g centrifugation at 4 °C for 15 min. Protein concentrations were determined by a BCA Protein Assay Reagent Kit (Thermo Fisher Scientific). Proteins (10 μg) were separated on 10% SDS/PAGE gels, and transferred onto PVDF membranes . After blocking with bovine serum albumin (BSA), the membranes were incubated with a primary antibody. Anti-CDX2 (#12306), anti-mTOR (#2972), anti-phospho-mTOR , anti-S6K1 (#9202), anti-phospho-S6K1 , anti-S6 (#2708), anti-phospho-S6 , anti-4EBP1 (#9644), anti-phospho-4EBP1 , and anti-c-Myc (#5605) antibodies were obtained from Cell Signaling Technology; anti-GSK-3β (sc-9166) and anti-Cyclin D1 (sc-753) were purchased from Santa Cruz Biotechnology; anti-β-catenin (ab6302), anti-Axin2 (ab109307) and anti-eIF4E antibodies were obtained from Abcam; and anti-β-actin (AP0060), anti-rabbit IgG (BS13278) and anti-goat IgG (BS30503) antibodies were obtained from Bioworld Technology. Proteins were visualized using the Beyo ECL Plus chemiluminescence detection kit . ECL signals were detected by a FluorChem M imaging system . Band density was calculated using Image Analysis Software . Results were normalized to β-actin or the total level.Cells were lysed in RIPA buffer, containing 1% Triton-X, 20 mmol/L Tris-HCl (pH = 7.4), 5 mmol/L EDTA and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and then followed by 14,000× t-test was conducted to determine the differences between 2 groups using SAS , and Duncan’s multiple-range test to determine differences among groups. Differences were considered statistically significant when p < 0.05.Data are expressed as the mean ± SEM. Student’s


Copy number alterations (CNA) and loss of heterozygosity (LOH) represent a large proportion of genetic structural variations of cancer genomes. These aberrations are continuously accumulated during the procedure of clonal evolution and patterned by phylogenetic branching. This invariably results in the emergence of multiple cell populations with distinct complement of mutational landscapes in tumor sample. With the advent of next-generation sequencing technology, inference of subclonal populations has become one of the focused interests in cancer-associated studies, and is usually based on the assessment of combinations of somatic single-nucleotide variations (SNV), CNA and LOH. However, cancer samples often have several inherent issues, such as contamination of normal stroma, tumor aneuploidy and intra-tumor heterogeneity. Addressing these critical issues is imperative for accurate profiling of clonal architecture.We present CLImAT-HET, a computational method designed for capturing clonal diversity in the CNA/LOH dimensions by taking into account the intra-tumor heterogeneity issue, in the case where a reference or matched normal sample is absent. The algorithm quantitatively represents the clonal identification problem using a factorial hidden Markov model, and takes an integrated analysis of read counts and allele frequency data. It is able to infer subclonal CNA and LOH events as well as the fraction of cells harboring each event.The results on simulated datasets indicate that CLImAT-HET has high power to identify CNA/LOH segments, it achieves an average accuracy of 0.87. It can also accurately infer proportion of each clonal population with an overall Pearson correlation coefficient of 0.99 and a mean absolute error of 0.02. CLImAT-HET shows significant advantages when compared with other existing methods. Application of CLImAT-HET to 5 primary triple negative breast cancer samples demonstrates its ability to capture clonal diversity in the CAN/LOH dimensions. It detects two clonal populations in one sample, and three clonal populations in one other sample.CLImAT-HET, a novel algorithm is introduced to infer CNA/LOH segments from heterogeneous tumor samples. We demonstrate CLImAT-HET’s ability to accurately recover clonal compositions using tumor WGS data without a match normal sample.The online version of this article (doi:10.1186/s12920-017-0255-4) contains supplementary material, which is available to authorized users. Cancer is a disease that develops through accumulation of various genetic variability . The cloCancer-associated studies have been greatly promoted by continuous advances in experimental technologies for landscape of cancer genomes –12. WithIn this study, we present CLImAT-HET, a novel algorithm based on the framework of CLImAT , for infIn contrast to related approaches in the literature, our method presents several distinct characteristics. APOLLOH  is desigWe perform a comprehensive evaluation of CLImAT-HET using simulated and real WGS data. By quantitative benchmarking on simulated datasets, we demonstrate that CLImAT-HET has the capacity to accurately infer cellularity of clonal clusters and subclonal CNA/LOH segments, and shows significant advantages when compared with existing approaches. We apply CLImAT-HET to 5 primary triple negative breast cancer (TNBC) samples to show its ability to identify clonal diversity in the CNA/LOH dimensions.N SNPs is represented by read counts dN1:, meanwhile B allele frequency is represented by B-allele read depth bN1: and total read depth TN1:. Following the procedures adopted in CLImAT [dN1:, bN1: and TN1: using an integrated HMM, and iteratively examine the model complexity using BIC for different number of clonal clusters, and finally output clonal/subclonal CNA and LOH segments as well as the cellularity of each clonal cluster.The pipeline of CLImAT-HET is illustrated in Fig. n CLImAT , read coβ), and another harbors the aberration event with cellularity of β. To avoid extensive local parameters, we further assume all the aberration events can be clustered into a finite number of K groups, of which the kth group corresponds to the kth clonal population with respective cellularity of βk (β1˂β2˂…˂βK), and tumor purity is equal to βK. Based on these assumptions, the model parameters can be effectively inferred by borrowing statistical approaches.It is intractable to precisely depict the genome-wide aberration status of tumors containing multiple subclones, therefore we adopt a simple assumption that the observed signals at a genomic locus are generated from underlying three types of cell populations: normal (non-tumor) cells, tumor cells with normal genotype, and tumor cells harboring the aberration event of interest Fig. . Thus, cc and the kth clonal cluster, we assume B-allele read depth is binomial distributed with the conditional probability defined as follows:We define a set of states to depict copy number status of tumor genomes  of normal cells respectively, nc and uc represent the copy number and BAF of tumor cells respectively. In addition, we assume read counts is negative binomial (NB) distributed with the conditional probability defined as follows:λ is mean read counts associated with normal copy, and λc,k is formulated as:In formula (1), we only consider heterozygous genotypes in each aberration state. The mean of B allele copy number The meanings of all other parameters involved in above formulas are the same as the ones described in CLImAT, and we do not go into detail here.c and clonal cluster k, therefore we implement CLImAT-HET as a factorial HMM with C × K hidden states by combining tumor genotypes and clonal clusters, where C is the number of aberration states defined in Table S1 and K is the number of clonal clusters. The HMM thus has two underlying Markov chains with one chain depicting aberration state sequence and another delineating corresponding clonal clusters , where π is the initial state probability distribution, A is the state transition matrix, β is the cellularity of all clonal clusters, λ denotes the copy neutral read counts, and p refer to the success probability as a parameter of NB distributions. For the expectation step of EM algorithm, we calculate the expectation of the partial log-likelihood function of read counts and B-allele read depth respectively, and formulated as:γi,c,k is the posterior probability of the ith SNP being in aberration state c and the kth clonal cluster, and is calculated using forward-backward algorithm [n-1 to n as follows:LLn is the value of the log-likelihood function in the nth iteration. If the value of Inc is less than a specific threshold (1 × 10−4), then the parameter updating procedure is stopped.The conditional probabilities described above rely on two latent variables, aberration state ers Fig. . We empllgorithm . For thelgorithm  to updatK, the EM algorithm is implemented as follows: 1) start with initial parameters θ0 =  and calculates the joint-posterior probability γi,c,k, 2) update θ1 =  using Newton–Raphson method, 3) repeat steps (1) and (2) until a specified number of iterations are reached or the convergence criterion is met. The converged parameters θ0 =  to find the globally optimal solution.Given the number of clonal clusters α > 0 is the regularizing term, m is the number of free parameters to be estimated and N is the number of SNPs. Our goal is to find an optimal value of K that leads to the model with the minimum value of BIC. A feasible solution is to perform an exhaustive search for possible values but it is practically intractable. Alternatively, CLImAT-HET starts with the initial assumption of tumor homogeneity (K = 1) and then iteratively increases clonal cluster number by one (K = K + 1) until the BIC value of the model no longer decreases. The optimal model is thus the one in the final iteration. For the computational convenience, we do not directly calculate the BIC of the model, but the difference of BIC between two adjacent models. Suppose that the number of clonal clusters is n in the nth iteration, and the BIC difference between the current model and the previous model is defined as follows:mn are the increment of the likelihood and the number of free parameters from model n-1 to model n, respectively. Δmn is measured by formula as follows:As a vital parameter of CLImAT-HET model, the number of clonal clusters is determined using BIC. The BIC of a model is defined as follows:The iteration is stopped once the BIC difference is greater than zero. After the model learning is finished, aberration information including copy number, tumor genotype, aberration type and clonal cluster for each SNP can be inferred from the hidden state with the maximum posterior probability. At the same time, segmentation of all SNPs based on hidden states is performed to output clonal/subclonal CNA and LOH segments.ith segment as follows:bij and Tij are B-allele read depth and total read depth of the jth heterozygous SNP in the ith segment, and dij is the corresponding read counts, c and the kth clonal cluster, respectively. Furthermore, the scores for all segments are scaled to 0 ~ 100.Similar to the method described in CLImAT, we define a reliability score for the An efficient implementation of CLImAT-HET by using C/Matlab is freely available at GitHub , our preWGS data from 5 primary TNBC samples described in a previous study  are analn) and four tumor genomes , and then generate mixtures by mixing five genomes at predefined proportions. As illustrated in Figure S1  and 0.4 (pa + pb); similarly, the mixture “a_020b_035c_025n_020” will yield three clonal clusters with cellularity of 0.35 (pb), 0.25 (pc) and 0.8 (pa + pb + pc), while the mixture “a_010b_010d_025n_055” contains three clonal clusters with cellularity of 0.25 (pd), 0.35 (pb + pd) and 0.45 (pa + pb + pd).For each simulated sample, the underlying cellularity of clonal clusters are computed based on the relationship between the tumor genomes making up the mixture. For example, the mixture “a_010b_030n_060” is made up by genomes Two advanced methods, OncoSNP-SEQ  and CLImFor the simulated samples, copy number and genotype profiles of all segments predefined in simulation experiment are used as the golden standard for evaluation. Accuracy is calculated for each method by comparing the predictions with the ground truth. We consider a segment to be accurately identified only if any predicted segment covers the 75% size of the segment and have exact matching of total and major copy number with the segment. Accuracy is defined as the proportion of accurately identified segments among all segments.In this section, we perform a comprehensive assessment of CLImAT-HET on both simulated and real datasets in terms of inferring cellularity of clonal clusters and identifying CNA/LOH segments.We employ a simulation study to evaluate the accuracy of our estimates of cellularity of clonal clusters, tumor purity and predictions of CNA/LOH segments. Our simulated data is generated based on a real normal sample HCC1954-BL downloaded from CGHub as described in the Methods. We run CLImAT-HET, OncoSNP-SEQ and CLImAT to infer CNA/LOH segments and associated cellularity.p-value < 5 × 10−5, MAE < = 0.02) with the underlying cellularity across all simulated samples  for all the mixtures relative to the ground truth tumor purity is observed for CLImAT-HET , and CLImAT also achieves good performance .To assess the accuracy of cellularity estimations of CLImAT-HET, we compare the estimated cellularity to the ground truth cellularity for each clonal cluster. For each sample, the underlying cellularity of clonal clusters are computed based on the tumor genomes making up the mixture (more details in the Methods). CLImAT-HET is able to correctly infer the number of clonal clusters and corresponding cellularity for all the simulated mixtures. An example of cellularity estimations on a simulated sample “a_035b_015d_025n_025” with three clonal populations is shown in Fig. les Fig. , b and cHET Fig. . OncoSNPNext, we proceed to assess the abilities of CLImAT-HET, OncoSNP-SEQ and CLImAT in inferring tumor genotypes. We consider a segment to be accurately identified if and only if both the total and major copy numbers are accurately identified, and the sizes of the segments are not considered. For each simulated sample, total and major copy number profiles of all segments predefined in simulation experiment are used as the golden standard for evaluation. CLImAT-HET is able to accurately infer the total and major copy numbers of each segment for all the simulated mixtures. An example result from the simulated sample “a_035b_015d_025n_025” is shown in Fig. We also examine CLImAT-HET on 5 primary triple negative breast cancer samples, which are sequenced at approximately 30× coverage and also assayed by Affymetrix SNP6.0 array . ASCAT  is a widK = 2 as the optimal number of clonal populations since the BIC difference becomes to be positive when the number of clonal populations increases to 3.The subclonal prediction results of CLImAT-HET on sample SA223 are shown in Fig. K = 3 as the optimal number of clonal populations.For sample SA227, CLImAT-HET predicts it as heterogeneous with three distinct clonal populations, and the subclonal prediction results are shown in Fig. For samples SA220, SA224 and SA225, the measures of goodness of fit returned by ASCAT are 0.95, 0.94 and 0.98 respectively, indicating these samples are probably homogeneous. Accordingly, CLImAT-HET analysis of these samples shows that there is also no significant evidence for existence of subclonal events, and the inferred cellularity of the single clonal population is 0.62, 0.52 and 0.75 respectively, which are in good concordance with the tumor purities predicted by CLImAT and ASCAT Table . The copCLImAT-HET is a novel statistical method for inferring subclonal CNA and LOH segments from WGS data of heterogeneous tumor samples. It is developed based on our previously published algorithm CLImAT, and take into account the issue of intra-tumor heterogeneity. Compared with CLImAT that only handles homogeneous tumor samples, CLImAT-HET can efficiently deal with both homogeneous and heterogeneous tumor samples. The read counts and read depths data generated from multiple cell populations is quantitatively represented, and proper emission models are adopted in the HMM. Furthermore, we integrate a BIC module into the CLImAT-HET framework to effectively determine the underlying number of subclonal populations. These features enable CLImAT-HET’s advantages in handling complex WGS data. First, the appropriate decomposition of mixed signals improves the CNA/LOH detection performance. Second, CLImAT-HET is more sensitive to the aberration events represented in minor cell populations when compared to existing methods as shown in simulated study. Third, CLImAT-HET is able to accurately infer the cellularity each subclonal cluster. Finally, the evaluation results on both simulated and real WGS data demonstrate the advantages of our algorithm.CLImAT-HET also has limitations due to its adopted modeling assumptions. The assumption that only one aberrant genotype exists at each genomic locus will not hold if distinct subclonal populations have different aberrant genotypes at the same locus. However, it is difficult to distinguish among multiple subclones that have variable genotypes. The emission models of CLImAT-HET need to be extended to account for these situations, and the joint analysis of read counts and read depths signals may output multiple solutions. Identifying distinct haplotypes harboring linked mutations might be a good way to identify multiple mutations at the same locus in different populations.In summary, we demonstrate that CLImAT-HET represents remarkable advantages in inferring subclonal CNA/LOH segments from tumor WGS data. The high performance of CLImAT-HET benefits from delicate representation of copy number profiles of distinct cell populations, and efficient statistical methods for inference of the global parameters. We expect that CLImAT-HET will complement the arsenal of bioinformatics tools developed for investigating the role of tumor tumourigenesis and progression.


As cellular models of DA neurons, we used the proliferating or RA-differentiated human neuroblastoma cell line SH-SY5Y. In both DA neuron-like cultures, ANP is able to positively affect the Wnt/β-catenin signaling, by inducing β-catenin stabilization and nuclear translocation. Importantly, activation of the Wnt pathway by ANP exerts neuroprotective effect when these two cellular systems were subjected to neurotoxic insult (6-OHDA) for mimicking the neurodegeneration of PD. Our data support the relevance of exogenous ANP as an innovative therapeutic molecule for midbrain, and more in general for brain diseases for which aberrant Wnt signaling seems to be involved.In the last decades increasing evidence indicated a crucial role of the Wnt/β-catenin signaling in development of midbrain dopaminergic (mDA) neurons. Recently dysregulation of this pathway has been proposed as a novel pathomechanism leading to Parkinson’s disease (PD) and some of the molecules participating to the signaling have been evaluated as potential therapeutic targets for PD. Atrial natriuretic peptide (ANP) is a cardiac-derived hormone having a critical role in cardiovascular homeostasis. ANP and its receptors (NPRs) are widely expressed in mammalian central nervous system (CNS) where they could be implicated in the regulation of neural development, synaptic transmission and information processing, as well as in neuroprotection. Until now, the effects of ANP in the CNS have been mainly ascribed to the binding and activation of NPRs. We have previously demonstrated that ANP affects the Wnt/β-catenin signaling in colorectal cancer cells through a Frizzled receptor-mediated mechanism. The purpose of this study was to investigate if ANP is able to exert neuroprotective effect on two  The proia nigra . Until nia nigra , seem toia nigra .In this context, deepening of knowledge on molecular mechanisms underlying PD insurgence and progression is crucial for discovering innovative molecular targets useful for developing more effective therapeutic strategies. In the last years, numerous studies have been published, that aimed to identify innovative PD biomarkers, also useful as therapeutic targets, and to demonstrate the neuroprotective role of endogenous and exogenous molecules The Wnt pathway has a key role in a large part of biological processes and regulates, in the CNS, all aspects of neuronal functions including differentiation, synapse formation, neurogenesis and neuroprotection . In the in vitro and in vivo  is a 28aa peptide that belongs to a family of cardiac and vascular-derived hormones having a critical role in cardiovascular homeostasis mainly by regulating blood volume and pressure . In the  in vivo  has made in vivo . ANP, or in vivo . ANP, as in vivo . Interes in vivo  and that in vivo . It has  in vivo , and thaeceptors . Until neceptors .in vitro models of PD, and if this effect could be associated with activation of the Wnt/β-catenin pathway. Even if reliable cellular systems to study pathophysiological mechanisms of PD and to test innovative therapeutics are limited, the human neuroblastoma cell line SHSY5Y, proliferating or differentiated by retinoic acid (RA), is currently the most used in vitro models of DA neurons (ANP is synthesized as an inactive precursor (pro-ANP) that is converted to the mature active peptide after proteolytic cleavage by the membrane-associated serine protease Corin, which extracellular region encloses the two Frizzled-like cysteine-rich domains Fzd1 and Fzd2, receptors for the Wnt signaling . Corin h neurons . Therefo neurons .L-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 μg/ml) and cell cultures were maintained at 37°C, in a humidified atmosphere of 5% CO2. For passaging, cells were detached from culture flasks with 0.05% trypsin and 0.002% EDTA solution. All media and supplements for cell cultures were acquired from Hyclone .SHSY5Y cell line was obtained from the American Type Culture Collection  and was validated by the purchaser cell bank. Cells were grown as monolayer in Eagle’s Minimum Essential Medium (α-MEM) plus HAM’s F12 (1:1), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 4/cm2 and 6 × 104/cm2 and cultured for 24 h prior to treatments. Cells were treated with 100 nM ANP , a concentration selected as the lowest effective and not toxic dose in preliminary dose-response experiments  cells were seeded at densities ranging from 4 × 104/cm2 and maintained in culture for 24 h to obtain monolayer at about 75% confluence. Cells were then treated with 10 μM retinoic acid (RA) in absolute ethanol and maintained in low serum medium (1% FBS) for a total of 9 days. To replenish RA, the differentiating agent was added at day 2, day 5, and day 8 after seeding, by replacing cell culture medium with new fresh medium without detaching the cell monolayer, and at day 10 the differentiated cultures were used for the experiments or analyzed. ANP treatment on SHSY5Ydiff was performed as described above for SHSY5Ywt cells.The differentiated phenotype (SHSY5Ydiff) was obtained based on protocols previously reported by other Authors , 2017. IFor mimicking the neurodegeneration of PD, SHSY5Ywt and SHSY5Ydiff cells were exposed for 24 h to 50 and 100 μM of 6-OHDA (prepared in 0.1% ascorbic acid in DMSO), respectively. These concentrations have been selected as the lowest effective doses in preliminary dose-response experiments . Cell viability was evaluated by Trypan blue dye exclusion method. Mitotic index (MI) evaluation was carried out as previously described . QuantitTable 1. The specificity of each antibody has been preliminary verified by Western blot, by considering the production of a single band matched to the proper molecular weight. Primary antibodies were revealed with Alexa Fluor 488-conjugated anti-mouse or anti-rabbit IgG (Molecular Probes). Samples were analyzed by using the LEICA TCS SP5 confocal microscope.For confocal microscopic analyses, SHSY5Ywt or SHSY5Ydiff cells were grown or RA-differentiated on the ibiTreat μ-Slide 4 well . Immunocytochemical analyses were performed on cells fixed with 2% paraformaldehyde (Sigma–Aldrich). After permeabilization with 0.2% Triton X-100 (Sigma–Aldrich), immunofluorescence staining was done using the primary antibodies against β-catenin, Nestin, Tubulin-β3, NeuN and TH, described in Table 1. Primary antibodies were revealed with peroxidase-conjugated secondary antibody . Densitometric analysis was done using the ImageJ processing program1. Values, normalized to β-Actin or GAPDH, were reported as uncalibrated Optical Density (OD) or as fold vs untreated control. Data were from at least three independent experiments and presented as the mean ± SD.After washing with ice-cold PBS, cells were lysed using a 50 mM Tris-HCl buffer at pH 8.0. Specifically, the buffer solution contained 150 mM of NaCl, 1% of NP-40, 10% of glycerol, 0.1 mM of EGTA, 0.5 mM of EDTA, 50 mM of NaF, 1 mM of Na3OV4, and a protease inhibitor cocktail (Sigma–Aldrich). Lysates were clarified by centrifugation and proteins were quantified using the Bradford reagent . 15–20 μg of each cell extract was separated using SDS/PAGE ranging from 8 to 12%, and then transferred to nitrocellulose membrane . Membranes were incubated for 1 h at room temperature with 5% BSA in Tris-buffered saline-Tween , probed with the specific antibodies reported in Table 2. The amplification conditions were: 1 min at 95°C, 40 cycles of 10 s at 95°C and 40 s at 60°C. Cq values were determined from the system software using ‘single threshold’ mode. The relative expression level for each gene were calculated from these Cqs using experimentally determined amplification efficiencies, and then normalized for the reference gene GAPDH. Results were reported as fold of induction vs untreated control. In all experiments each sample was analyzed in triplicate, and no-template controls and no-reverse transcription controls were inserted.Transcription levels of β-catenin (β-cat), LEF1 and TH genes were assessed by qPCR in untreated controls and in SHSY5Ywt cells challenged with 100 nM ANP for times ranging from 3 to 24 h. Total RNA was extracted using TRIzolTM Reagent , and treated with RNase-free DNase (Promega), according to the manufacturer’s instructions. After extraction with a mix of Phenol:Chloroform:Isoamyl Alcohol  and precipitation with ethanol, 1 μg of total RNA was reverse-transcribed using random primers with ImProm II Reverse Transcription system (Promega) according to the instructions of the manufacturer. qPCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad) using 250 nM of the specific primers, by the CFX Connect Real-Time PCR Detection System (Bio-Rad). Sequences of primers used are reported in t-test, and p < 0.05 was considered as statistically significant. All data were from at least three independent experiments and presented as means ± SD.Statistical analysis was performed using the two-tailed Student’s Figure 1). Confocal microscopy  and Western blot analysis  showed that SHSY5Ywt cells expressed Tubulin-β3 and NeuN (specific for mature neurons) as well as TH and Nurr1 (specific for DA neurons), together with detectable levels of Nestin . This confirms that SHSY5Ywt cells possess the features of dopaminergic neurons and are appropriate for studying neurotoxicity or neuroprotection in experimental PD, as also reported by others , with β-catenin that localized almost exclusively at the cell membrane , and this indicated that SHSY5Ywt cells were potentially responsive to molecules affecting the Wnt pathway.SHSY5Y cells in basal condition (SHSY5Ywt) have been characterized for the expression and intracellular distribution not only of neuronal markers  and of the DA neuron specific markers TH and Nurr1, but also of two crucial molecules participating to the Wnt/β-catenin signaling, the Fzd1 receptor and β-catenin . RA treatment for a total of 9 days gave rise to cells exhibiting neuronal differentiation with neurite outgrowth , low proliferation rate, as demonstrated by lower levels of c-Myc , and block in G1 phase, as demonstrated by cytofluorimetric analysis  and by higher levels of p21 , than those recorded in the untreated SHSY5Y cells. SHSY5Ydiff cells were then compared with SHSY5Ywt for the neuronal markers , DA neuron specific markers , neuronal survival factors involved in neuroprotection against oxidative stress , and Wnt pathway related molecules (Fzd1 and β-catenin) . Confocal microscopy  and Western blot analysis  showed that SHSY5Ydiff cells, expressed higher levels of Tubulin-β3, NeuN, TH and Nurr1 , compared with SHSY5Ywt, indicating that the RA-differentiated cellular system was phenotypically more cognate to mature dopaminergic neurons than the not differentiated one. Moreover, expression levels of the survival factors DJ-1 and active (phosphorylated) AKT were also considerably increased in SHSY5Ydiff as compared with SHSY5Ywt  . In both cellular systems β-catenin localized almost exclusively at the cell membrane , while Fzd1 receptor was expressed at higher levels  in SHSY5Ydiff compared to SHSY5Ywt (1.6-fold of increment). This indicated that SHSY5Ydiff cells could be potentially responsive to molecules affecting the Wnt pathway, similarly to SHSY5Ywt.SHSY5Ydiff cells were obtained as described in Section “Materials and Methods” and schematized in nsistent . For thiIn order to verify whether the treatment with the natriuretic peptide affected the Wnt signaling also in these cellular systems as previously demonstrated for other cells , SHSY5YwFigures 4A,B) showed that ANP, similarly to Wnt1a, induces a significant increase of total β-catenin (p = 0.0098 vs untreated control), already after 3 h of treatment, and a concomitant decrease of β-catenin phosphorylation at Thr41 (pβ-catenin Thr41), one of the molecular event upstream β-catenin ubiquitination and degradation by the proteasome. Moreover, the decrease of pβ-catenin correlated to an increase of GSK phosphorylation at Ser9 (pGSK-3βSer9), the inactive form of this enzyme  showed that ANP also caused a redistribution of β-catenin from cell-cell junction sites to cytoplasmic and nuclear compartments, between 6 and 24 h of treatment. Taken together, these results indicate that, in SHSY5Ywt cells, ANP positively affects the Wnt signaling by inducing β-catenin stabilization and nuclear translocation. This is further supported by the up-regulation of the Wnt-signaling dependent transcriptional factor LEF 1, as measured by RT-qPCR analysis after 24 h of ANP treatment, when a significant increase of transcription of the Nurr1-regulated gene TH was also recorded . Conversely, at 3 and 6 h of ANP treatment, β-catenin mRNA resulted unmodified or down-regulated , indicating that the high levels of the total protein concomitantly recorded by WB  were actually due to a reduction of β-catenin degradation induced by the natriuretic peptide, rather than to a decrease transcription of β-catenin gene.In SHSY5Ywt cells, WB analysis .SHSY5Ydiff cells were also responsive to treatment with 100 nM ANP, as demonstrate by the β-catenin cytoplasmic accumulation and nuclear translocation observed between 6 and 24 h of treatment, similarly to what observed under treatment with the Fzd1 ligand Wnt1a .In order to establish the optimal concentration of 6-OHDA to be used in the experiments of neuroprotection, we examined, in a preliminary dose-response test, the susceptibility of both SHSY5Ywt and SHSY5Ydiff to the neurotoxin  showed that 6-OHDA treatment drastically decreased the number of adhering cells and destroyed the neurite network in both proliferating and differentiated SHSY5Y cells, while exposure to ANP did not influenced cell adhesion and viability nor neurite integrity. Pre-treatments with 100 nM ANP were able to substantially prevent detachment of cells from the substrate, also partially preserving the neuritic network , and significantly (p < 0.01) reduced the percentage of dead cells induced by 6-OHDA in both cellular models .Based on results from the preliminary dose-response test, SHSY5Ywt and SHSY5Ydiff were exposed to 50 and 100 μM of 6-OHDA, for times up to 24 h. To verify the ability of ANP in preventing the cytotoxicity induced by 6-OHDA, cells were pre-treated with 100 nM of the natriuretic peptide, 30 min or 24 h before 6-OHDA addition to cell culture medium. Phase contrast microscopy  showed that the challenging with 6-OHDA for 24 h dramatically decreased total β-catenin expression (p < 0.001), compared to both untreated control and ANP-treated cells , and concomitantly increased β-catenin phosphorylation and degradation . Moreover, 6-OHDA significantly (p < 0.01) reduced the expression levels of the neuronal marker Tubulin-β3 and of the DA neuron specific markers TH and Nurr1  as well as of the survival factors involved in neuroprotection against oxidative stress AKT/phospo-AKT and DJ-1 . Pre-treatment with ANP, by inducing β-catenin stabilization, partially or completely restored the expression of all markers analyzed to levels near to those recorded in the untreated control at T0 . It is noteworthy that 24 h of treatment with ANP alone significantly up-regulated the expression levels of the survival and anti-apoptotic factor DJ-1 , the product of the PARK3 gene, that has a neuroprotective role against oxidative stress and is dysfunctional or low expressed in PD patients . In particular, already after 7 h of treatment, the neurotoxin was able to significantly (p < 0.001) decrease total β-catenin expression, and concomitantly increase β-catenin phosphorylation and degradation  , also reducing, between 7 h and 24 h, the DA neuron specific markers TH and Nurr-1 , while the survival factor and PD-related protein DJ-1 was not significantly modified. Differently to what recorded the proliferating model, in RA-differentiated cells the neurotoxin induces a significant increase in pAKT levels at both times examined  , and this could additionally explain the higher resistance to the neurotoxin-induced stress exhibited by the SHSY5Ydiff compared to SHSY5Ywt. Nevertheless, also in SHSY5Ydiff pre-treatment with ANP partially or completely reversed the neurotoxin-induced changes of the markers analyzed already after 7 h of treatment . In particular, at this time point, in ANP pre-treated samples we recorded a significant (p < 0.001) increase in total β-catenin and a concomitant decrease in β-catenin phosphorylation and degradation, compared to cells treated with 6-OHDA alone . A significant (p < 0.01) increase of Nurr-1 expression was also recorded at 24 h in ANP pre-treated samples. At 24 h, TH, which expression is regulated by Nurr-1 . Moreover, in samples pre-treated with ANP for 24 h and subjected to the neurotoxin challenging, we recorded an additional increment in AKT phosphorylation levels compared with 6-OHDA alone, and this further sustains the neuroprotective role exerted by the natriuretic peptide . The neuroprotective efficacy of the natriuretic peptide was also recorded in both SHSY5Ywt and SHSY5Ydiff models subjected to pre-treatment with ANP for 30 min, and this suggests that the molecular events that reinforce cell survival are triggered soon after its addition to culture medium .Similar results were obtained when we used as model of DA neurons the SHSY5Ydiff cells, where the effects induced on the molecular markers were assessed at 7 and 24 h of 6-OHDA treatment , and this strongly suggests that the ANP-induced neuroprotection against 6-OHDA challenging was strictly related to canonical Wnt signaling activation. The involvement of Wnt/β-catenin pathway activation in the neuroprotective ability of ANP is also sustained by the significant increment in Nurr1 expression in samples pre-treated with the natriuretic peptide before 6-OHDA challenging, recorded after 24 h of treatment in both cellular models (Table 3). In fact, it has been very recently reported that β-catenin, after nuclear translocation, binds on the upstream promoter region of Nurr1 and increases its transcription, thus directly regulating Nurr1 gene expression  similarly to what we have previously demonstrated for colorectal cancer cells . Thus, ANP seems to be able of acting as a Wnt agonist or antagonist, depending on the cell type and/or pathological situations, and this strongly increases the attractiveness of this natriuretic hormone as an innovative therapeutic approach in a wide range of diseases. Actually, ANP could function as a Wnt modulator capable of successfully reverting aberrant Wnt signaling in different pathological situations, by restoring the balancing between Wnt-OFF and Wnt-ON state. This could also be one of the function in the brain of endogenous ANP, that, together with the two other natriuretic peptides BNP and CNP, is widely expressed in mammalian CNS , and are encouraging for future studies aimed to deep this issue.It is noteworthy that, besides the neuroprotective ability demonstrated by the natriuretic peptide, in SHSY5Y proliferating neuroblastoma cells the effects induced by ANP on Wnt signaling caused the inhibition of proliferation, as demonstrated by the MI decrement recorded in cells treated with concentration ≥ 100 nM . The rationale for this assumption resides in the fact that, the two Frizzled-like cysteine-rich domains, Fzd1 and Fzd2, enclosed in the extracellular region of Corin, are structural domains crucial for pro-ANP processing  has been already approved by the Ministry of Health and Welfare of Japan (de facto, it has already passed the toxicological screening for the extension of the therapeutic application of this molecule.In this work we clearly demonstrate, for the first time, that ANP possesses neuroprotective ability on DA neurons by directly modulating the Wnt/β-catenin pathway. We purchase strong of Japan  as a druEven if further studies, aimed to demonstrate the neuroprotective effectiveness of this natriuretic peptide in animal models of PD, will be required, these results indicate that modulation by ANP of Wnt/β-catenin signaling in neuron cells, and in particular in mDA neurons, could be an innovative approach for PD treatment, and represent a possible starting point for future clinical applications.ACB and EP carried out the setting and characterization of the cellular models, performed the treatments of cell cultures, Western blot analysis and immunocytochemistry, and helped to draft the manuscript. FA performed the experiments of immunofluorescence, cell viability and growth and cell cycle. SR carried out RT-qPCR. MC supervised the RT-qPCR experiments and revised critically the manuscript. GN and GS helped to draft the manuscript and revised it critically. PP performed the cytofluorimentric analysis and revised critically the manuscript. AS designed the study, supervised all experiments, analyzed the data, performed morphological and confocal microscopic analyses, carried out the statistical analysis and draft the manuscript. All authors read and approved the final manuscript.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.