Wnt/β-catenin is a crucial repressor of adipogenesis. We have shown that E2 promoter binding factor 1 (E2F1) suppresses Wnt/β-catenin activity through transactivation of β-catenin interacting protein 1 (CTNNBIP1), also known as inhibitor of β-catenin and TCF4 (ICAT) in human colorectal cancers. However, it remains unknown whether ICAT is required for E2F1 to promote differentiation by inhibiting β-catenin activity in pre-adipocytes. In the present study, we found that 1-methyl-3-isobutylxanthine, dexamethasone, and insulin (MDI)-induced differentiation and lipid accumulation in 3T3-L1 pre-adipocytes was reversed by activation of β-catenin triggered by CHIR99021, a GSK3β inhibitor. Intriguingly, we observed a reduced protein level of E2F1 and ICAT at a later stage of pre-adipocytes differentiation. Importantly, overexpression of ICAT in 3T3-L1 pre-adipocytes markedly promote the adipogenesis and partially reversed the inhibitory effect of CHIR99021 on MDI-induced adipogenesis and lipid accumulation by regulating adipogenic regulators and Wnt/β-catenin targets. Moreover, pre-adipocytes differentiation induced by MDI were markedly inhibited in siE2F1 or siICAT transfected 3T3-L1 cells. Gene silencing of ICAT in the E2F1 overexpressed adipocytes also inhibited the adipogenesis. These data indicated that E2F1 is a metabolic regulator with an ability to promote pre-adipocyte differentiation by activating ICAT, therefore represses Wnt/β-catenin activity in 3T3-L1 cells. We also demonstrated that ICAT overexpression did not affect oleic acid-induced lipid accumulation at the surface of Hela and HepG2 cells. In conclusion, we show that E2F1 is a critical regulator with an ability to promote differentiation and adipogenesis by activating ICAT in pre-adipocytes. The alarming increase in the incidence of obesity among adults and children worldwide has prompted extensive researches on molecular mechanisms responsible for the synthesis and catabolism of triglycerides (TG) in white adipose tissues . A compe−/− mice have a reduced fat accretion, increased insulin sensitivity, and a decreased circulating level of cholesterol [E2 promoter binding factor 1 (E2F1) is a transcriptional factor involved in cell cycle progression, cell differentiation, and apoptosis . Severallesterol ,15,16. Alesterol , indicatIn our previous study on E2F1-driven transactivation of downstream targets and its function in cancer cells, we have shown that inhibitor of β-catenin and TCF4 (ICAT), also known as the β-catenin interacting protein 1 (CTNNBIP1), is a direct transcriptional target of E2F1 . Importa3T3-L1 pre-adipocytes, 293T cells, Hela, and HepG2 cells were obtained from the American-Type Culture Collection . Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), and penicillin/streptomycin were obtained from Gibco BRL . Primary antibodies against β-catenin, PPARγ, C/EBPα, CCND1 (Cyclin D1), and c-MYC were purchased from Cell Signaling Technology . E2F1 antibody was purchased from Santa Cruz Biotechnology . CTNNBIP1 (ICAT) antibody was purchased from Abcam . Peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Huaxingbio Biotechnology Co . The pIRES2-EGFP and pLent-EF1a-FH-CMV-GFP expression vector, packaging plasmid psPAX2 and envelope plasmid pMD2.G were all purchased from miaolingbio . BODIPY493/503, Lipofectamine RNAiMAX Reagent and Lipofectamine 3000 were purchased from Invitrogen . CHIR99021 (GSK3β inhibitor), serum TG determination kit, insulin, dexamethasone (Dex), 1-methyl-3-isobutylxanthine (IBMX), oil red O, and all other reagents not indicated were purchased from Sigma Chemical Co. .2 incubator. When 60–70% confluence was reached, cells were passaged using trypsin, and passages 5–15 cells were used in the present study.Cells were maintained in DMEM supplemented with 10% FBS, 100 units/mL penicillin G sodium and 100 mg/mL streptomycin sulfate. Monolayer of 3T3-L1 pre-adipocytes were induced to differentiate into mature adipocytes as previously described . BrieflyThe recombinant lentivirus was produced as described previously . BrieflyThe full-length human ICAT cDNA was sub-cloned by standard PCR into BamHI and EcoRI-restricted pIRES2-EGFP vector. All constructs were verified by DNA sequencing. Transient transfections of Hela and HepG2 cells were performed using Lipofectamine 3000, according to the manufacturer’s instructions. Briefly, 5 μg of pIRES2-EGFP-ICAT vector or empty pIRES2-EGFP vector were transfected into cells cultured at sub-confluent density in 6-well dishes. The signal of GFP was monitored by fluorescence microscope  24 h post-transfection. To get the stable ICAT-expressing cell line, Hela and HepG2 cells were maintained in non-selective medium for 2-days post-transfection, then plated in 800 μg/mL G418 medium for 3 weeks, with frequent changes of medium to eliminate dead cells and debris until distinct colonies can be visualized. Individual colonies then are trypsinized and transferred to flasks for further propagation. Stable ICAT overexpressing cells were also sorted by GFP signals through fluorescence activated cell sorting selection, data were analyzed using the Cell Quest software  and expanded for in vitro studies. Cells stably expressed ICAT were grown to 70% confluence on the 6-well plates and treated with 500 μmol/L oleic acid complexed to albumin at a molar ratio of 8:1 for 3 h.Procedures for immunofluorescent staining were essentially the same as previously described. Cells were rinsed twice in PBS, fixed with 4% paraformaldehyde solution for 1 h, permeabilized with 0.1% triton X-100 in PBS for 15 min on ice, blocked with 5% goat serum in PBS for 1 h followed by incubation with primary antibody , washed three times with PBS, and incubated with fluorescently labeled secondary antibody (1:50 dilution for 1 h) followed by Hoechst (1:1000). Images for morphological analysis were acquired under a fluorescence microscope.−ΔΔCt method was used to determine the fold changes in mRNA levels of each sample, as compared to the reference sample.Total RNA was extracted from cells using the Trizol reagent . Reverse transcription PCR was performed using the PrimeScript RT Reagent Kit  as instructed by the manufacturer and cDNA was used as a template in the subsequent reactions. Real-time PCR was performed using SYBR Premix Ex Taq II  and the ABI-Prism 7500 Sequence Detection System  according to the instruction from the manufacturer. The primer sequences (5′–3′) used are listed in 3VO4, 1.0 mmol/L NaF, and protease inhibitor tablet , followed by sonication for three times with 10 s/ time. The whole-cell lysates were centrifuged at 12,000 rpm for 10 min to collect the supernatant. The protein concentration of the supernatant was determined using the Pierce BCA protein Assay Kit  with bovine serum albumin as standard. Equal amounts of proteins were separated using SDS-page gels and transferred to polyvinylidene difluoride (PVDF) membranes . The membranes were blocked in 5% fat-free milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature, and then were incubated with indicated primary antibodies overnight at 4 °C. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, the chemiluminescent signal was detected using Super-Enhanced Chemiluminescence Kit .Cells were harvested for the analysis of protein abundance by Western blot as previously described . Cells w2O or BODIPY493/503 (stock concentration 1 mg/mL and working solution 1:1000 dilution) for 15 min at room temperature followed by washing for three times with PBS and viewed with a microscope. BODIPY493/503-stained lipid droplets were viewed through a fluorescence microscope. To quantify intracellular lipids, the oil red O-stained lipid droplets were dissolved with 100% isopropanol for 10 min. The absorbance of extracted dye was then measured at 520 nm.Lipid droplets in cells were stained by oil red O or BODIPY493/503. Cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h on ice, followed by washing with 60% isopropanol. Then stained with oil red O working solutions containing 6 mL stock solution (5 g/L in isopropanol) and 4 mL double-distilled HTG contents in adipocytes were measured using an assay kit as described previously . BrieflyFor E2F1 or ICAT knockdown in 3T3-L1 pre-adipocytes, 100 pmol of each siRNA oligonucleotides  were transfected into cells plated in 6-well dishes using Lipofectamine RNAiMAX Reagent as described by instructions. Cells transfected with non-targeting siRNA (NC) were used as control. The pre-adipocytes stably overexpressed E2F1 were transfected with 100 pmol siICAT or siNC. Cells were harvested to detect the protein expression or processed for designated assays 48 h post-transfection.p < 0.05. A value according to the post hoc ANOVA statistical analyses. The results were considered statistically significant when p < 0.05.Data were expressed as means ± SEM and were statistically analyzed by GraphPad PRISM5 . Both normality and homogeneity tests were examined before the statistical analysis in our study. Differences between two groups were assessed using the unpaired two-tailed Student t test. Data sets that involved more than two groups were assessed using ANOVA, followed by Tukey post-hoc test. In the figures, data with different superscript letters are significantly different at p < 0.05). Both the mRNA level (p < 0.05) in differentiated cells, as compared with un-differentiated cells. In agreement with the phenotype changes, mRNA level of fatty acid binding protein (AP2), a well-known adipocyte marker, was upregulated (p < 0.05) (p < 0.05) at day 3 of differentiation and were reduced to an undetectable level at the later stages of adipocyte differentiation  (p < 0.05) , a GSK3β inhibitor, which has been reported to activate the canonical Wnt/β-catenin pathway in 3T3-L1 pre-adipocytes . Adipoge < 0.05) E,F in 3T < 0.05) E,F. In op < 0.05). To identify a repressing effect of ICAT on Wnt/β-catenin activity in 3T3-L1 pre-adipocytes, the pre-adipocytes stably overexpressed ICAT or the empty vector were treated with or without CHIR99021, a GSK3β inhibitor. As shown, Western blot analysis showed that CHIR99021 treatment led to remarkable upregulation of β-catenin and its downstream targets, including c-MYC and CCND1, as well as significantly downregulation of protein abundances for PPARγ and C/EBPα (p < 0.05) (p < 0.05)  D,E. Thes < 0.05) A,B, as w < 0.05) C and int < 0.05) D. Intere < 0.05) . Althoug < 0.05) D,E, as w < 0.05) . This fip < 0.05) (p < 0.05) without affecting that of E2F1 (p < 0.05) by gene silencing of E2F1 or ICAT. Western blot analysis showed that protein levels of C/EBPα and PPARγ were reduced, while those of c-MYC and CCND1 were upregulated (p < 0.05) (p < 0.05) by gene silencing of ICAT (ICAT has been reported to be a direct target of E2F1 that is responsible for the suppression of β-catenin activity in cancer cells . To test < 0.05) A, confir of E2F1 B, valida of E2F1 D and lip of E2F1 E were dr < 0.05) C by siE2 < 0.05) D,E. The  of ICAT F, confirHela and HepG2 cells can directly accumulate lipid droplets in the cells with the induction of oleic acid ,28,29. IIn the present study, we found a novel function of E2F1/ICAT on promoting pre-adipocytes differentiation and lipid accumulation in 3T3-L1 cells. Depletion of E2F1 or ICAT by siRNA led to reduced differentiation and decreased TG contents by inhibiting expression of C/EBPα and PPARγ in MDI-treated cells. Overexpression of ICAT in 3T3-L1 pre-adipocytes markedly promoted the adipogenesis induced by MDI. In addition, this promotive effect was also observed in CHIR99021-treated cells, in which Wnt/β-catenin was activated by inhibiting GSK3β, a negative regulator of Wnt/β-catenin signaling, therefore repressing differentiation of pre-adipocytes. An inhibitory effect of CHIR99021 on adipogenesis was partially attenuated by lentivirus-mediated ICAT overexpression in 3T3-L1 cells. In addition, the adipogenesis in the stable E2F1 overexpressed adipocytes were markedly reduced by ICAT knockdown. This effect of E2F1/ICAT on differentiation was mainly mediated by regulating Wnt/β-catenin signaling. These findings revealed a crosstalk between E2F1/ICAT signaling and Wnt/β-catenin on pre-adipocyte differentiation .Adipogenesis is a critical process implicated in the development of metabolic disease. Both clinical and experimental data have indicated that small molecules or compounds with the ability to interfere in the adipogenesis process might be a potentially therapeutic agent for obesity ,31. ConsICAT has been identified as a direct transcriptional target of E2F1 , throughIn the present study, we found that the protein levels of E2F1 were reduced at 24 h treatment, which was dramatically enhanced on the day 3 of differentiation, and then reduced to an undetectable level as the cell differentiated into mature adipocytes. This result was consistent with a previous study . The shiAdipogenesis is associated with decreased activation of β-catenin signaling. To investigate a promotive effect of ICAT on adipogenesis by repressing β-catenin, 3T3-L1 pre-adipocytes transfected with ICAT or empty vector were treated with GSK3β inhibitor to upregulate β-catenin. As expected, GSK3β inhibitor treatment led to enhanced β-catenin and blocked cell differentiation. This effect was partially reversed by ICAT overexpression as shown by alterations in lipid droplets and TG contents. PPARγ and C/EBPα are critical regulators for cell differentiation, which were negatively regulated by CCND1 and c-MYC, respectively ,41,42, tOf note, we found that siE2F1 transfected cells had a higher inhibitory effect on MDI-induced differentiation, as compared with siICAT transfected cells. This result is due to the following reasons. First, besides ICAT as described herein, E2F1 can regulate pre-adipocytes differentiation by inducing other targets, such as Perp-1 ,45 and PIt has been reported that E2F1 transcriptional activity is enhanced in obese subjects and non-alcoholic fatty liver disease . Our dat


Although Wnt/β-catenin signaling is generally conserved and well understood, the regulatory mechanisms controlling context-specific direct Wnt target gene expression in development and disease are still unclear. The onset of zygotic gene transcription in early embryogenesis represents an ideal, accessible experimental system to investigate context-specific direct Wnt target gene regulation. We combine transcriptomics using RNA-seq with genome-wide β-catenin association using ChIP-seq to identify stage-specific direct Wnt target genes. We propose coherent feedforward regulation involving two distinct classes of direct maternal Wnt target genes, which differ both in expression and persistence of β-catenin association. We discover that genomic β-catenin association overlaps with Foxh1-associated regulatory sequences and demonstrate that direct maternal Wnt target gene expression requires Foxh1 function and Nodal/Tgfβ signaling. Our results support a new paradigm for direct Wnt target gene co-regulation with context-specific mechanisms that will inform future studies of embryonic development and more widely stem cell-mediated homeostasis and human disease.  •Combining RNA-seq and β-catenin ChIP-seq identifies direct Wnt target genes•Two distinct classes of direct maternal Wnt/β-catenin target genes can be discerned•We propose coherent feedforward regulation of gene expression of the second class•Maternal Wnt target gene expression of both classes requires Nodal/Foxh1 signaling Biological Sciences; Molecular Biology; Developmental Biology Xenopus has been used as an experimental model for the elucidation of germ layer specification  is initially controlled solely by these maternal factors, but maternal control is handed over to the zygotic genome following the synthesis of new gene products. How genes are differentially regulated by transcription factors to specify tissue-specific progenitor cells during this transition is an area of active investigation  and noggin (nog) , plays drastically different roles before and after the maternal-to-zygotic transition  . Within in (nog) , which tin (nog) . Contextin (nog) ; i.e., zin (nog)  or Fgf sin (nog) . Remarkain (nog) .Here we investigate the regulation of direct gene targets of maternal Wnt/β-catenin signaling at the genome-wide level. Different from the later zygotic direct Wnt8a/β-catenin target genes, we find these direct maternal targets are co-regulated by Foxh1-mediated Nodal/Tgfβ signaling. Our results further define two distinct classes of direct maternal Wnt target genes, which differ both in persistence of β-catenin association and temporal expression, with early genes involved in controlling expression of later ones in an apparent feedforward regulatory loop.sia1 and nodal3) at midblastula stage  analysis. Remarkably, our knockdown and rescue experimental design identify transcripts of only ten genes significantly regulated by maternal Wnt signaling at the early onset of ZGA (midblastula). All turn out to encode paralogs of l3, 5, 6 A. Since gsc and nog, which had previously been shown to be Wnt regulated  analysis. β-Catenin indirectly associates with genomic DNA sequences by binding sequence-specific DNA binding transcription factors, principally of the LEF/TCF protein family e.g., . We perfWe then compared this genome association of maternal Wnt-regulated β-catenin before with the genome association of zygotic Wnt-regulated β-catenin well after ZGA  and class 2   . As withc, chrd) G, 23%, wc, chrd) H, 18%, wc, chrd) . In concfoxh1 knockdown and a pharmacological Nodal/Tgfβ signaling inhibitor  and of the second class .We directly validated the requirement of Foxh1 function and Nodal/Tgfβ signaling activity for regulation of direct maternal Wnt/β-catenin target genes using a nhibitor . foxh1 knockdown I and inhnockdown K caused Since we had previously shown that loss of context-defining Bmp or Fgf signaling had no effect on β-catenin recruitment to zygotic Wnt8a/β-catenin target loci , we testsia1 and nodal3 , and direct zygotic Wnt8a/β-catenin target genes, such as hoxd1 and ventx1 . Our previous analysis of direct zygotic Wnt8a/β-catenin target genes had revealed at least two contexts .Stefan Hoppler .Raw sequencing data generated for this study have been deposited in the ArrayExpress database at EMBL-EBI under the accession number GSE53654, GSE72657, and GSE85273.Previously published datasets used in this study are available from Gene Expression Omnibus at NCBI under the accession numbers All methods can be found in the accompanying


The field of gene therapy has experienced an insurgence of attention for its widespread ability to regulate gene expression by targeting genomic DNA, messenger RNA, microRNA, and short-interfering RNA for treating malignant and non-malignant disorders. Numerous nucleic acid analogs have been developed to target coding or non-coding sequences of the human genome for gene regulation. However, broader clinical applications of nucleic acid analogs have been limited due to their poor cell or organ-specific delivery. To resolve these issues, non-viral vectors based on nanoparticles, liposomes, and polyplexes have been developed to date. This review is centered on non-viral vectors mainly comprising of cationic lipids and polymers for nucleic acid-based delivery for numerous gene therapy-based applications. Gene therapy has gained considerable attention in the last few years for treating myriads of devastating diseases. Gene therapy relies on the introduction of nucleic acids or their synthetic analogs  or genome editing proteins , in the cells to regulate gene expression with minimal off-target toxicity. Thus, gene therapy resolves the problem at its source. Though many advances have been made in the field of gene therapy, the delivery of nucleic acid analogs (NAA), as well as genome editing proteins to the target site, continues to be an unresolved issue for the broad utility of gene therapy-based applications. The delivery must be assisted with synthetic biocompatible nanocarriers to overcome the biological barriers and to deliver the cargo at the site of action . A summaLiposome formulations have been used widely for the delivery of small molecules and macromolecule-based therapeutics . LiposomLiposomal vectors based on DOTMA were the first generation of lipid-based vectors utilized to deliver plasmid DNA in vitro. However, DOTMA-based formulations also caused cellular toxicity along with the activation of the immune system because of their cationic charge . Hence, Optimal amounts of cationic lipids are required for high encapsulation efficiency that can lead to an increase in cationic surface charge and subsequently cause toxicity. Hence, ionizable lipids containing an amino head group with an acid dissociation constant (pKa) below 7 are employed ,31. Low The first demonstration of mRNA therapy was performed in 1992 where the intrahypothalamic delivery of vasopressin mRNA led to the temporary reversal of diabetes in rats for 5 days . mRNA-baIn another study, novel lipid libraries were generated for erythropoietin (EPO) mRNA delivery. The formulation components included C12-200  amino) ethyl)n(2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethyl) azanediyl) bis(dodecan-2-ol) as an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a helper lipid, cholesterol (CHO), and C14-PEG2000 as a lipid-anchor PEG. Using a design of experiment (DOE) approach, different libraries of varying C12-200:mRNA weight ratio, molar compositions of lipid components, and phospholipids were tested. The optimized C35 formulation contained C12-200:DOPE:CHO:C14-PEG2000 (35:16:46.5:2.5) as lipid components with a C12-200:mRNA weight ratio of 10:1. The C35 formulation displayed a sevenfold increase in EPO protein expression as compared to the conventional formulation. Further, LNPs were tested to achieve the highest serum EPO levels after systemic delivery in mice. The optimized C35 formulation demonstrated threefold increase in EPO-based luciferase expression in the liver as compared to control groups henicosa-12,15-dienoate (DMAP-BLP) based lipid was used in the LNP formulation. A molar ratio of DMAP-BLP:DSPC:CHO:PEG-lipid (50:10:38.5:1.5) was used to generate LNPs containing IgEsig-prM-E mRNA. The intramuscular administration of LNPs with booster doses at 8 and 14 weeks in immunocompetent mice led to increased serum neutralizing titers . Immunized mice also displayed superior survival post-infection with the ZIKA virus as compared to the control group. Furthermore, modified vaccines based on Japanese encephalitis viral IgE sequence (JEVsig-prM-E LNPs) administered at a dose of 2–10 μg displayed neutralizing titers of approximately 1/100,000 with minimal viremia.LNPs have also been explored for the delivery of mRNA-based vaccines. There has been a considerable interest in developing a vaccine for the ZIKA virus ,39,40. RCaenorhabditis elegans, where RNA controls the gene expression by targeting mRNA strands nonadecanoate (L101) was used to formulate biodegradable and effective LNP formulations. Biodegradable LNPs were made with a molar ratio of L101:DSPC:CHO:PEG-DMG (60:8.5:30:1.5) encapsulating an siRNA targeting proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 is a protein majorly synthesized in the liver that binds to the LDL receptor and regulates the transport of lower-density lipoprotein (LDL) particles. Irreversible binding of the PCSK9 protein to the LDR receptor leads to their loss of function to internalize the LDL particles. This process leads to elevated LDL levels in the plasma, followed by an increasing risk for cardiovascular disease ,55. PCSKTM), a second generation 2′-O-methoxyethyl modified ASO, received global approval for the treatment of hTTR in 2018 [Antisense oligonucleotides (ASOs) are synthetic nucleic acids that hybridize and bind to mRNA by Watson–Crick base pairing and lead to the formation of an ASO–mRNA heteroduplex. Antisense strategy was first reported by Zamecnik et al., where they explored a 13 nucleotide sequence to target 35S RNA of Rous sarcoma virus, which led to the inhibition of viral replication . Althoug in 2018 .PS are synthetic nucleic acids that possess a modified chemical structure wherein the oxygen atom is replaced with sulfur in the phosphodiester linkage, which prevents their enzymatic degradation by nucleases and improves the bioavailability . LNA disFurthermore, the encapsulation of ASOs, as mentioned above in carrier systems, grants them further protection from nucleases and improves their cellular uptake and biodistribution. In general, ASOs demonstrate their efficacy by one of the following mechanisms: (1) recruitment of RNase H at an mRNA–ASO heteroduplex site, which results in the cleavage of target mRNA , (2) theHowever, the therapeutic delivery of ASOs always exerts an enormous challenge for their broader clinical application. LNPs have made promising strides in the delivery of therapeutically active ASOs. Yang et al. developed an LNP-based formulation for ASO delivery . They geBCL-2, which is an anti-apoptotic gene overexpressed in a variety of cancers. An initial screen of LNP formulation was performed on A549  cell lines. LNPs delivered G3139 efficiently and downregulated BCL-2 expression to approximately 40% and approximately 83% for mRNA and protein, respectively. G3139–GAP LNPs, in combination with Paclitaxel, were tested in vivo in xenograft mice. Systemic treatment with G3139-GAP LNPs yielded the highest median survival time (approximately 57.3 days) at a dose of 5 mg/kg in mice along with highest reduction in BCL-2 expression in tumors as confirmed by immunohistochemistry staining compared to control groups [In conjunction with the optimal delivery strategy, ASOs have shown tremendous potential for targeting oncogenic mRNA for cancer therapy . Cheng el groups .Carrier systems containing cationic polymers have an added advantage of formulating smaller uniform particle size, which leads to improved transfection efficiency. Cationic polymers tend to condense and pack the negatively charged nucleic acids . Poly-L-The primary foundation of PACE polymers is based on the presence of branched amino groups to aid in endosomal disruption and have a cleavable ester moiety that can readily hydrolyze in biological conditions . PACE-baPoly (β-amino esters) are biodegradable polymers that show higher transfection efficiency owing to endosomal escape and show lower toxicity as compared to PEI cationic polymers. However, to reduce the overall surface charge of PBAEs, hyperbranched PBAEs (hPBAEs) were developed. hPBAEs of hDD90-118 were made to encapsulate luciferase mRNA and delivered in mice by inhalation. hPBAEs-treated mice showed optimal luciferase protein levels (101.2 ng/g) after 24 h of inhalation .6 fold increase) properties as compared to regular PACE in vitro. In addition, aPACE efficiently delivered mRNA coding for erythropoietin (EPO) in vivo and produced high levels in the blood for up to 48 h without inducing any systemic toxicity. This study explains the overall versatility of PACE polymers wherein their chemistry could be optimized for a wide range of mRNA delivery-based therapeutic applications. Further, a library of PACE polymers with different end groups was tested to assess their superior endosomal escape and transfection efficiency. It was noted that mRNA encapsulation efficiency and endosomal escape plays an essential role in determining the transfection efficiency of PACE polymers [Similarly, poly(amino-co-ester) (PACE) has been extensively used for the mRNA delivery. In general, polymeric materials for gene delivery are synthesized by the “bottom–up” approach. However, the “bottom–up” approach can be difficult and possesses numerous challenges, especially on a commercial scale-up level. Saltzman lab has developed next-generation actuated PACE (aPACE) by the “top–down” approach , which ipolymers . aPACE cTissue transplantation is a vital therapeutic area that has been explored for treating various devastating diseases. Though successful, still, the host adaptive immunity to the grafted tissues is a major challenge for the transplantation-based therapies. Human endothelial cells containing class II transactivator (CIITA) major histocompatibility complex (MHC) molecules regulate immune activation and therefore are considered as important components of the host rejection response. Hence, the treatment of graft endothelial cells with siRNA targeting CIITA would reduce the expression of MHC class II molecules and thereby lead to immunosuppression with better allograft acceptability. To achieve this, PACE-based formulations were explored as a carrier system for siRNA targeting of CIITA. A series of PACE polymers were synthesized based on increasing the composition of 15-pentadecanolide (PDL) to achieve higher hydrophobicity, which generated solid nanoparticles and were tested in the HUVEC  cell line for cytotoxicity. PACE 90 (containing 90% PDL content) nanoparticles were chosen for further ex vivo study as they displayed minimal cytotoxicity. It has been demonstrated that the single ex vivo transfection of PACE containing siRNA weakens the MHC class II expression in human arteries for approximately 4 to 6 weeks after transplantation into immunodeficient mice. This study concludes the advantage of PACE polymers to modulate the release and excellent encapsulation efficiency of siRNA .siRNA delivery has been limited due to challenges such as poor in vivo stability and membrane penetration. The upregulation of Nogo-B protein is seen as a biomarker for hepatic fibrosis and alcoholic liver disease. The subsequent knockdown of Nogo-B protein would thereby lead to healthy liver function, suggesting a suitable starting point for their siRNA-based therapy. A range of PACE polymers were synthesized to achieve the maximum transfection of pLucDNA (plasmid Luciferase DNA), and cytotoxicity was evaluated in HEK293 and HUVEC cell lines followed by in vivo studies. PACE 70 (solid PACE nanoparticles made from 70% 15-pentadecanolide (PDL)) showed increased uptake in Kupffer cells, liver sinusoidal endothelial cells, and hepatic stellate cells when administered systemically. Subsequently, PACE 70 siRNA nanoparticles injected in spleen showed >60% knockdown of Nogo-B protein in the liver. The direct injection of nanoparticles in the spleen was conducted as it displays the same biodistribution of nanoparticles in the liver when compared with intravenous administration .Cationic carriers have been extensively deployed for the delivery of ASOs. Chitosan is a naturally occurring cationic biodegradable polymer used for the delivery of nucleic acids. It was demonstrated that chitosan-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles possess positive surface charge that assist to condense nucleic acid cargo and display their burst release kinetics at pH 7 with an acceptable safety profile . In thisCaenorhabditis elegans [MicroRNAs (miRNA) were first identified in 1993 in  elegans . miRNA b elegans ,101. It  elegans . In part elegans . Further elegans . This stSurface modifications are often employed on gene delivery vectors to ensure cell/site-specific targeting and to improve the stability of nucleic acid cargo . The catAntibody-based therapy has seen a major revolution as polyspecific monoclonal antibodies are capable of targeting multiple tumor antigens with specificity without affecting normal bystander cells . More thIn addition to the coating of delivery systems, direct conjugation with chemical scaffolds such as cholesterol to synthetic nucleic acid mimics such as siRNAs is also performed to increase their specificity as well as decrease the enzymatic degradation after in vivo delivery ,112. CovTM), the second siRNA drug approved by the FDA for the treatment of acute intermittent porphyria (AIP) developed by Alnylam Pharmaceuticals contains three GalNAc residues covalently linked to the siRNA targeting delta-aminolevulinic acid synthetase 1 [The GalNAc approach is widely used for siRNAs targeting liver hepatocytes. The GalNAc site specifically targets asialoglycoprotein receptor (ASGPR), which is overly expressed in hepatocytes . Givosirhetase 1 . A numbehetase 1 ,119, Lumhetase 1 , Revusirhetase 1 , Fitusirhetase 1 ,123 and hetase 1 ,125,126—hetase 1 , GSK3389hetase 1 , IONIS-Fhetase 1 , and IONhetase 1  are GalNOn the other hand, the first-generation DPC technology utilized an endosomolytic polymer PBAVE (poly(butyl amino vinyl ether) where PEG-GalNAc  is conjugated on one end and siRNA cargo is conjugated at other end by reversible disulfide linkage. In DPC technology, PEG chains provide enzymatic stability during systemic circulation and GalNAc residues ensure hepatocyte uptake through the asialoglycoprotein receptor. In an acidic environment, the PBAVE polymer leads to endosomal disruption and the release of siRNA in the cytoplasm . The secFok1 endonuclease [Fok1 endonuclease [Recent advances in genome editing technology possess a vast potential to treat genetic disorders by correcting mutated genes by using cells’ own repair and recombination machinery . Major tnuclease . Similarnuclease . On the nuclease ,139. HowMahiney et al. employed chitosan-coated PLGA nanoparticles to deliver ZFN-mRNA with a donor template in surfactant protein B deficient mice by intrathecal administration. The delivery of ZFN-mRNA led to a correction of genes and improved survival for mice with low immune response . Conway Overall, numerous non-viral based strategies have been employed to improve nucleic acid delivery for next-generation gene therapy. Cationic lipids and polymers have made significant progress in the delivery of various classes of nucleic acids for therapeutic purposes . LikewisHowever, ligand-targeted delivery for cationic lipids and polymers still needs to be explored to increase their applicability at the clinical level. The pros and cons of lipid-based vectors and cationic polymeric vectors are summarized in


Keratoconus (KC) is a corneal dystrophy characterized by progressive ectasia that leads to severe visual impairment and remains one of the leading indications for corneal transplantation. The etiology is believed to be multifactorial and alterations have been documented in the biomechanical, biochemical and ultrastructural characteristics of the cornea. While the exact site of disease origin is still debated, changes in the corneal epithelium are believed to occur even before the disease is clinically manifested. In this study we investigate the possible role of β-catenin as mechanotransducer in KC corneal epithelium. The sheets of corneal epithelium removed from keratoconic eyes when they underwent collagen crosslinking as a therapeutic procedure were used for this study. The healthy corneal epithelium of patients undergoing Laser Refractive Surgery for the correction of their refractive error, served as controls. Immunoblotting and tissue immunofluorescence studies were performed on KC epithelium to analyse the expression and localization of β-catenin, E-cadherin, ZO1, α-catenin, Cyclin D1, α-actinin, RhoA, and Rac123. Co-immunoprecipitation of β-catenin followed by mass spectrometry of KC epithelium was performed to identify its interacting partners. This was further validated by using epithelial tissues grown on scaffolds of different stiffness. Histology data reported breaks in the Bowman’s layer in KC patients. We hypothesize that these breaks expose the epithelium to the keratoconic corneal stroma, which, is known to have a decreased elastic modulus and that β-catenin acts as a mechanotransducer that induces structural changes such as loss of polarity (Syntaxin3) and barrier function (ZO1) through membrane delocalization. The results of our study strongly suggest that β-catenin could be a putative mechanotransducer in KC epithelium, thus supporting our hypothesis. Such regulation is based on mechanotransductional processes which initiate distinct signalling pathways1. The cell specific and molecular basis of mechanosensing and the sequence of biochemical reactions that ensue, have been the focus of recent research3. Several molecules have been proposed as possible mechanosensors such as integrins, ion channels, G-protein-coupled receptors, and cell–cell junctional proteins4.The mechanical properties of biological tissues are essential for their physiological functions6. RNA sequencing studies on KC corneal epithelium have reported collagen degradation, and downregulation of TGF β, Hippo, Hedgehog, Notch1 and Wnt signalling pathways8. The potential role of long non coding RNA has also been explored in KC9. While the exact site of origin of the disease is still debated, changes in corneal epithelium, basement membrane, Bowman’s layer and stroma have been reported11. Tear proteome analysis by electrophoretic separation and Mass spectrometric (MS) techniques have identified decreased expression of cystatin family of proteins  and increased expression of lipocalin family of proteins (tear lipid binding proteins), Cathepsin B and higher level of cytokines12. Recent research has also highlighted the influence of endocrine function in regulating metabolism and inflammatory pathways in KC suggesting KC to be a partially systemic disease13. Metabolites including hormones such as prolactin-induced protein have been proposed to be important hormonally regulated biomarkers in KC14. While the exact etiology of the disease remains an enigma, the final common pathway is believed to be a decrease in the elastic modulus of the stroma. The lower stiffness of stroma initiates a cascade of events that results in morphological and biochemical changes that affect the shape and the optical function of the cornea. Morphological changes in the corneal epithelium have been well documented and are considered to be one of the earliest detectable changes observed in KC15. Based on the hypothesis that these changes are representative of a mechanotransduction process, we believe that studying the signalling pathway in the epithelium of KC patients would offer a unique opportunity to unveil the molecular basis of mechanotransduction in corneal epithelium.Keratoconus (KC) is a bilateral progressive visually debilitating clinical condition characterized by abnormal thinning and protrusion of the cornea in response to biomechanical instability of the corneal stroma. Several studies have shown that genetic, biochemical and biomechanical factors are involved in the pathogenesis of this disease16, responds significantly to extracellular matrix stiffness17. The central molecule in Wnt/β-catenin pathway is β-catenin which is an evolutionarily conserved protein and exists in three different subcellular locations: membrane bound, cytosolic, and nuclear18. The membrane bound β-catenin has adhesion function whereas its nuclear form plays an important role in transcriptional regulation. In the membrane, β-catenin binds with both E-cadherin and α-catenin which in turn interact with actin19. The α-/β-catenin interaction is regulated by small GTPases—RhoA, Rac1 and Cdc42, which in turn regulate actin polymerization20. In corneal limbal epithelial stem cells, the nuclear localization of β-catenin is reported to mediate cell proliferation. In contrast, the central corneal epithelium exhibits membrane localization of β-catenin indicating reduced cellular proliferation21.Wnt/β-catenin pathway, which is known to control diverse cell functions7. However, to the best of our knowledge, the role of β-catenin in KC has not been explored so far. We hypothesize that β-catenin plays an important role as a mechanotransducer and that the detection of its localization could potentially be used for early diagnosis of KC. In this study, we analysed the localization and expression of β-catenin in KC epithelium. We also studied the expression of its interacting proteins such as E-cadherin, α-catenin, α- actinin, ZO1(Zonula occludens1). Scanning electron microscopy (SEM) and Actin staining revealed the structural changes in KC epithelium. β-catenin Co-IP in corneal epithelium of mild KC and healthy control identified novel interacting partners. It also explained the possible role of β-catenin in KC epithelium where canonical Wnt pathway is known to be inactive. Further, the response of β-catenin to different elastic modulus was studied using Poly dimethysiloxane (PDMS) and gelatin hydrogel with control corneal epithelium. The results further validated our hypothesis that β-catenin does have a role in mechanotransduction.Wnt signalling has been reported to be altered in KC epitheliumP > 0.05) across KC epithelium and compared it with control epithelium, we did not observe any significant changes in total β-catenin expression in KC epithelium across the grades  was observed compared to control epithelium , was observed compared to control epithelium    Fig. A. The lo01) Fig. C. Furthe05) Fig. D. PossibP < 0.05) in severe KC epithelium compared to the control whereas Rac 123 levels were significantly downregulated (P < 0.05)in severe KC epithelium   in SEM imaging whereas the severe KC epithelial showed a thin layer. The basal cells of severe KC epithelium exhibited larger surface than the control epithelium Fig. A. The boP < 0.05) was observed suggesting that the KC epithelium is likely to be more migratory  down regulated  in plasma membrane whereas in mild KC, only 18% of the identified proteins were localized to plasma membrane Fig. D.P < 0.05)  Fig. A. Next, 05) Fig. B indicat05) Fig. A.Figure 25. β-catenin localization on epithelial graft grown on PDMS showed membrane localization whereas on gelatin hydrogel it showed both membrane and cytoplasmic localization similar to the epithelium of mild KC  confirmed that in KC epithelium, the localization of β-catenin has been altered and is cytosolic and nuclear. However, there is no difference in the total β-catenin protein expression. On further investigation of the interacting partners of membrane bound β-catenin, we observed significant down regulation of E-cadherin and α-catenin expression in KC corneal epithelium. The role of E-cadherin could  possibly be compensated by increased Pan cadherin expression. Additionally, membrane bound E-cadherin was lost indicating the loss of cell–cell adhesion. To maintain a polarized epithelium, actin cytoskeleton co-operates with E-cadherin- and integrin-based cell–cell or cell–matrix adhesions38. However, this protein may not be directly involved in disease progression of KC as β-catenin was unaltered in the diseased condition. β-catenin is a central molecule in Wnt signaling pathway and the transcriptional activity of β-catenin is also modulated by direct regulation of its subcellular localization by a variety of interacting partners39. In mild KC epithelium in the absence of the Wnt signaling, the loss of interaction between β-catenin and α-catenin might lead to lowered ubiquitylation and stabilization of β-catenin. Similarly, the ubiquitylation of native β-catenin was strongly inhibited in α-catenin depleted cells leading to stabilization of the protein40. Additionally, the known interacting proteins Collagen alpha-1 chain and Laminin subunit alpha-4 could also alter the localization of β-catenin. Furthermore, in response to collagen 1 coating, actin reorganization was altered in corneal epithelial cells. β-catenin localization was altered in response to matrix stiffness (PDMS to gelatin hydrogel) mimicking KC. Additionally, Tenascin C was found in both control and mild KC epithelium, it was upregulated in KC due to the presence of the β-catenin in the cytosol and possibly in nucleus. Tenascin C has an antiadhesive activity and has been reported to be regulated by β-catenin41. Therefore, we summarize that β-catenin might sense the altered stiffness in KC when the Bowman’s layer is ruptured. It is established that various layers of cornea exhibit different elastic moduli25 and as the disease progresses the epithelium is exposed to matrix of different elastic moduli.The localization of β-catenin is also dependent on Wnt signallingIn summary, our study revealed distinct changes in the localization of β-catenin in the corneal epithelium of patients with all clinical grades of Keratoconus. These changes appear to be directly influenced by changes in the elastic modulus of the substrate, and were associated with the downregulation of E cadherin and α-catenin that were also observed. The total expression of β-catenin was unchanged, suggesting a loss of interaction between β-catenin and α-catenin as demonstrated by the co-immunoprecipitation studies. Additionally, there was loss of ZO-1 tight junction protein and upregulation of α-actinin. Overall, these changes may contribute to the altered structure and polarity of the corneal epithelial cells and may be expected to make them less adhesive and more migratory in nature. Based on the consistency of our observations, we propose that β-catenin could be considered as a potential mechanotransducer in the corneal epithelium .This study, which conformed to the tenets of the Declaration of Helsinki, was reviewed by the local ethics committee and approved by the institutional review board of Vision Research Foundation, Sankara Nethralaya, Chennai India (Ethics No. 659-2018-P).The tissues used for the study were taken from patients after obtaining their signature and informed consent.42. The clinician used an epikeratome to remove the epithelium as a single sheet while performing Photorefractive keratectomy (PRK). Care was taken not subject it to any mechanical stress. The control tissues to be used for cell culture were collected in Defined Keratinocyte-Serum Free medium (SFM) (Thermofisher Cat.No-10744019) and were processed immediately after the collection. However, the control and the KC epithelium tissues which were subjected for immunoblotting, Tissue immunofluorescence and Co-immunoprecipitation (Co-IP) was collected in DMEM F-12(Gibco) medium with sodium Pyruvate (Sigma) and transported to the laboratory. Concurrently, Polydimethylsiloxane (PDMS) (Sylgard 184 Silicone Elastomer kit) and gelatin (Bovine skin Type B Sigma) hydrogel of different elastic modulus were prepared as described to serve as substrate for culture of epithelial tissues from normal control43.All study subjects were clinically examined at the department of Cornea, Medical Research Foundation, Chennai, India and the clinical details are presented in Table g for 10 min at 4 °C. The supernatant was collected, Proteins were estimated using BCA protein assay kit (Thermo Fisher Cat No-23227). For Western blot, equal concentration of tissue lysates were loaded onto the gel and separated on a sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) at 100 V in electrophoresis buffer . The proteins were separated and transferred to PVDF membrane  using semi-dry transblot apparatus (Hoefer) at 1.50 mA/cm2. The membrane was then blocked for 1 h at 25 °C in 5% (w/v) non-fat dry milk powder (NFDM) in TBST . It was washed with TBST, incubated at 4 °C overnight with the following antibodies :E-cadherin, Pan cadherin, α-catenin, β-Catenin, from Cadherin-Catenin antibody sampler kit  β-Catenin for Co-IP , β-Catenin for immunoblotting (Abcam-ab22656) ZO1, Wnt5ab, RhoA, Rac1/2/3, α-Actinin  β-actin, Tenascin C and Cyclin D1 , Syntaxin 3 . Each antibody was 1000-fold diluted in either 5% (w/v) BSA (Hi Media Cat No-MBO3) or NFDM in TBST. After overnight incubation, the membrane was washed thrice for 5 min with TBST and further incubated in corresponding HRP-conjugated Anti-rabbit and Anti-mouse secondary antibody). The secondary antibodies used were diluted to 10,000- fold in 5% NFDM (w/v) in TBST. After incubation, the membrane was again washed thrice for 5 min with TBST. HRP activity was detected using HRP substrate (Bio-Rad Cat No-1705061) in Bio-Rad Gel Documentation system (Protein Simple).Tissue collected was dissolved in RIPA (Radioimmunoprecipitation assay) buffer (EMD Millipore Cat No-20-188), sonicated in ice for 15 s, and centrifuged at 13,000×. The tissues were imaged using Zeiss Microscope using 2 different channels.Tissues collected from patients were fixed in 4% Paraformaldehyde and washed with Phosphate buffered saline (PBS).Tissues were permeabilized with 0.5% triton-100 followed by PBS wash. The tissues were then blocked with 1%BSA prior to overnight incubation with primary Antibody. The detection was done using Cy3.5 secondary antibody and counterstained with Hoechest (Thermo fisher Cat No-33342).Further actin fibers of the tissues were stained with 100 nM of Phalloidin-488  after permeabilization. The tissues were incubated for 45 min in dark and Counter-stained with HoechstU Upright Microscope from Nikon Instruments Inc).Formalin-fixed paraffin-embedded (FFPE) tissue blocks of control and KC epithelium were made and processed for immunohistochemical analysis. Immunohistochemistry (IHC) was performed on Bench Mark GX (Ventana Roche) Automated devices for the Bio-SB antibody at M/s S.M Surgipath, Chennai, using β-Catenin (Abcam-ab22656) and YAP (CST-14074) antibodies. β catenin protein was visualized with HRP conjugated anti mouse antibody (Sigma Aldrich). The peroxidase was detected with 3,3′ diaminobenzidine (Sigma Aldrich) each section was counter stained with Mayer’s Haematoxylin (sigma Aldrich). All stained sections were analysed under a light microscope (Eclipse Ni-Control tissues and mild KC tissues were pooled separately after collecting from patients for performing co-immunoprecipitation(Co-IP). Endogenous co-immunoprecipitation(Co-IP) was performed to identify the novel interacting partner of β-catenin. For performing co-immunoprecipitation(Co-IP), around 500 μg of protein lysate was required as reported earlier. Hence, we pooled lysates from 4 Control samples and designated as C1. Similarly, C2 and C3 lysates were prepared. For KC also, lysates from 4 mild KC samples were pooled and designated as K1. Similarly K2 and K3 lysates were also prepared. Tissue lysates were prepared using RIPA buffer. 400 μg lysates, were precleared using 40 μl of Protein-A Magnetic beads (New England Biolabs Cat No-S1425S) to remove non-specific components from the lysate. After preclearing 4 μg of β-catenin antibody  and IgG antibody were incubated overnight at 4 °C. Further, 40 μl of Protein A/G Magnetic bead suspension were added and incubated with precleared lysate, agitated for 1 h at 4 °C. Magnetic field was then applied to pull the beads to the side of the tube and 3 washes were performed using Wash buffer. Further the bead pellet was suspended in 5X sample loading buffer.3. The gel was washed with washing solution  until the Coomassie die was completely removed and incubated at room temperature for 15 min with gentle agitation. The gel was then dehydrated using 100% acetonitrile(ACN). Then ACN was removed, and the dry gel was kept at room temperature for 10–20 min in a vacuum evaporator. The gel was rehydrated for 30 min at 56 °C using reduction solution . Further, alkylating solution  was added and incubated for 30 min in the dark at room temperature. After sequential washing with 25 mM NH4HCO3, 25 mM NH4HCO3/50%ACN, and 100% ACN, gel pieces were dried and rehydrated with 12.5 ng/mL trypsin  solution in 25 mM ammonium bicarbonate on ice for 30 min. The digestion was continued at 37 °C overnight. The tryptic peptides were extracted from gel pieces with extraction solution  and sonicated in ultrasonic water bath for 10 min. Further extracted peptides were resuspended in 50% acetonitrile, 0.1% TFA. The samples were spin down and spotted 0.5µL volume on alpha-cyano-4-hydroxycinnamic acid matrix  of MALDI plate. The spots were allowed to dry and further plate was loaded into Voyager.The immunoprecipitated product was loaded on 10% SDS Polyacrylamide gel. The electrophoresis of the sample was carried out using Tris–Glycine buffer at 100 V. After the run was completed the proteins were stained with Coomassie blue stain (Sigma Cat No-G1041).The gel band from IgG Lane, β-catenin IP from control tissue and β-catenin IP from mild KC tissue were excised into 1 mmScanning electron microscopy , was used to analyse the morphology of epithelial tissue collected after surgery. The tissues were fixed with 3.7% glutaraldehyde (Sigma Cat No- G5882) in PBS for 15 min in aluminium-coated coverslip. After washing twice with PBS, the fixed tissues were dehydrated with an ascending sequence of ethanol . After evaporation of ethanol, the samples were left to dry at room temperature for 24 h on a glass substrate, and then analyzed by SEM after gold–palladium sputtering .43.Polydimethylsiloxane (PDMS) gel was prepared using polymer and curing agent in a ratio of 1:10 and incubated at a temperature of 80 °C for 24 h in 24 well plate. Further, the PDMS were coated with type-1collagen (Sigma Cat No -C4243).Hydrogels were prepared using type B gelatin (Sigma G9391). 50 mg/ml of gelatin concentration with 0.8 µg/mL of glutaraldehyde as crosslinker were used for preparing hydrogel and incubated at 4 °C for 4 h for gelation and cross-linking. The cross-linked gelatin hydrogels were immersed in a 50 mM glycine aqueous solution under agitation for 1 h to block the residual aldehyde groups of glutaraldehyde, followed by two washes with double-distilled water for 1 h−1 and the strain of 0.1% at 25 °C. G′ is the storage modulus for measuring the gel-like behaviour of a system, whereas G″ is the loss modulus for measuring the sol like behaviour of the system.The stiffness of the hydrogels were measured using Microsquischer  instrument. The compressive method was employed using 3X3mm plate bound to microbeam. Different forces  were used for obtaining the force Vs time and force Vs displacement curves. To measure the stiffness, stress (Force/Area) and % strain(tip displacement) were calculated. The graph of stress vs strain was plotted, and linear region was considered to calculate the slope to find the young’s modulus. The rheology (young’s modulus) of hard PDMS gel was measured using MCR301 rheometer (Anton Paar). As a positive control, we used cadaveric stroma along with PDMS. Dynamic oscillatory strain sweep experiments were completed on the PDMS/Cadaveric stroma to find the limit of viscoelastic property. The sample size of 1 cm radius was used between the compressive plates. The size and thickness of the sample was maintained. The dynamic time sweep was conducted at the frequency of 1 rad s45. After 24 h of incubation, tissues were fixed and immunofluorescence imaging was performed.Corneal epithelial cells were cultured in Defined Keratinocyte-Serum Free medium (SFM) (Thermofisher Cat.No-10744019).The tissues collected from control patients was cultivated on type 1 collagen coated 1:10 Polydimethoxy siloxane (PDMS) of desired elastic modulus and 50 mg/ml of Gelatin Hydrogel in Defined Keratinocyte-SFM medium (Thermofisher Cat.No-10744019).Adherence of the tissues to culture plate was assured by a gravitational force from viscoelastic solution added on top of the tissues  as reported earlierCorneal section was deparaffinized in Xylene and then rehydrated in 100% and 95% ethanol. After washing, it was stained with Hematoxylin (HI Media GRM 236). The section was differentiated with 1% HCl in 70% ethanol, then washed with water, followed by blueing with weak ammonia. The slide was stained with eosin and then dehydrated slides were mounted.p Values < 0.05, 0.01, and 0.001 respectively.The corneal epithelium was collected from documented KC patients. All experiments were performed on at least 7 independent patients samples. In the manuscripts ‘n’ represents individual patient samples. The statistical significance of the differences was analysed by one-way analysis of variance (ANOVA) followed by Bonferroni Post-hoc test when comparisons were done between control, mild, moderate and severe KC using Graph Pad software for the samples mentioned in the Figs. Supplementary Information.


These results support the notion that a cyanobacterial ancestral protein laid the foundation for the evolution of photorespiratory GOX enzymes in modern eukaryotic phototrophs.Photorespiration has been shown to be essential for all oxygenic phototrophs in the present-day oxygen-containing atmosphere. The strong similarity of the photorespiratory cycle in cyanobacteria and plants led to the hypothesis that oxygenic photosynthesis and photorespiration co-evolved in cyanobacteria, and then entered the eukaryotic algal lineages up to land plants via endosymbiosis. However, the evolutionary origin of the photorespiratory enzyme glycolate oxidase (GOX) is controversial, which challenges the common origin hypothesis. Here, we tested this hypothesis using phylogenetic and biochemical approaches with broad taxon sampling. Phylogenetic analysis supported the view that a cyanobacterial GOX-like protein of the 2-hydroxy-acid oxidase family most likely served as an ancestor for GOX in all eukaryotes. Furthermore, our results strongly indicate that GOX was recruited to the photorespiratory metabolism at the origin of Archaeplastida, because we verified that Glaucophyta, Rhodophyta, and Streptophyta all express GOX enzymes with preference for the substrate glycolate. Moreover, an “ancestral” protein synthetically derived from the node separating all prokaryotic from eukaryotic GOX-like proteins also preferred glycolate over  Oxygenic photosynthesis is among the most important biological processes on Earth, because it produces the vast majority of organic carbon and nearly all of the atmospheric oxygen. This process is thought to have evolved approximately 2.5 billion years ago in cyanobacteria, and was later conveyed into a eukaryotic host cell via endosymbiosis, giving rise to plastids ,2,3. The2 by Rubisco . In addition to the carboxylation of ribulose 1,5-bisphosphate (RuBP) with CO2, Rubisco also catalyzes the oxygenation of RuBP, being the first step in a process termed “photorespiration”, leading to the appearance of the potent enzyme inhibitor 2-phosphoglycolate (2PG) [2 and 0.04% CO2 [2. It has been estimated that in a crop, C3 plant photorespiration might decrease the yield by approximately 30% under present day atmospheric conditions [All organisms performing oxygenic photosynthesis fix inorganic carbon as COte (2PG) ,9. Despi.04% CO2 . As 2PG .04% CO2 ,12,13, i.04% CO2 . Howevernditions . Thus, pnditions ,17.2-containing atmosphere with low CO2 . In con2 . LOX and GOX (here summarized as GOX-like proteins) belong to the group of 2-hydroxy-acid oxidases, display highly similar primary and tertiary structures  [Spirogyra pratensis (sequence is shown in Cyanophora paradoxa was corrected (see below). The complete gene with a corrected exon/intron structure and the complete protein coding sequence is shown in To evaluate the GOX phylogeny, we considered a broad spectrum of phyla, including non-photosynthetic species. The well-characterized At-GOX2 from 3g14415)  was used3g14415)  searchesTo reanalyze the phylogenetic origin of the plant and animal GOX-like proteins, we took sequences from a broad spectrum of phyla into consideration, for example, we also included related proteins from the chromalveolate taxa . In totaThe midpoint-rooted Bayesian tree  is well Similar to the previous study by Esser et al. , we alsoOverall, our phylogenetic analyses show that eukaryotic GOX-like proteins are more closely related to those of cyanobacteria than to those of any other prokaryotic group, including Proteobacteria and Archaea. As it is known that phylogenetic analysis can be biased when homologous proteins evolved into enzymes with varied metabolic functions, we set out a comprehensive biochemical evaluation of GOX-like proteins among Archaeplastida.l-lactate, that is, they rather represent LOX than GOX enzymes. Therefore, we analyzed the substrate preference of GOX-like proteins from all groups of Archaeplastida. To this end, we overexpressed selected cDNAs in E. coli to obtain recombinant proteins for biochemical characterization. In addition to the previously obtained data for GOX and LOX proteins from Cyanobacteria, Rhodophyta, Chlorophyta, and land plants [Cyanophora paradoxa and the streptophyte (sister clade of land plants) alga Spirogyra pratensis were biochemically characterized using substrate concentrations of glycolate and l-lactate ranging from 0.1 to 200 mM. We found that both recombinant proteins can oxidize glycolate and, to a lesser extent, l-lactate  to glycolate than to l-lactate as a substrate. Although the Vmax values are only slightly higher for glycolate than for l-lactate, the catalytic efficiency (kcat/Km) is 20 to 33 time higher with glycolate as substrate, which is similar to AtGOX2 substrate preferences. For example, many of these enzymes preferentially oxidize d plants ,38, the -lactate , which io AtGOX2 . Based olycolate . This fil-lactate over glycolate [Arabidopsis revealed that the three amino acid residues in the active site  and a slightly higher kcat/Km with glycolate as a substrate -2-hydroxy-acid oxidase protein family. Besides the important role of GOX in the photorespiration of plants, where it oxidizes glycolate to glyoxylate  library [Arabidopsis At-GOX2 (At3g14415) sequence. The full-length cDNA sequence from Spirogyra (Spirogyra GOX (Sp-GOX) was deposited in GenBank under the accession number AVP27295.1.The complete sequence of the  library , which wpirogyra  was compC. paradoxa was identified by BLAST searches with plant and algal GOX proteins. The originally annotated Cyanophora protein of the genomic Contig54585 consists of 316 amino acids, which is considerably shorter than the GOX protein from Arabidopsis (367 amino acids). Initially, we used this cDNA to synthesize the Cyanophora gox gene; however, we failed to obtain an enzymatic active protein, most likely because of the missing C-terminus. Subsequent BLAST searches including EST sequences identified two different EST sequences (EG947183.1 and ES232585.1), which partially overlap with the gox gene in the Cyanophora genome database . The newly obtained cDNA for the Cyanophora gox gene  was also used for gene synthesis (Thermo Fisher), but we again failed to produce an enzymatic active protein. Finally, we re-sequenced the entire gox gene from Cyanophora gDNA using new primer sets . The gox gene of Spirogyra and the gene encoding the ancestral GOX protein were also cloned into pASG-IBA43plus. However, the N-terminal His-tag was used for purification with Ni-NTA Sepharose, according to the supplier’s protocol . The obtained E. coli strain was grown in an LB medium to an OD750 of 0.6. The gene expression was initiated with the addition of 200 µg/L anhydrotetracycline for 4 to 16 h at 20 to 30 °C. A soluble protein of the ancestral N3-GOX could be only obtained when cultivating E. coli cultures at 18 °C for 16 h after induction with anhydrotetracycline. E. coli cells were harvested by centrifugation and re-suspended in buffer A , and 0.1 mM Flavin mononucleotide (FMN)) in case of His-tag fusion proteins or buffer W  in case of Strep-tag proteins. The protein extraction was done by ultrasonic treatments  on ice. After centrifugation , the cell-free protein extract was directly loaded onto Ni-NTA or Strep-tactin columns. The His-tagged proteins were washed using buffer A supplemented with 40 to 80 mM imidazole, whereas His-tagged GOX was eluted with buffer A supplemented with 200 mM imidazole. For Strep-tagged proteins, the washing steps were performed using the buffer W, and the proteins were eluted by buffer W, supplemented with 2.5 mM desthiobiotin. Subsequently, the eluted proteins were desalted using PD-10 columns , and finally dissolved in 20 mM Tris/HCl pH 8.0 containing 1 mM DTT and 0.1 mM FMN. The purity of the eluted proteins was checked by SDS-PAGE and staining with Coomassie Brilliant Blue , as described by Hackenberg et al. [2 in 1 min at 30 °C. The protein concentrations were estimated according to the literature [The GOX or LOX activity was measured by the detection of Og et al. . One uniterature . l-lactate and other related substrates according to the requirements of the specific host. The monophyly of eukaryotic and cyanobacterial GOX-like proteins points to a cyanobacterial origin of all eukaryotic GOX-like proteins, including the photorespiratory GOX of phototrophic eukaryotes. Biochemically, these proteins operate as GOX rather than LOX in the Archaeplastida. The slight preference for glycolate of the synthetic ancestral N3-GOX suggests that glycolate oxidation could have been the ancestral function, and during the course of evolution, this protein has changed its preference to glycolate or 2-fixing cyanobacteria optimized the ancestral GOX-like protein to operate as LOX, which helps consume oxygen, protecting nitrogenase [max of glycolate oxidation.The likely scenario for the evolution of GOX among eukaryotes and particularly of the photorespiratory GOX of Archaeplastida is summarized in rogenase . Among trogenase . After trogenase . The incHence, the early acquisition of a peroxisomal GOX-like protein by the hypothetical host cell ,76,77 ca


The resulting resins have glass transition temperatures and strengths similar to those of conventional epoxy resins. The influences of GA content, DMP-30 content, and temperature on the dissolution rate were studied. The decomposed epoxy oligomer (DEO) is further used as a reaction ingredient to prepare new resins. It is found that the DEO can improve the toughness of epoxy resins significantly. This work provides a simple method to prepare readily recyclable epoxy resins, which is of low-cost and easy to implement.Epoxy-anhydride resins are widely used in engineering fields due to their excellent performance. However, the insolubility and infusibility make the recycling of epoxy resins challenging. The development of degradable epoxy resins with stable covalent networks provides an efficient solution to the recycling of thermosets. In this paper, 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30) is incorporated into the epoxy-glutaric anhydride (GA) system to prepare high-performance epoxy resins that can be recycled below 200 °C at ordinary pressure via ethylene glycol (EG) participated transesterification. The tertiary amine groups in DMP-30 can catalyze the curing reaction of epoxy and anhydride, as well as the transesterification between ester bonds and alcoholic hydroxyl groups. Compared with early recyclable anhydride-cured epoxy resins, the preparation and recycling of diglycidyl ether of bisphenol A (DGEBA)/GA/DMP-30 systems do not need any special catalysts such as TBD, Zn(Ac) Further, industrial epoxy thermosets and composites can be dissolved in TBD-alcohol solvents via catalyst-activated TERs [The development of vitrimers containing dynamic covalent bonds (DCBs) makes it possible to recycle thermosets under mild conditions ,23,24,25ted TERs ,41.2 [2 [In order to activate TERs between ester bonds and alcoholic hydroxyl groups, sufficient high-efficiency catalysts such as Zn(Ac)2 ,42, Sn, and low strengths. Hao et al. [Tg and strength. The resulting resins can be easily hydrolyzed using a phosphotungstic acid aqueous solution. Here, the tertiary amines in TEOA played a dual role, which catalyzed both the curing reaction and the TERs between ester bonds and hydroxyl groups. This work also provides a new strategy to develop recyclable high-performance epoxy resins with low cost from industrial raw materials.Recently, researchers developed a new type of catalyst-free epoxy vitrimers by introducing abundant hydroxyl groups or covalently bonded tertiary amines as the internal catalyst of TERs ,47,49,50o et al.  incorpor2) used in early recyclable epoxy vitrimers reported in the literature, the introduction of DMP-30 avoids the disadvantages of high cost, toxicity, and poor miscibility with the epoxy-anhydride system. In this paper, EG is selected as the solvent for dissolution because of its high boiling point and rich hydroxyl groups phenol (DMP-30), which can effectively reduce the curing temperature and speed up the curing process ,52. It cl groups . The disW: 340.41 g/mol) was purchased from Sigma Aldrich . The curing agent glutaric anhydride  and catalyst 2,4,6-tris(dimethylaminomethyl)phenol  were ordered from Macklin Biochemical Co., Ltd. . The release agent JD-909 was obtained from Jiadan Mold Release Agent Co., Ltd. . Organic solvents used for swelling and dissolution tests including N,N-dimethylformamide , 1,2,4-trichlorobenzene , xylenes (AR grade), tetrahydrofuran , heptane (AR grade), and ethylene glycol  were provided by Macklin Biochemical Co., Ltd. . All the chemical reagents were used directly without any purification.The epoxy oligomer diglycidyl ether of bisphenol A  to determine the curing conditions of the DGEBA/GA/DMP-30 curing system. In each case, a sample weighing ~10 mg was sealed in a 40 µL aluminum crucible. The sample was first stabilized at 0 °C for 10 min and then heated from 0 °C to 250 °C at the heating rate of 10 °C/min under a nitrogen atmosphere.−1 for 32 scans with a resolution of 4 cm−1. The band of carbon-carbon stretching vibration of phenyl (c-cνph)( = 1607 cm−1) was used as a reference to normalize the obtained spectra.In order to monitor the functional groups of samples during the curing and recycling process, FTIR spectra were recorded by a Nicolet iS50 FTIR spectrometer  in attenuated total refraction (ATR) mode. Before testing, the sample was irradiated under an infrared lamp to remove the moisture. The dried sample was scanned from 650 to 4000 cmE’) and damping factor (tan δ) were collected during the temperature scan. The Tg value was assigned at the maximum of tan δ.DMA experiments were conducted on a DMA242E instrument  to obtain the thermal-mechanical behaviors of epoxy resins. The samples were cut into sheets with a dimension of 20 mm × 5 mm × 1 mm. During the experiments, the tensile mode was used at the frequency of 1 Hz, and the temperature increased isothermally from ambient temperature to 150 °C at the rate of 3 °C/min. Storage modulus (ν) was calculated by Equation (1):E’ is the storage modulus of the resin in the rubbery plateau region at Tg + 50 °C, R is the gas constant, and T refers to the absolute temperature in Kelvin.The crosslink density . The samples were cured in dumbbell-like molds designed according to ASTM D638, and the effective size of the sample is 50 mm × 13 mm × 3~5 mm. The tensile rate was 1 mm/min for all the tests, and at least three specimens were tested in each sample.TGA measurements were performed on a simultaneous thermal analyzer  in the TGA mode to evaluate the thermal stability of epoxy resins. The sample weighing ~10 mg was placed into an alumina pan and heated from ambient temperature to 800 °C at a heating rate of 10 °C/min under a nitrogen atmosphere with a gas flow rate of 50 mL/min.m0) of 5 mm in length, 3 mm in width, and 2 mm in thickness was immersed in the organic solvent at room temperature for 96 h or 180 °C for 12 h, then taken out and weighed (ms). The swollen sample was then placed in a vacuum oven at 180 °C for 12 h to eliminate the solvent. The residual mass (md) of the dried sample was recorded.The solubility of epoxy resins in DMF, TCB, xylenes, THF, and heptane at ambient temperature or high temperature was investigated. The sheet sample . The selected temperature was around at the boiling point of EG and far less than the pyrolysis temperature of epoxy resins. At different time intervals, the sample was taken out, cleaned with 95% ethanol, and weighed immediately to record the mass change. The disappearance of solid epoxy resins meant the complete dissolution of samples. A mixture solution composed of epoxy oligomer and extra EG was derived.The recycling of decomposed epoxy oligomer (DEO) was achieved by heating the dissolved solution at a higher temperature (200 °C) in an open environment for ~12 h, where residual EG is evaporated. There were two disposal methods for recycled DEO. On the one hand, the DEO was cured directly at 180 °C for ~24 h to obtain the re-crosslinked network. Another strategy was to mix the DEO with the raw epoxy materials to prepare new resins. The specific synthesis process was as follows. The DEO  and molten DGEBA were mixed at 130 °C for 10 min under magnetic stirring. After getting a homogeneous mixture, GA and DMP-30 were added successively in a determined amount and continuously stirred at 80 °C to obtain a uniform dispersion. After vacuuming, the reactive mixture was poured into the metal mold and cured with the same conditions as the original epoxy resins.The catalyzed curing of epoxy-anhydride systems is usually complicated ,54. Herec=oνester)( = 1732 cm−1) did not increase after curing at 150 °C for 2 h ( = 914 cm−1). With the increase of GA content, the intensity of the ester bond peak increased, suggesting a higher content of ester bond in the crosslinked network. As the curing reaction tended to be complete with increasing the content of the curing agent [−1 appeared in the FTIR spectra when the ratio of epoxide to acyl, r = 1:1, while it disappeared when r = 1:2. Abundant hydroxyl groups are necessary for the TERs [The chemical structures of five kinds of epoxy resins before and after curing were characterized by FTIR spectra . After cng agent ,58, a vithe TERs ,51, but the TERs ,62,63,64Tgs obtained from the peak temperature of tan δ curves were changed from 46.39 °C to 87.73 °C  and elastic modulus (1390.24 MPa). Epoxy1 with the least amount of GA showed the lowest tensile strength (42.24 MPa) and elastic modulus (1155.05 MPa). The elongation at break increased gradually with r. The difference in the mechanical properties of epoxy resins can be attributed to the difference in crosslink density and the number of unreacted groups in networks [networks ,67,68,69modulus 10.24 MPa.networks ,59,70, tT5% (the temperature at 5% weight loss) values ranged from 357 °C for r = 1:1.00 to 385 °C for r = 1:2.00, while the T10% (the temperature at 10% weight loss) values ranged from 390 °C to 401 °C. The lower thermal stability of the resin with r = 1:1.00 could be associated with the degradation of unreacted epoxy monomer in the network. The derivative thermogravimetric (DTG) curves showed that the Tmax (the temperature corresponding to the maximum weight loss rate) of each sample was ~425 °C. In the subsequent dissolution and recycling experiments, the temperature of 140–200 °C was used to avoid the influence of thermal degradation.In order to determine the appropriate dissolution and recycling temperature which was far lower than the pyrolysis temperature of epoxy resins, the thermal stability of epoxy-anhydride networks was determined using TGA . The epoIn order to explore the solubility of epoxy resins, Epoxy1 was immersed in DMF, TCB, xylenes, THF, and heptane respectively at ambient temperature for 96 h. The color of these solutions did not change , indicat−3 g/min (Epoxy1) to ~4.5 × 10−4 g/min (Epoxy5) with the increase of GA dosage. Epoxy1 can be completely dissolved after soaking in EG at 180 °C for ~70 min, while that for Epoxy5 with the highest crosslink density was ~20 h  showed a remarkable decrease with the increase of DEO content: 2.91 mmol/cm3 for Epoxy5 vs 1.28 mmol/cm3 for REP5-20  reclaimed through evaporating excess EG can be reused to prepare new epoxy resins. Taking Epoxy5 as an example a, the fr REP5-20 . Figure formance . As showat break . Common at break ,75. The Tg (87.73 °C), tensile strength (65.59 MPa), and elastic modulus (1390.24 MPa). The preparation and recycling of these resins did not need other expensive and toxic organic salt or strong base catalysts such as Zn(Ac)2, TBD, etc. DMP-30 played a dual role of participating in the curing process and activating the TERs during the recycling. Five kinds of epoxy resins with different crosslink densities were prepared by adjusting the ratio of epoxy monomer and anhydride. The thermal and mechanical properties of the resins varied with GA content. The influences of crosslink density, DMP-30 content, and temperature on the dissolution rate were then studied. Specifically, epoxy resins with higher crosslink density had a lower dissolution rate in EG. The increase of DMP-30 content and temperature could advance the initiating point of dissolution and speed up the decomposition of epoxy resins. Finally, the decomposed epoxy oligomer reclaimed through evaporating excess EG was used as a reactive ingredient to prepare new epoxy resins with high modulus and ductility. The method proposed in this work is low-cost and easy to implement, which provides a new strategy for the modification, classification, and recovery of epoxy resins. Further work is needed to explore whether the recycling mechanism is suitable for other epoxy-anhydride-tertiary amine catalyst systems. This work may impact the applications of epoxy resins in detachable adhesives, removable electronic encapsulation, non-destructive surface coatings, recyclable composite materials, and so on.In this work, DGEBA, GA, and DMP-30 were used to synthesize recyclable high-performance epoxy resins. Compared with early degradable epoxy vitrimers, the resins prepared in this work exhibited a higher


CXC chemokine receptor type 4 (CXCR4), which is highly expressed in cancer stem cell populations in tumor patients. However, the mechanisms of nobiletin in cancer stem cells (CSCs) remain elusive. This study was aimed to explore the potential target and mechanisms of nobiletin in cancer stem cells using bioinformatics approaches. Nobiletin treatment on MDA-MB 231 cells reduces the expression of Gene expression profiles by public COMPARE predicting the sensitivity of tumor cells to nobiletin. Functional annotations on gene lists are carried out with The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8, and WEB-based GEne SeT Analysis Toolkit . The protein-protein interaction (PPI) network was analyzed by STRING-DB and visualized by Cytoscape. ESR1, NCOA3, and RPS6KB1 and one significant module were filtered out and selected from the PPI network. Microarray analyses reveal many genes involved in protein binding, transcriptional and translational activity. Pathway enrichment analysis revealed breast cancer regulation of estrogen signaling and Wnt/ß-catenin by nobiletin. Moreover, three hub genes, i.e. Nobiletin might serve as a lead compound for the development of CSCs-targeted drugs by targeting estrogen and Wnt/ß-catenin signaling. Further studies are needed to explore the full therapeutic potential of nobiletin in cancer stem cells. Recent studies have shown that the ability of tumors to develop and propagate depends on a small population of cells called cancer stem cells (CSCs) . CSCs are responsible for resistance to chemotherapy and radiotherapy . Conventional chemotherapy has proved to be able to reduce tumor size; however most of the tumor relapsed because the population of CSCs that were able to survive and grow into tumor bulk . The CSC-targeted therapy will target the CSCs population whose slowly growth , and thus the effectiveness of cancer therapy will be achieved. Collectively, CSC-targeted therapy is needed to prevent relapse after chemotherapy. Flavonoid compounds have been shown to overcome chemoresistance  and to inhibit CSCs . One potential flavonoid compound to be developed as CSC-targeted drugs is nobiletin . PreviouIn this study, we used comprehensive bioinformatics analysis to explore nobiletin cytotoxicity and mechanism in CSCs. Analysis of the public library from the COMPARE database was done to produce a list of drugs that have similarities with nobiletin, as well as a gene list that was influenced by nobiletin on the NCI 60 cell line panel. From the microarray data, functional annotations are then carried out to predict molecular mechanisms, functions and roles of these genes. Furthermore, an analysis of protein-protein interaction was performed from the gene list. Hence we provide information about the possible molecular mechanisms of the nobiletin and its molecular targets against cancer stem cells.Data collection and processingCytotoxicity and mRNA arrays data were obtained from the NCI 60 DTP website (http.dtp.nci.nih.gov) . COMPARE analysis with the public library produces a list of drugs that have similarities with nobiletin, as well as a list of gene expressions on the NCI 60 cell line panel . The similarity pattern is expressed as the Pearson correlation coefficient. In this study, the list of compounds and genes was limited to the Pearson correlation coefficient <-0.5 and> 0.5.Functional and pathway enrichment analysisGene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out by The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 , with p<0.05 was selected as the cutoff value. Moreover, pathway enrichment was also conducted busing Overrepresentation Enrichment Analysis (ORA) from WEB-based GEne SeT AnaLysis Toolkit  with FDR<0.05 was selected as the cutoff value . Construction of PPI network and module analysisProtein-protein interaction (PPI) network was constructed with STRING-DB v11.0 . Confidence scores >0.7 were considered significant. PPI network was visualized by Cytoscape software. Genes with a degree score more than 5, analyzed by CytoHubba plugin, were selected as hub genes.COMPARE analysis reveals mRNA target list and standard agent50 value  while FRAT1 shows the Pearson correlation coefficient of 0.543. Both FRAT1 and FRAT2 are regulatory genes of Wnt/ß-catenin signaling. ESR1 shows the Pearson correlation coefficient of 0.525. Level of mRNA expression analysed by COMPARE showed 108 genes regulated by nobiletin , which 1Gene ontology analysis of potential nobiletin target genesESR1 and NCOA3), and play a role in the molecular function of protein binding, transcriptional and translational activity, as well as steroid hormone activity, e.g. ESR1. Gene ontology analysis was classified into biological process, cellular component and molecular function . There aKEGG pathway enrichment, protein-protein interaction (PPI) network construction and module selectionESR1, NCOA3 and RPS6KB1 .This study analyzed the molecular mechanism of nobiletin in CSCs using bioinformatics approaches. A pharmacological network analysis using bioinformatics approach can help to explain the potential target and mechanism of compounds in several diseases . Analysis of cytotoxicity with a public database of COMPARE showed that nobiletin exhibits cytotoxicity at the same level in NCI-60 cells panel showing by similar IC50 value. Nobiletin cytotoxicity does not depend on particular tissue. The low ICCOMPARE analysis identified 11 standard agents which have a correlation with nobiletin . TamoxifFRAT1 and FRAT2 are genes with positive, while VANGL1 is genes with negative Pearson correlation coefficient, respectively. Those genes also involve in the Wnt/ß-catenin signaling pathway. The frequently rearranged in advanced T-cell lymphomas 1 (Frat 1) and 2 (Frat 2) are positively regulator of the Wnt signaling pathway by stabilizing ß-catenin through the association with GSK-3 . Upon binding to GSK3, Frat prevents the phosphorylation and accompanying degradation of ß-catenin and allows the activation of downstream target genes . Wnt/ß-catenin signalling may be aberrantly activated through Frat1 overexpression in ovarian serous adenocarcinomas . The expression of Frat is also positively correlated with the degree of tumor differentiation and the abnormal cell expression of ß-catenin in lung cancer . Overexpression of Frat1 and abnormal expression of β-catenin were found to represent a poor prognosis for the non-small cell lung cancer patients . Frat1 demonstrates oncogenic properties in prostate cancer by inhibiting GSK 3β against β-catenin and thus promoting cell growth , while Frat2 mediates the oncogenic activation of Rac by mixed lineage leukemia fusions . VANGL1 encodes a transmembrane protein that interacts with Frizzeld a receptor of Wnt  and negatively regulates canonical Wnt/β-catenin signaling in mammalian cells. FRAT1 and FRAT2 are tumor promoting genes whereas VANGL1 is a tumor suppressor gene which involved in the Wnt/ß-catenin signaling pathway and thus posses as a molecular target of nobiletin. COMPARE analysis showed that KEGG pathway enrichment analysis revealed that estrogen and Wnt/ß-catenin signaling are regulated by nobiletin. There is only a few studies on the role of estrogen in BCSCs. A study demonstrated that estrogen treatment reduces mammosphere formation from estrogen receptor-positive breast cancer cells . Other studies showed that estrogen signaling blocking by tamoxifen induces chemoresistance due to EGFR and estrogen receptor cross talk . Expression of Wnt/ß-catenin signaling pathway-regulated genes correlates with estrogen receptor expression . Activation of Wnt/ß-catenin signaling and CSCs properties are associated with advanced progression of ER-positive breast cancer . The Wnt/ß-catenin pathway is considered to be one of the most important pathways in the regulation of CSCs . A study showed that Wnt/ß-catenin and estrogen signaling pathways cross-talk in vivo through functional interaction between ERα and β-catenin . Therefore it is interesting to further explore the effect of nobiletin in estrogen and Wnt/ß-catenin signaling as well as its cross-talk in CSCs. Pathway enrichment analysis with KEGG also showed the mTOR pathway regulated by nobiletin even the p-value is slightly greater than the cut off (p= 0.0548). The PI3K/Akt/mTOR signaling pathway is important for CSCs maintenance and could be a promising target for development of CSC-target drugs . Rapamycin and triciribine target CSC population, and inhibits migration and invasion on glioblastoma and neuroblastoma cells . COMPARE analysis showed that triciribin is one of the compounds with the highest similarity to nobiletin, and therefore the effect of nobiletin in mTOR signaling is also potential to be further explored.ESR1, NCOA3, RPS6KB1 ESR1 encodes estrogen receptor alpha which regulates estrogen signaling upon estrogen binding. Abnormal estrogen signaling leads to the development of a variety of diseases, such as cancer, metabolic and cardiovascular disease, neurodegeneration, inflammation, and osteoporosis . NCOA3 encodes nuclear receptor coactivator 3, a member of the nuclear receptor co-activator family known to be overexpressed in breast cancer and essentially involved in estrogen-mediated cancer cell proliferation . Overexpression of NCOA3 promotes breast cancer chemoresistance to tamoxifen  and paclitaxel . NCOA3 also drives the formation of cancer stem-like cells and supports tumor outgrowth in breast cancer models . Moreover, NCOA3 is a selective co-activator of ERα-mediated transactivation of PLAC1, novel cancer-associated placental in MCF-7 breast cancer cells . RPS6KB1 encodes ribosomal protein S6 kinase B1 which plays a key role in regulating protein translation and progression of hepatocellular carcinoma , prostate cancer  and small cell lung cancer . S6K1 also activates ERα and promotes the proliferation of estrogen receptor-positive breast cancer cells . Taken together, those three genes regulate estrogen signaling in breast cancer and could be evaluated for further studies of marker and target genes of nobiletin in breast cancer stem cells.There are three hub genes identified from PPI networks, i.e. Previous studies showed the role of nobiletin in estrogen, Wnt/ß-catenin and mTOR signaling. In estrogen signaling, nobiletin prevents bone loss due induced by estrogen deficiency in rats  and inhibits lower cytotoxicity on MCF-7 estrogen receptor-positive breast cancer cells than on SKBR3 HER2 positive and MDA-MB 468 triple-negative breast cancer cells . Moreover, treatment of nobiletin in lower dose decreases activity and expression of aromatase on MCF-7 cells . In Wnt/ß-catenin signaling, nobiletin inhibits its signaling pathway in hypoxia stimulated Caki-1 and 786-O renal cell carcinoma , and inhibits invasion via inhibition of AKT/GSK3ß/ß-catenin pathway in glioblastoma cells . Nobiletin shows inhibition of mTOR signaling on MDA-MB-468 triple-negative breast cancer cells . On the mTOR signaling pathway, nobiletin also protects cadmium-induced neurotoxicity induced by cadmium  and increases the sensitivity of colorectal cancer to oxiplatin . Accordingly, those studies support the present study and enhance the development potential of nobiletin as CSCs-drugs by targeting estrogen, Wnt/ß-catenin and mTOR signaling. CXCR4 in breast cancer cells . Accordingly, nobiletin is potential to target CSCs by inhibiting estrogen and Wnt/ß-catenin signaling. This present study showed that nobiletin target estrogen signaling and Wnt/ß-catenin signaling. Protein interaction networks showed three hub genes regulates estrogen signaling. A previous study also showed functional interaction between estrogen and Wnt/ß-catenin signaling . Estradiol not only stimulates the estrogen signaling pathway but also increases the cancer stem cell (CSC) population in estrogen receptor-positive breast cancer cells . Treatment with hormone antagonist in estrogen receptor-positive breast cancer cells may repress their estrogen receptors and be resistant to hormone therapy . However, a recent study showed that tamoxifen-resistant cells exhibit increased stemness properties via activation of Wnt/ß-catenin signaling . The interaction of CXC chemokine receptor type 4 (CXCR4) with its ligand CXC motif ligand 12 (CXCL12) plays important roles in maintaining CSCs properties in tamoxifen-resistant breast cancer cells , nasopharyngeal CSCs , esophageal CSCs , and stimulates the angiogenesis in vascular endothelial cells through upregulation of the MAPK/ERK and PI3K/AKT and Wnt/β-catenin pathways. . A study showed that nobiletin decreases the expression of This present study has several limitations, including the mRNA data used for the PPI network. This might give different results because the expression of mRNA is not always correlated to the protein level. This study is also using bioinformatics approaches, therefore further in vitro and in vivo studies are needed to validate the results as well as to explore the full therapeutic potential of nobiletin on CSCs.ESR1, NCOA3, and RPS6KB1 and one significant module were filtered out and selected from the PPI network. Taken together, using a bioinformatics approach, we showed that nobiletin might serve as a lead compound for the development of cancer stem cells-targeted drugs by targeting targets estrogen and Wnt/ß-catenin signaling. In conclusion, we found that tamoxifen, triciribine phosphate and 4-ipomeanol are standard drugs with the highest score of Pearson correlation coefficient to nobiletin. Moreover, many genes involved in protein binding, transcriptional and translational activity. Importantly, pathway enrichment analysis revealed breast cancer regulation of estrogen signaling and Wnt/ß-catenin by nobiletin. In addition, three hub genes, i.e.